JP2001512036A - Compositions and methods for measuring antiviral drug susceptibility and resistance, and compositions and methods for screening antiviral drugs - Google Patents

Abstract

  (57) [Summary] The present invention provides a method for determining susceptibility to an HCV or HCMV antiviral agent, comprising: (a) introducing a resistance test vector comprising a patient-derived segment and an indicator gene into a host cell; (b) ) Culturing the host cell obtained in step (a); (c) measuring the expression of the indicator gene in a target host cell; and (d) measuring the expression of the indicator gene obtained in step (c). Comparing the expression of the indicator gene measured when performing steps (a) to (c) in the absence of the antiviral agent, the steps (a) to (c), In step (b) to step (c) or step (c), a method is provided for causing a test concentration of the HCV antiviral agent to be present. The present invention also provides a method for determining HCV or HCMV antiviral drug resistance in a patient, comprising: (a) initially determining the antiviral drug susceptibility in said patient according to the method described above, and determining a segment from said patient. (B) obtaining antiviral drug susceptibility in the same patient at a later time; and (c) antiviral drug measured in steps (a) and (b). Comparing the susceptibility at the later time point to the susceptibility at the initial time point, wherein the reduction in the antiviral susceptibility at the later time point indicates the development or progression of antiviral drug resistance in the patient. provide. The invention also provides a method for assessing the biological effect of a candidate HCV or HCMV antiviral drug compound. A resistance test vector containing a segment derived from a patient containing the HCV or HCMV gene and an indicator gene, and a host cell infected with the resistance test vector are provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This application is related to US Ser. No. 08 / 903,5, filed Jul. 30, 1997.
No. 07, claiming priority, the content of which is incorporated herein by reference.

[0002]

BACKGROUND OF THE INVENTION

Viral drug resistance The use of antiviral compounds in chemotherapy and chemoprevention of viral diseases is a relatively new development in the field of infectious diseases, especially when compared to over 50 years of experience with antibacterial antibiotics. Designing antiviral compounds is not easy. This is because viruses present a number of unique problems. Viruses replicate within cells and often require the use of host cell enzymes, macromolecules, and organelles for the synthesis of virus particles. Therefore, safe and effective antiviral compounds must be able to distinguish between the specific function of cells and the specific function of viruses with a high degree of efficiency. In addition, due to the nature of viral replication, assessment of the in vitro susceptibility of virus isolates to antiviral compounds must be performed in a complex culture system consisting of living cells (eg, tissue culture). The results from such assay systems vary widely depending on the type of tissue culture cells used and the conditions of the assay.

[0003] Given the high rate of viral replication and mutation frequency, drug resistance of the virus is an important issue. Drug-resistant mutants were first identified in Poxulurus for thiosemicarbazone (Appleyard and Way (1966) Brit. J. Exptl. Pathol. 47,144
-51), a poliovirus for guanidine (Melnicks et al. (1961) Science 134,5).
57) Influenza A virus for amantadine (Oxford et al., (1970) Nat
ure 226, 82-83; Cochran et al., (1965) Ann. NY Acad Sci 130, 423-429), and the virus in herpes simplex for iododeoxyuridine (Jweta et al., (1970) Ann. NY Acd
Sci 173, 282-291). Approximately 14 to date for various antivirals
Zero HIV drug resistance mutations have been identified to date (Mellors et al., (1995
Intnl. Antiviral News, supplement and Condra, JH et al., (1996) J Virol 70,827.
0-8276). For various antiviral agents, approximately 20 human cytomegalovirus (H
CMV) Drug resistance mutations have been identified to date (Biron (1996) Antiviral
Chemotherapy, 4,135-143).

[0004] The small and efficient genome of the virus has aided extensive research into the molecular genetics, structure and replication cycle of most important human viral pathogens. As a result, both the activity and resistance to antiviral drugs have been more accurately characterized than for any other class of drugs (Richman (1994) Trends
Microbiol, 2,401-407). The probability of the appearance of a resistant mutant is at least four factors: 1) the frequency of viral mutations; 2) the intrinsic mutability of the viral target for a specific antiviral agent; 3) the selective pressure of the antiviral agent; And 4) a function of the size and rate of virus replication. With respect to the first factor, in single-stranded RNA viruses whose genome replication lacks a proof-reading mechanism, the frequency of mutation is approximately 3 × 10 ⁻⁵ per base pair per replication cycle (Holland et al., (1).
992) Curr.Topics Microbial Immunol. 176, 1-20; Mansly et al., (1995) J Virol. 69, 508.
7-94; Coffin (1995) Science 267, 483-489). Thus, the human immunodeficiency virus (
As in the case of HIV) or hepatitis C virus (HCV), a single 10 kilobase genome would be expected to contain an average of one mutation for every three progeny virus genomes. For the second factor, the inherent mutability of the viral target site in response to a specific antiviral agent can significantly affect the probability of resistant mutants. For example, zidovudine (AZT) is another approved thymidine analog, d4T.
(E) in vitro and in vivo more easily than H
Select mutations in IV reverse transcriptase.

[0005] A likely unavoidable consequence of one action of an antiviral drug is that it confers effective selective pressure on viral replication to select drug-resistant mutants (Herrmann et al., ( 1977) Ann NY Acad Sci 284,632-7). With respect to the third factor, as drug exposure increases, the selective pressure on the replicating virus population increases, facilitating the more rapid emergence of drug-resistant mutants. For example, a higher dose of AZT
They tend to select drug-resistant viruses more quickly than at lower doses (Richman et al.,
(1990) J. AIDS. 3,743-6). This selection pressure for resistant mutants increases the probability of such mutants occurring as long as significant levels of viral replication are maintained.

[0006] A fourth factor, the size of the viral population and the rate of replication, has major consequences for the probability of emergence of resistant mutants. Many viral infections are characterized by high levels of viral replication at high viral speeds (Perelson et al., (1996) Scien
ce, 271,1582-1586; Nowak et al., (1996); PNAS 93,4398-4402). This is especially true for chronic infections with HIV and hepatitis B and C. The probability of the appearance of AZT resistance is increased in HIV-infected patients with decreasing CD4 lymphocyte counts associated with increasing levels of HIV replication (supra).

[0007] Higher levels of virus increase the probability of pre-existing mutants. The emergence of resistant populations has been shown to result from the survival and selective increase of pre-existing subpopulations that appear randomly in the absence of selective pressure. All viruses have a baseline mutation rate. Calculations of approximately 10 ¹⁰ new virions generated daily during HIV infection (Ho et al., (1995) Nature 373, 123-126) show that mutation rates of 10 ^-4 to 10 ^-5 per molecule indicate that HIV infection is It ensures that almost any single point mutation at any point in time is pre-existing. There is accumulating evidence that drug-resistant mutants actually exist in a subpopulation of HIV-infected individuals (Na
jera et al., (1994) AIDS Res Hum Retroviruses 10,1479-88; Najera et al., (1995) J Vi
rol. 69, 23-31; Havlir et al., (1996) J. Virol. 70, 7894-7899). It has also been well described that approximately 10 ^-5 proportions of drug-resistant piconavirus mutants already exist (Ah
mad et al., (1987) Antiviral Res. 8, 27-39).

Hepatitis C Virus (HCV) Hepatitis C virus (HCV) infection occurs worldwide and before it is identified,
Transfusion was the leading cause of associated hepatitis. Anti-H in blood donors from around the world
Serum prevalence of CV has been shown to vary between 0.02% and 1.23%. Also,
HCV is also a common cause of hepatitis in individuals exposed to blood products. Estimates of 150,000 annual HCV infections in the United States alone over the past decade (Alter 1993, Infect. Agents Dis. 2, 155-166; Houghton 1996, in Fields Vir
ology, 3rd edition, pp. 1035-1058).

[0009] Hepatitis C virus (HCV) is a member of the Flaviviridae family of viruses,
It is a positive-strand, non-segmented RNA virus with a lipid envelope. Other members of this family are pestiviruses (eg, bovine virus diarrhea virus, or BVDV, and classical swine fever virus, or CSFV), and flaviviruses (eg, yellow fever virus and dengue virus). See Rice, 1996 in Fields Virology, Third Edition, pp. 931-959. Although the molecular investigation of HCV replication, and thus the understanding of the function of its encoded protein, has greatly advanced through virus isolation and sequencing of the viral genome, the production of native or recombinant HCV from molecular clones has been efficient. Low levels of replication have been hampered by the lack of cell culture, however, some cells were infected with viruses from HCV-infected humans or chimpanzees, or some were transfected with RNA derived from HCV cDNA clones. Has been observed in cell lines.

[0010] HCV is closely associated with the endoplasmic reticulum and replicates in infected cells in the cytoplasm (FIG. 1).
reference). A new positive sense RNA is released and translation is initiated via an internal initiation mechanism (Wang et al., 1993, J. Virol. 67, 3338-3344; Tsukiyama-Kohara et al., 19
92, J. Virol. 66, 1476-1483). Internal initiation is dictated by a cis-acting RNA element at the 5 'end of the genome; some reports indicate that this internal ribosome entry site, or the full activity of the IRES, is Suggesting that it is observed in the first 700 nucleotides of the reading frame (ORF), which corresponds to the first 123 amino acid range (Lu and Wimme
r, PNAS 93, 1412, 1996). All of the protein products of HCV are produced by proteolytic cleavage of large (3010-3030 amino acids depending on the isolate) polyprotein, which comprises three proteases: a host signal peptidase, a viral self-cleaving metalloproteinase. NS2, or one of the viral serine proteases NS3 / 4A (see FIG. 2). The combined action of these enzymes is related to the structural proteins (C, E1 and E2) and the nonstructural proteins (NS2,
NS3, NS4A, NS4B, NS5A, and NS5B). NS
5B is a viral RNA-dependent RNA polymerase (RDRP) responsible for converting input genomic RNA into negative strand copies (complementary RNA or cRNA), which cRNA is more positive by NS5B Serves as a template for transcribing sense genome / messenger RNA.

[0011] Several laboratories and laboratories have attempted to identify and develop anti-HCV drugs. Currently, the only effective treatment for HCV is alpha-interferon, which can only control the amount of virus in the liver and blood (viral load) in relatively few infected patients (Houghton 1996, in F
ields Virology, 3rd edition, pp. 1035-1058). However, the availability of the molecular structure of the HCV serine protease, NS3 / 4A (Love et al., 1996, Cell 87, 331-342; Kim et al., 1996, Cell 87, 343-355), and protease inhibitors in the treatment of HIV-1 infection Given the success of using, there should be a feasible and viable alternative. In addition to HCV protease inhibitors, inhibitors that can specifically interfere with HCV replication include virus-specific, such as internal initiation dictated by IRES, RDRP activity encoded by NS5B, or RNA helicase activity encoded by NS3. Target activity.

As a result of their high error rate of RDRP, RNA viruses can be particularly adapted to many new growth conditions. Most polymerases in this class are estimated at one error rate at 10,000 nucleotides copied. Abbreviation
At a genome size of 9.5 kb, it appears that at least one nucleotide position in the HCV genome is mutated each time the genome is copied. Thus, it appears that drug resistance develops during chronic exposure to antiviral agents. As in the case of HIV, a rapid and convenient assay for drug-resistant HCV would greatly improve the probability of successful antiviral therapy given the selection of drugs and the non-overlapping pattern of drug-resistant genotypes. . Mutations associated with resistance can sometimes be rapidly identified by growing the virus in cell culture in the presence of the drug, a fairly successful approach used for HIV-1. . However, at present, there is no convenient cell culture system for HCV. Therefore, it is not possible to determine the exact nature of the genetic alterations that confer drug resistance in vitro. Thus, in the absence of a known list of resistance-related mutations, the preferred resistance assay relies on phenotypic reading rather than genotypic reading.

There are currently no established drug resistance assays for HCV available. However, some investigators suggest that chimeric replication, or reporter gene expression,
A chimeric virus system containing the HCV NS3 protease was devised to be dependent on S3 activity. These systems include NS3 function and its cofactor, NS4A.
Have been devised to study various embodiments of and to provide a prototype for cell-based drug screening assays.

[0014] Hirowatari et al. (Anal. Biochem. 225: 113, 1995) describe NS2-NS3 linked to the endoplasmic reticulum.
An expression vector was constructed for synthesizing -tax1 fusion protein (tax1 is a transcriptional trans-activator from HTLV-I retrovirus). NS3
Upon cleavage by (or NS2), the tax1 transactivator is released and translocates to the nucleus where it acts to activate the expression of the HTLV-1 LTR regulated reporter gene (CAT).

[0015] Halm et al. (Virology 226: 318, 1996) constructed a chimeric poliovirus containing HCV NS3 upstream of the poliovirus polyprotein; replication of the chimera is dependent on NS3 protease activity. This is because the release of the native N-terminus of the poliovirus polyprotein is essential for poliovirus replication. However,
This chimera does not contain the NS4A protein of HCV which has been shown to modify the activity of NS3.

(J. Virol. 71: 1417, 1997) constructed a chimeric Sindbis virus containing HCV NS3 / 4A upstream of a Sindbis virus polyprotein; replication of the chimera was NS3 protease activity. Depends on. This is because the release of the natural N-terminus of the Sindbis virus polyprotein is essential for inhibiting Sindbis virus replication.

In Vitro Expression System Resistance test vectors rely on cell culture for transfection, replication, and expression of the indicator gene. Others have described systems for transfecting Huh cells with intact HCV RNA synthesized in vitro from cDNA constructs and have demonstrated that replication can occur (Yoo et al., (1995). ) J. Virol. 69, 32-38). In addition, infection with HCV-infected humans or viruses present in chimpanzee serum or plasma has identified several cell lines that support HCV replication (Va
lli et al., (1997) Res. Virol. 148, 181-186; Shimizu et al. (1992) PNAS 89, 5477-5481; Shi.
(1993) PNAS 90, 6037-6041; Shimizu and Yoshikura (1994) J. Virol. 68, 84.
06-8408; Shimizu et al. (1994) J. Virol. 68, 1494-1500; Mizutani et al. 1996) J. Virol. 70
, 7219-7223). Currently, these systems are limited to those detected by susceptibility methods such as RT / PCR and do not appear to allow for efficient production of the indicator gene (IG). As new cell lines and transfection methods have been discovered, improved methods will be available soon. One example of a possible improvement would be to transfect RNA as an RNP complex prepared by in vitro transcription in the presence of purified NSSB so that transcription immediately begins uptake into cells. This strategy applies to influenza viruses (Enami and P
alese (1991) J. Virol. 65,2711-2713), rabies virus (Schenell et al. (1994) EMSC
J. 13, 4195-4203), and vesicular stomatitis virus (Lawson et al., (1995) PNAS 92, 447).
7-4481) was applied to negative strand RNA viruses.

Since flavivirus genomic long RNA is infectious, HCV vectors
It can be in the form of a cDNA construct containing a T7 RNA polymerase at the terminus and a promoter for the T7 polymerase terminator sequence at the 3 terminus. Thus, RNA can be synthesized in large quantities in vitro and transfected into cells. Another approach is to transfect cells directly with a DNA construct that contains a strong eukaryotic promoter (such as the CMV IE promoter). Potential advantages of transfection of RNA, rather than DNA, include: That is, transfection of RNA circumvents potential cell type-specific limitations of promoter activity; DN
Although translation after A transfection must be preceded by transcription and RNA processing events that cause a delay of hours or days before reaching maximum expression levels, translation of recombinant proteins is usually Occurs within minutes to hours following transfection of highly active RNA. When transfecting RNA, representation of the pseudoviral species is easier.
This is because sufficient amounts of RNA can be synthesized from uncloned RNA products by including the sequence of bacteriophage RNA polymerase in the 5 'PCR primer. This approach has been shown to maintain the diversity of poliovirus pseudospecies (Chumakov, J. Virol. 70: 7331-7334, 1996). R
The generation of the correct 5 'and 3' ends on NA is due to the promoter sequence at the 5 'end and the restriction sendnuclease recognition site at the 3' end for the viral sequences (for DNA template linearization prior to transcription, ) Can be more easily achieved by in vitro transcription. However, the 3 'end can also be generated precisely via replacement of the self-cleaving RNA ribozyme sequence present in the cDNA construct (see, eg, Chorrira et al. (199).
4), J. Biol. Chem. 269, 25856-25864).

A third transfection strategy that possesses some of the advantages of RNA transfection is the T7 RNA polymerase promoter at the 5 ′ end, and the 3 ′ end
DNA transfection of a construct containing a T7 RNA polymerase terminator into cells expressing T7 RNA polymerase. Polymerase expression can be achieved by a variety of means, the most efficient of which is probably infection of cells transfected with recombinant T7 polymerase / vaccinia virus (Fuerst et al., (1986)). PNAS 83,8122-8126).

Human Cytomegalovirus (HCMV) Human Cytomegalovirus (HCMV) is endemic throughout the world, and infection rates appear to be relatively constant throughout the year, rather than seasonally. . Humans are the only known reservoir for HCMV, and natural transmission occurs through direct or indirect contact between individuals. 0.2% to 2 born in the United States
.2% of infants are infected in utero. The other 8% to 60% become infected during the first 6 months of life as a result of infection acquired during childbirth or following lactation in the breast. Breast milk transmission is the most common aspect of HCMV transmission worldwide due to the high incidence of reactivation of HCMV infection in the breast. In most developing countries, children
40% to 80% are transmitted before puberty. In other parts of the world, 90% to 100% of the population are infected during childhood.

[0021] Human cytomegalovirus (HCMV) is a member of the Herpesviridae family. A typical herpes virion is an approximately 100-110 nm diameter icosadel tahedral capsid containing linear double-stranded DNA and 162 capsomeres with pores running down the long axis, an amorphous form surrounding the core. Consists of a "tegument" and an envelope containing a viral glycoprotein spike on its surface. Virion sizes are in the range of 120-300 nm due to differences in the thickness of the segmented layers. 3 below
There are two subgroups of herpes virus.

1. Alphaherpes virus; HSV, VZV. 1. Variable host range, relatively short regeneration cycles, rapid expansion in culture, effective destruction of infected cells, sensory ganglia. Beta herpes virus; HCMV. Limited host range, long regeneration cycles, slow progression of infection in culture. Infected cells are expanded and carrier cells are easily established. The virus is maintained in latent formation in sensory ganglia, lymphoid cells, kidney and other tissues.

[0023] 3. Gamma herpes virus; EBV. An extremely narrow experimental host range replicates in lymphoblastoid cells, causing lytic infection in some types of epithelial and fibroblastoid cells.

There are eight known herpes viruses. That is, human herpes virus 1 (herpes simplex virus 1, HSV-1) and human herpes virus 2 (herpes simplex virus 2)
, HSV-2), human herpes virus 3 (herpes zoster virus, VZV),
Human Herpesvirus 4 (Epstein-Barr Virus, EBV), Human Herpesvirus 5 (Human Cytomegalovirus), Human Herpesvirus 6, Human Herpesvirus 7, and Human Herpesvirus 8. The herpes virus genome consists of linear double-stranded (ds) DNA in virions that cyclize and concatamerize upon release from the viral capsid in the nucleus of infected cells (see FIG. 10). The herpesvirus genome ranges in size from 120 to 230 kilobase pairs (kbp). The genome is polymorphic in size within individual populations of the virus (up to 10 kbp difference). This variation is due to the presence of terminal and internal repeated sequences. Herpesviruses can be divided into six groups, AF, based on their overall genomic organization. HSV and HCM
V enters group E, where sequences from both ends repeat in reverse and are internally juxtaposed, dividing the genome into two components, L (long) and S (short), and each consisting of SEQ U _L and U _S uniquely, which is close by the inverted repeat (Fig. 10). In these viruses, both components can be reversed with respect to each other, and the form of DNA extraction from virions consists of four equimolar populations in which the relative orientation of the two components differs (see FIG. 11). .

HCMV is a beta-herpesvirus and is unique among beta-herpesviruses in that it falls into the class E genome type. The genome of HCMV is approximately 230 kb in length, is fully sequenced (EMBL sequence database accession # X17403), and has 208 predicted open reading frames (ORFs) that are greater than 100 amino acids in length. (Mocarski, ES (1996) ch. 76
In Fields Virology, BNFields, DMKnipe, 3rd edition, edited by PMHowley et al. 2447-2492; Chee et al. (1990) Curr cop Microbiol Immunol 154: 125-170). ORFs are named by their position within unique and repeated regions of the viral genome (TRL, UL, IRL, IRS and US) and are numbered sequentially.

In the naturally occurring population of viruses, the genome exists in four isomers (see FIG. 11). In HCMV, as in HSV, the LS junction can be deleted, thereby freezing the genome into one of four isomers without affecting the ability of the virus to grow in cultured cells. I do.

The HCMV genome contains a variable number of terminal repeats “a” and “a ′” that are present in variable numbers in direct orientation at both ends of the linear genome. The variable number of “a” repeats is L−S
Present at the junction in the opposite direction. The number of "a" sequences at these positions is 1-1.
The range is 0, with 1 being dominant. The size of "a" in HCMV ranges from 700 to 900 bp. The "a" sequence is responsible for cleavage and packing signals. The packing signal is two highly conserved short sequence elements located within pac-1 and pac-2 labeled "a". pac-1 and pac-2
The 220 bp fragment carrying both elements is sufficient to carry a site for cleavage / packaging and inversion on the recombinant CMV construct. The ends of the linear genome are generated by cleavage events that release a single 3 'overhanging nucleotide at either end of the genome. Genome further adjacent to a unique sequence U _L and U _S an L and S components of the genome, "b" and "b '" (or TR
L and IRL) and large inverted repeats called "c" and "c '" (or IRS and TRS) (see FIG. 10).

The HCMV replication cycle is relatively slow compared to other herpesviruses. Viral replication involves the ordered expression of a continuous set of viral genes. These sets are expressed at different time points after infection and include the α (immediate), β1 and β2 (delayed), and γ1 and γ2 (late) sets based on post-infection time points accumulating their transcripts. . DNA replication, genomic mutations, and virion polymorphism are coordinated at each step through the temporal regulation of the appropriate gene product. Expression of the gene product is rapid. Gene expression is delayed for 24-36 hours. Progeny virions begin to accumulate 48 hours after infection and reach maximal levels at 72-96 hours. In fibroblasts that allow replication, DNA replication can be detected as early as 14-16 hours after infection. HCMV stimulates host DNA, RNA and protein synthesis. HCMV replicates most rapidly in actively dividing cells, and HCMV replication is inhibited by pretreating the cells with a host cell metabolic reducing agent. HC
The MV genome circularizes shortly after infection. This ring produces a concatameric form of DNA, and genomic inversion occurs within the concatameric form of DNA. Most of the replicating DNA is larger than unit length, with terminal fragments deleted based on Southern blot analysis.

Targets for Drug Resistance Drugs currently used to treat HCMV (ganciclovir (GCV), foscarnet, cidofovir) are mutated due to mutations in two viral genes: UL97 phosphotransferase And it is known to select UL54 viral DNA polymerase.

UL97: phosphotransferase 707 amino acids (aa) (2121 bp). Mutations associated with GCV resistance were identified as aa #: 460, 520, 590, 591, 592, 593, 594, 595, 59
6, 600, 603, 607, 659, 665. The phosphotransferase protein has the following two functional domains. 1) The amino terminal 300 aa encodes the regulatory domain, and 2) the carboxy terminal 400 aa defines the catalytic domain. All known drug resistance mutations are found in the catalytic domain (approximately 1.2 kb sequence). In HSV, the thymidine kinase gene product (TK) is expressed by GC in cells.
The cause of V phosphorylation and resistance to GCV in HSV is associated with a mutation in the thymidine kinase gene. HCMV has homology to the HSV thymidine kinase gene. The gene homologous to UL97 in HSV (UL13) is a protein kinase.

UL54: viral DNA polymerase, 1242a.a. (3726 bp). Mutations in this gene can result in resistance to GCV and other nucleotide analogs (including cidofovir) and foscarnet. Mutations associated with Foscarnet resistance include aa # 700 and 715. Mutations associated with GCV resistance include aa # 301, 412, 501, 503 and 987. The mature protein has four recognition domains: 1) 5'-3 'exoRNAse H, 3'-5' exonuclease, a proposed catalytic domain, and an accessory protein binding domain.

A new therapy under development is a drug targeting CMV protease (U
L80) and DNA mutant enzymes ("terminase").

[0033] GCV-resistant HCMV was recovered from the central nervous system of patients with HCMV-related neuropathy who received long-term GCV maintenance therapy. HCMV resistant strains were selected, which were
S is preferentially located. Although it is often not possible to culture virus from cerebrospinal fluid (CSF), PCR can be used to amplify HCMV DNA.

[0034] Primary isolates of CMV can replicate slowly. In addition, there is a significant delay in the growth rate of some drug-resistant clinical isolates. In a mixed virus population, the resistant virus population may be masked by the susceptible population. Thus,
Assay results that depend on virus growth will not be reliable.

For virus culture, most assays use blood and urine. Because it is easy to get them. However, the virus from these compartments is not affected by specific tissues where disease is taking place, especially vitreous fluid and Csf.
) Can not represent the virus. Although there are a few amino acid residues that are relatively frequently modified among drug-resistant strains of herpes virus recovered from patients, the wide distribution of mutations in most strains makes rapid genetic screening methods impractical. Be realistic. Importantly, biological testing for antiviral susceptibility of HCMV is more informative as the susceptibility phenotype of drugs derived from individual genetic alterations is complex and variable.

Currently Available Virus Resistance Assays The definition of viral drug susceptibility is generally defined as the concentration of antiviral agent at which a given percentage of viral replication is inhibited (eg, the IC ₅₀ for an antiviral agent is the ₅₀ % of viral replication). % Is the concentration that is inhibited). Thus, reduced viral drug susceptibility is a hallmark that the antiviral agent has already been selected for the mutant virus that is resistant to the antiviral agent. Viral drug resistance is generally defined as the decrease in viral drug susceptibility in a given patient over time. In a clinical sense, viral drug resistance is evidenced by antiviral drugs that are less effective or no longer clinically effective in patients.

Several types of assays are available for detecting and measuring the antiviral drug susceptibility of HCMV. The two most commonly used methods are plaque reduction assays and DNA hybridization assays. Currently, plaque reduction assays are considered standard. Both assays require HCMV isolation and passage in cell culture. Generally, it takes 4 to 6 hours to get the results from the assay.

[0038] Now, sensitized plaque reduction assays can be performed directly on clinical specimens, including blood, urine, bronchoalveolar lavage, and cerebrospinal fluid. Two assays modified from the standard plaque reduction assay detect either the CMV immediate early antigen or the late antigen. The procedure is substantially identical to the standard plaque reduction assay, except that the virus is tested directly without prior passage and the incubation time is reduced to 96 hours (Gerna et al., ( 1995) J.
Clin. Microbiol. 33, 738-741). The limitation of these assays is that they can only be performed in patients with high levels of viremia. Again, the essential step in the detection of drug resistant isolates is still the culture of the virus.

Another approach is to detect specific viral DNA mutations associated with drug resistance. In this assay, PCR primers are used to amplify the viral DNA, and the viral DNA is genotyped using restriction sites present in the mutant DNA but not the wild-type DNA. 2 with a total of 3 or 4 restriction digests
Analysis of one PCR product detects 78-83% of UL97 mutants that are resistant to ganciclovir (some mutations in UL97 encoding phosphotransferase result in resistance to ganciclovir). (Chou et al., (1995), J. Infect. Dis. 172, 239-242). A major limitation of this assay is that infrequent or new resistant mutants are not identified. Also,
No DNA polymerase mutant (UL54), which is indicative of high levels of ganciclovir resistance and high probability of multidrug resistance, is detected.

It is an object of the present invention to provide a drug sensitivity and resistance test that can indicate whether a viral population in a patient is resistant to a given given drug. Another object of the present invention is to provide a test that allows a physician to replace one or more drugs in a treatment guideline for a patient who has become resistant to a given drug or drug after a previous course of treatment. Is to do. It is a further object of the present invention to provide a test that allows for the selection of an agent that is effective in treating a viral infection. Yet another object of the present invention is to provide for clinical and research applications,
It is to provide a safe, standardized, available, fast, accurate and reliable assay for drug sensitivity and resistance. Yet another object of the present invention is to provide tests and methods for assessing the biological effects of candidate drug compounds acting on specific viral genes and / or viral proteins, particularly with respect to viral drug resistance and cross-resistance. To provide. It is another object of the present invention to provide means and compositions for evaluating viral drug resistance and susceptibility. This and other objects of the invention will be apparent from the entire specification.

[0041]

DETAILED DESCRIPTION OF THE INVENTION

To better understand the invention described in the specification, the following description is provided.

The following flow chart shows various vectors and host cells that can be used in the present invention. It is not intended to be all-inclusive.

Vector Indicator gene cassette + virus vector (functional / non-functional indicator (genomic or subgenomic) gene) ↓ Indicator gene virus vector (functional / non-functional indicator gene) ↓ + patient sequence acceptor site ↓ + patient-derived Segment ↓ Resistance test vector (segment derived from patient + indicator gene) Host cell Packaging host cell-transfected with packaging expression vector Resistance test vector host cell-Packaging host cell transfected with resistance test vector Target host cell A host cell to be infected by the resistance test vector virus particles produced by the resistance test vector host cell. Components of the resistance test vector system containing the indicator gene can be delivered to the target host cell at the time of infection, or can be stably integrated into the target host cell chromosomal DNA.

Resistance Test Vector A “resistance test vector” refers to a D including a patient-derived segment and an indicator gene.
It refers to one or more vectors that together contain NA or RNA. When the resistance test vector contains two or more vectors, the segment derived from the patient can be contained in one vector, and the indicator gene can be contained in a different vector. For clarity, a resistance test vector comprising two or more vectors is referred to herein as a resistance test vector system, but should also be understood as a resistance test vector. The DNA or RNA of the resistance test vector can thus be contained in one or more DNA or RNA molecules. In one embodiment, the resistance test vector is created by inserting a segment from the patient into the indicator gene viral vector. In another embodiment, the resistance test vector is created by inserting a segment from the patient into a packaging vector, while the indicator gene is contained in a second vector, eg, an indicator gene viral vector. As used herein, “patient-derived segment” refers to various means such as cells of an infected patient (eg,
For example, molecular cloning or polymerase chain reaction of a segment population from a patient using viral DNA or complementary DNA (cDNA) prepared from viral RNA present in peripheral blood mononuclear cells, PBMC), serum or other bodily fluids. PCR) refers to one or more viral segments obtained directly from a patient by using amplification. If the viral segment is "directly obtained" from the patient, it is obtained without passage of the virus by culture, or when culturing the virus, the selection of mutations in culture is minimized by minimizing the number of passages. Exclusion. The term "viral segment" refers to the gene product (
For example, any functional viral sequence or viral gene that encodes a protein). As used herein, the term “functional viral sequence” refers to enhancers, promoters, polyadenylation sites, sites of action of trans-acting factors, internal ribosome entry sites (IRES), translation frameshift sites, packaging sequences, integrated sequences. Or any nucleic acid sequence (DNA or RNA) having a functional activity such as a splicing sequence. If the drug targets more than one functional viral segment or viral gene product,
A segment from the patient corresponding to each of the viral genes will be inserted into the resistance test vector. In the case of a combination therapy in which two or more antiviral agents targeting two different functional viral sequences or viral gene sequences are to be evaluated, a patient-derived segment corresponding to each functional viral segment or viral gene product will have a resistance test vector. Will be inserted. Depending on the particular construct to be used as described herein, the segment from the patient may be a unique restriction site or a specific location (e.g., a patient sequence acceptor) in an indicator gene viral vector or, for example, a packaging vector. Site).

As used herein, the term “patient-derived segment” includes segments from humans and various animal species. Such species include, but are not limited to, chimpanzees and other primates, horses, cows, cats and dogs.

A patient-derived segment can also be inserted into a resistance test vector using any of several alternative cloning techniques. For example, introduction of a class II restriction site into both the plasmid backbone and the patient-derived segment, or uracil DNA glycosylase primer cloning, or site-specific recombination, or cloning by exonuclease overhang cloning.

[0047] The patient-derived segment can be prepared by either molecular cloning or gene amplification, or modification thereof, as described below, by inserting the acceptor site of the patient-derived sequence into the end of the patient-derived segment to be inserted into the resistance test vector. Can be obtained. For example, in a gene amplification method such as PCR, a restriction site corresponding to a patient sequence acceptor site can be inserted at the end of a primer used in a PCR reaction. Similarly, in molecular cloning methods such as cDNA cloning, the restriction site can be incorporated at the end of a primer used in first or second strand cDNA synthesis, or can be cloned in a method such as DNA primer repair. Or, regardless of uncloned DNA,
The restriction site can be incorporated into a primer used in the repair reaction. Patient sequence acceptor sites and primers are used to
ntation). The set of resistance test vectors with the designed patient sequence acceptor site provides for the redisplay of patient-derived segments that would be underrepresented in one resistance test vector alone.

The resistance test vector system introduces a patient sequence acceptor site by modifying an indicator gene viral vector (described below), or a packaging vector, amplifies or clones a patient-derived segment, and is amplified or cloned. The sequence is prepared by correctly inserting it into the indicator gene viral or packaging vector at the patient sequence acceptor site. Next, a resistance test vector system constructed from the indicator gene virus vector is used to construct a genomic virus vector or subgenomic virus vector and an indicator gene cassette (
Each of them is derived from the following). A resistance test vector system constructed from the packaging indicator vector is now derived from a genomic or subgenomic packaging vector and an indicator gene cassette (each of which is described below). The resistance test vector is then introduced into a host cell. Alternatively, a resistance test vector (also referred to as a resistance test vector system)
Introducing a patient sequence acceptor site into the packaging vector, amplifying or cloning the patient-derived segment, inserting the amplified or cloned segment exactly into the packaging vector at the patient sequence acceptor,
It is prepared by co-transfecting this packaging vector with the indicator gene viral vector.

In one preferred embodiment, the resistance test vector is introduced into a packaging host cell together with the packaging expression vector described below, wherein the drug resistance and susceptibility are referred to as “particle-based testing”. A resistance test based virus particle used in the test can be produced. In another preferred embodiment, a resistance test vector can be introduced into a host cell in the absence of a packaging expression vector to perform a drug resistance and susceptibility test, referred to herein as a "non-particle based test."

As used herein, “packaging expression vector” refers to packaging proteins (eg, structural proteins such as core and envelope polypeptides),
It provides factors such as trans-acting factors or genes required for replication defective viruses. In such a situation, the replication competent viral genome is compromised so that it cannot replicate on its own. This is because the packaging expression vector can give rise to the trans-acting or missing genes needed to rescue the defective viral genome present in cells containing the weakened genome, The genome means that it cannot save itself.

Indicator or Indicator Gene “Indicator or indicator gene” refers to a nucleic acid, DNA or RNA structure that encodes a protein, which can be directly or via a reaction, a measurable or recognizable feature, eg, a measurement. Produces colors or light of the possible wavelengths, or DNA or R
When the NA structure is used as an indicator, it results in a change or generation of a specific DNA or RNA structure. Preferred examples of indicator genes include the E. coli lacZ gene encoding beta-galactosidase, such as Photonis pyralis (firefly) or R.
luc gene encoding luciferase derived from any of enilla reniformis (sea pansy), alkaline phosphatase, E. coli phoA gene encoding green fluorescent protein, bacterial CAT gene encoding chloramphenicol acetyltransferase, and bacteria β-lactamase gene. Further preferred examples of indicator genes are secreted or cell surface proteins that are easily measured by assays such as radioimmunoassay (RIA) or fluorescence activated cell sorting (FACS), eg, growth factors, cytokines and cell surfaces. An antigen (eg, growth hormone, I1-2 or CD4, respectively)
). "Indicator gene" is understood to also include a selection gene, also termed a selectable marker. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase or E. coli gpt, or genes encoding resistance to the antibiotics hygromycin, neomycin, puromycin or zeocin. In the case of the above example of the indicator gene, the indicator gene and the patient-derived segment are different, that is, different genes to be distinguished. In some cases, patient-derived segments can also be used as indicator genes. In one embodiment, where the patient-derived segment corresponds to more than one viral gene that is the target of the antiviral agent, one of the viral genes also serves as an indicator gene. For example, the HCV protease gene can serve as an indicator gene by virtue of its ability to cleave a chromogenic substrate or, in turn, an inactive zymogen that cleaves a chromogen, producing a chromogenic reaction in each case. . In a second example, the HCMV phosphotransferase gene can serve as an indicator gene by its ability to phosphorylate a substrate and thereby up- or down-regulate its activity. In all of the above examples of the indicator gene, the indicator gene can be "functional" or "non-functional", but in each case, the expression of the indicator gene in the target cells ultimately depends on the effect of the patient-derived segment .

Functional Indicator Gene In the case of a “functional indicator gene”, the indicator gene is independent of the patient-derived segment,
The target, as defined below, could be expressed in a “packaging host cell / resistance test vector host cell” as defined below, but without the production of a functional resistance test vector particle and effective infection of the target host cell. It will not be able to be expressed in host cells. In one embodiment of a functional indicator gene, an indicator gene cassette comprising a gene encoding a control element and an indicator protein is provided in the indicator gene viral vector or packaging virus vector in a natural or exogenous enhancer / viral enhancer of the viral vector. Inserted in the same or opposite transcription orientation as the promoter. One example of a functional indicator gene in the case of HCV is to replace the indicator gene and its promoter (CMV IE enhancer / promoter) with the same or opposite transcription direction as the HCV enhancer-promoter, respectively, or to use the T7 associated with the viral vector. Phage RNA polymerase promoter (here T7
Promoter).

Non-Functional Indicator Gene Alternatively, the indicator gene may be a package transfected with a resistance test vector until the indicator gene is converted to a functional indicator gene via the action of one or more patient-derived segment products. May be "non-functional" in that it is not efficiently expressed in a host cell (referred to as a resistance test vector host cell). The indicator gene is rendered non-functional through genetic manipulation according to the present invention.

1. Exchanged Promoter In one embodiment, the promoter is in the same transcriptional orientation as the indicator gene,
At some point, not preceding or following the indicator gene coding sequence, the indicator gene is rendered non-functional by the location of the promoter. This misplaced promoter is referred to as the "replaced promoter." The non-functional indicator gene and its exchanged promoter are rendered functional by the action of one or more viral proteins. In one example of a non-functional indicator gene with an exchanged promoter in the case of HCMV, the promoter in the "b" region and the IRES of the indicator gene is identified as c
Put '/ and / or coding and stop region of a marker gene in the adjacent U _S region. Nonfunctional indicator gene in the resistance test vector, by repositioning the CMV IE promoter with respect to the indicator gene coding region, upon infection of target cells, it is converted into a functional indicator gene reversal of U _L / U _S junction You. Following reversal, the appropriately located indicator gene is expressed in the target cells.

[0055] The exchanged promoter can be a eukaryotic or prokaryotic promoter capable of being transcribed in the target host cell. In one example, a CMV IE promoter / enhancer region can be used. In a second example, the promoter will be small in size to allow insertion into the viral genome without disrupting viral replication. More preferably, a promoter will be used that is small in size and can be transcribed by a single subunit RNA polymerase introduced into the target host cell, such as a bacteriophage promoter. Examples of such bacteriophage promoters and their homologous RNA polymerases include:
Phages T7, T3 and Sp6 are included. Nuclear localization sequences (NLS) can be attached to RNA polymerases to localize RNA polymerase expression to the nucleus where they may be required to transcribe the repaired indicator gene. Such NLS can be obtained from any nuclear transport protein, such as the SV40T antigen. Using phage RNA polymerase, the EMC virus 5 'untranslated region (
An internal ribosome entry site (IRES), such as (UTR), can be added in front of the indicator gene, generally for transcription of the uncapped transcript.
In the case of HCMV, the exchanged promoter can be introduced at any position that does not destroy the cis-acting elements required for HCMV DNA replication. If there is inappropriate expression of the non-functional indicator gene by the transfection artifact (DNA ligation), a blocking sequence can be added to the end of the resistance test vector. In the HCMV example of the exchanged T7 promoter given above, such a blocking sequence may consist of a T7 transcription terminator positioned to block read-through transcription from DNA ligation.

[0056] 2. Exchanged coding region In a second embodiment, the 3 ′ coding region precedes, rather than after, the 5 ′ coding region because of the relative position of the 5 ′ and 3 ′ coding regions of the indicator gene. The gene is rendered non-functional. This misplaced coding region is referred to as the "replaced coding region." The orientation of the non-functional indicator gene should be the same as the orientation of the native or exogenous promoter / enhancer of the viral vector, since the mRNA encoding the functional indicator gene will be produced regardless of the orientation of the promoter. Or vice versa. The non-functional indicator gene and its exchanged coding region are rendered functional by the action of one or more patient-derived segment products. Examples of non-functional indicator genes with exchanged coding regions in the case of HCMV include a 5 'indicator gene coding region with an associated promoter in the b region of the HCMV genome and a 3' indicator gene coding region in the genome. c 'placed in the area and / or adjacent U _S region, the coding region has an opposite transcriptional direction. In both examples, 5 '
And the 3 'coding region can each also have an associated splice donor and acceptor sequence, which can be a heterologous or artificial splicing signal. Since the exchanged coding region prevents the formation of a functional transcript, the indicator gene can be functionally transcribed by an associated or viral promoter. The non-functional indicator genes in the resistance test vector are 5
'And 3' derived from the rearrangement of the indicator gene coding region, upon infection of target cells, and converted into a functional indicator gene by reverse rotation of the U _L / U _S bonding. Following transcription by the promoter associated with the 5 'coding region, the appropriately placed 5'
RNA with 'and 3' coding regions yields a functional indicator gene product.

[0057] 3. Negative Strand RNA Coding Region In a third embodiment, the indicator gene is rendered non-functional by the fact that it is expressed from RNA that is negatively sensed for the product of the gene encoded by the virus. Expression of luciferase from minigenomic RNA containing the luc gene in reverse requires negative strand RNA created during viral replication (
(Fig. 5).

Indicator Gene Virus Vector—Construction As used herein, “indicator gene virus vector” refers to a vector containing an indicator gene and its control elements and one or more viral genes. An indicator gene viral vector is an indicator gene cassette and a "virus vector" as defined below.
Assemble from. Indicator gene viral vectors can further include enhancers, splicing signals, polyadenylation sequences, transcription terminators, or other regulatory sequences. In addition, the indicator gene viral vector may be functional or non-functional. If the viral segment that is the target of the antiviral drug (for drug resistance and susceptibility testing is from the patient) is not included in the indicator gene viral vector, then in a second vector, which may be a packaging viral vector, Provided. "Indicator gene cassette" includes an indicator gene and a control element. "Viral vector" refers to a vector that contains some and all of the following: viral genes encoding gene products, regulatory sequences, viral packaging sequences. Viral vectors can further include one or more viral segments, one or more of which may be targets for antiviral agents. Two examples of viral vectors containing viral genes are referred to herein as "genomic viral vectors" and "subgenomic viral vectors." A “genomic viral vector” is a vector that contains a deletion of one or more viral genes and is capable of disabling viral replication but otherwise retaining mRNA expression and processing characteristic of the entire virus. It is.

In one embodiment of the HCV drug susceptibility and resistance test, the genomic viral vector comprises C, E1, E2, NS2, NS4, and NS5 (supra, 5
See page 2-53). In one embodiment of the HCMV drug susceptibility and resistance test, the genomic viral vector comprises a virus in which one or several genes have been deleted, such as JL54, UL80, UL97. "Subgenomic viral vector" refers to a vector that contains one or more viral gene coding regions that can encode a protein that is a target of an antiviral drug. In the case of HCV, a preferred embodiment is a subgenomic viral vector containing the HCV, NS2, NS3, NS4, NS5 genes (Figure 6). For HCMV, the preferred embodiment is UL54
Containing one or several viral genes, such as, UL80, UL90, etc.
It is a subgenomic virus vector containing an amplicon plasmid. Examples of viral clones used in viral vector construction include: Towne, Toledo, and AD16
9 Viral coding genes include natural enhancers / promoters,
Alternatively, it may be under the control of an exogenous virus or cell enhancer / promoter. A preferred embodiment for HCV drug susceptibility and resistance testing is to place the genomic or subgenomic viral coding region under the control of a T7 promoter. A preferred embodiment for HCMV drug susceptibility and resistance testing is to place the genomic or subgenomic viral coding region under the control of an endogenous HCMV promoter. In the case of an indicator gene, the viral vector containing one or more viral genes that are targets or encoding a protein that is the target of an antiviral agent (s), and thus encoding the vector, may have a patient sequence acceptor site. contains. The patient-derived segment is inserted into a patient sequence acceptor site in an indicator gene virus vector, a resistance test vector, as described below.

A “patient sequence acceptor site” is a site in a vector for inserting a patient-derived segment, which is: 1) a unique restriction site introduced into the vector by site-directed mutagenesis; A) a naturally occurring unique restriction site in the vector; or 3) a selected site into which a patient-derived segment can be inserted using alternative cloning methods (eg, UDG cloning, exonuclease overhang cloning). 4) It may be a site-specific recombination target site. In one embodiment, the patient sequence acceptor site is inserted into the indicator gene viral vector. The patient sequence acceptor site is preferably located within or near the coding region of the viral protein that is the target of the antiviral drug. The viral sequence used in the introduction of the patient sequence acceptor site preferably changes in the amino acid coding sequence found at that position,
Or it is chosen so that no conservative changes are made. Preferably, the patient sequence acceptor site is located within a relatively conserved region of the viral genome to facilitate the introduction of a segment from the patient. Alternatively, the patient sequence acceptor site is located between functionally important genes or regulatory sequences. The patient-sequence acceptor site should be located at or near a relatively conserved region in the Usurus genome to allow priming by the primers used to introduce the corresponding restriction site into the patient-derived segment Can be. To further improve the presentation of patient-derived segments, such primers can be designed as degenerate pools to accommodate viral sequence heterogeneity, or can be used to retain residues such as deoxyinosine (I) having multiple base pairing capabilities. Groups can be incorporated. A set of resistance test vectors having patient sequence acceptor sites defining identical or overlapping restriction site spacing are used together in drug resistance and susceptibility tests to contain internal restriction sites identical to a given patient sequence acceptor site. Thus, it is possible to provide presentation of segments from patients that would be underestimated in the resistance test vector alone.

Host Cell The resistance test vector is introduced into a host cell. Suitable host cells are mammalian cells. Preferred host cells are derived from human tissues and cells, which are primary targets for viral infection. In the case of HCV, these include human cells, such as liver cells, liver tumor cell lines and other cells. In the case of HCMV, suitable host cells include MRC5, HF, human foreskin fibroblasts and other cells. Host cells of human origin will ensure that the antiviral agent has effectively entered the cell and is converted by the cellular enzyme machine into a metabolically relevant form of the antiviral inhibitor. The host cell here
They are referred to as "packaging host cells,""resistance test vector host cells," or "target host cells." A "packaging host cell" is a host cell that provides a trans-acting factor and a viral packaging protein required by a replication-defective viral vector used herein, such as a resistance test vector to generate a resistance test vector viral particle. Say. The packaging protein can be provided by expression of a viral gene contained in the resistance test vector itself, the packaging expression vector (s), or both. Packaging host cells
A host cell that is transfected with one or more packaging expression vectors. When transfected with a resistance test vector, it is referred to as a "resistance test vector host cell", and is sometimes referred to as a packaging host cell / resistance test vector host cell. Name. Preferred host cells used as packaging host cells for HCV include huh7, HepG2. Preferred host cells used as packaging host cells for HCMV include MRC5 and HF. "Target host cell" refers to a cell to be infected by a resistance test vector virus particle produced by a resistance test vector host cell, in which expression or inhibition of the indicator gene occurs. Preferred host cells used as target host cells for HCV include HepG2 (Hiramatsu et al. (1997), J. Viral Hepatol. 4 (supplement 1), 61-67), Huh.
7 (Yoo et al., (1995), J. Virol. 69, 32-38), Vero (Valli et al. (1997) Res. Virol. 148).
, 181-186), Molt4Ma (Shimizu et al., (1992), PNAS 89, 5477-5481), HP BMa (Shimizu et al., (1993), PNAS 90, 6037-6041; Shimizu and Yoshikura (1994).
, J. Virol. 68,8406-8408; Shimizu et al., (1994), J. Virol. 68, 1494-1500), MT-2 (Mizutani et al., (1996), J. Virol. 70, 7219-7223). Is included. Preferred host cells used as target host cells for HCMV include MRC5 and HF.

Drug Sensitivity and Resistance Test The drug sensitivity and resistance test of the present invention can be performed on one or more host cells. Viral drug susceptibility is determined as the concentration of an antiviral agent at which a given percentage of the indicator gene expression is inhibited (eg, the IC ₅₀ for an antiviral agent is the concentration at which 50% of the indicator gene expression is inhibited). ). By using the method of the present invention, a virus segment that is a standard laboratory virus segment or a virus segment obtained from a patient who is na に 対 し て ve to the drug (ie, a patient who has never taken any antiviral agent). For, a standard curve for drug sensitivity of a given antiviral drug can be constructed. Correspondingly, viral drug resistance is measured over time as measured by comparing drug sensitivity to such a given standard or by increasing inhibition of indicator gene expression inhibition (eg, decreased indicator gene expression). The reduction of viral drug susceptibility for a given patient by taking sequential measurements in the same patient.

In the first type of drug susceptibility and resistance test, the first host cell prepared by transfecting a packaging host cell with the resistance test vector and the packaging expression vector (s) (resistance test vector host cell) This produces a resistance test vector virus particle. Next, a second host cell (target host cell) in which the indicator gene is measured is infected with the resistance test vector virus particle. Two such cell lines, including a packaging host cell transfected with the resistance test vector, and thus referred to as a resistance test vector host cell, and a target cell, are used in the case of a functional or non-functional indicator gene. The functional indicator gene is effectively expressed upon transfection of the packaging host cell, and the test of the present invention requires infection of the target cell with the supernatant of the resistance test vector host cell. A non-functional indicator gene with an exchanged promoter, exchanged coding region, or negative sense strand indicator RNA will not be efficiently expressed upon transfection of the packaging host cell, and will therefore infect the target host cell. Can be achieved by co-culturing with a resistance test vector host cell and a target host cell, or through infection of the target host cell with the resistance test vector host cell supernatant. In the second type of drug susceptibility and resistance testing, a single host cell (resistance test vector host cell) also serves as the target host cell. The packaging host cells are transfected to generate resistance test vector virus particles, and some of the packaging host cells are targeted for infection by the resistance test vector particles. Drug susceptibility and resistance testing using a single host cell type is possible with a viral resistance test vector that contains an exchanged promoter, an exchanged coding region, or a non-functional indicator gene with a negative sense strand indicator RNA. . Such an indicator gene is not effectively expressed upon transfection of the first cell, but is only effectively expressed upon infection of the second cell, and thus measures the effect of the antiviral agent being evaluated. Offer the opportunity. In the case of drug susceptibility and resistance tests using a resistance test vector containing a functional indicator gene, neither co-culture techniques using single cell types nor resistance and susceptibility tests can be used to infect target cells. Applies to HCMV). A resistance test vector containing a functional indicator gene requires two cell lines that use the filtered supernatant from the resistance test vector host cell to infect the target host cell.

In the case of HCV, in one embodiment of the invention, a genomic viral vector,
That is, pXHCV-luc; pXHCV-IRESluc; pXHCV / pxIRESluc; pXHCV / pXASIRESluc;
A particle-based resistance test is performed with a resistance test vector derived from pXluc-NSHCV / pXsHCV; pXBVDV (HCVNS3) luc; pXBVDV (HCVNSSB) luc. In the case of HCMV, one of the present inventions
In one embodiment, the packaging expression vector HCMVβ _2,7 FIG /
Genomic virus vector co-transfected with ΔgeneX or HCMVΔgeneX, ie, pA-CMV-VS-geneX; pA-CMV-CS-geneX; pA-CMV-VS-
geneX- (NF-IG) PP / pA-CMV-CS-geneX- (NF-IG) PP; pA-CMV-VS-geneX- (NF-IG) PCR / pA
-CMV-CS-geneX- (NF-IG) PP; pA-CMV-VS-geneX- (NF-IG) PCR / pA-CMV-CS-geneX- (NF-
IG) PCR: Perform a particle-based resistance test using a resistance test vector derived from pA-CMV-VS-geneX-F-IG / pA-CMV-CS-geneX-F-IG.

In the case of particle-based susceptibility and resistance tests, a resistance test vector viral particle is produced by transfecting a packaging host cell with a resistance test vector and a packaging expression vector. The resistance test vector viral particles are then used to infect a second host cell in which expression of the indicator gene is measured. In a second type of particle-based sensitivity and resistance test, a single host cell type (resistance test vector host cell) serves both purposes; transfecting some of the packaging host cells in a given culture and The test vector gives rise to virus particles and some of the host cells in the same culture are targets for infection by the thus produced resistant test vector particles. Resistance testing using a single host cell type is possible with resistance testing vectors that include a non-functional indicator gene with an exchanged promoter. Because such indicator genes are effectively expressed upon infection of permissive host cells, they are not effectively expressed upon transfection of the same host cell type, and therefore, the efficacy of the antiviral agent under evaluation. Because it provides an opportunity to measure For similar reasons, a resistance test using two cell types involves co-culturing the two cell types as an alternative to infecting the second cell type with virus particles obtained from the supernatant of the first cell type Can be done by doing so.

The packaging host cell is transfected with the resistance vector and the appropriate packaging expression vector (s) to obtain a resistance test vector host cell. Individual antiviral agents for HCV, including protease inhibitors, IRES inhibitors, and polymerase inhibitors and combinations thereof, are packaged at the appropriate range of concentrations at the time of their transfection into packaging host cells. Add to individual plates. 24 to 48 hours after the transfer, the target host cells are co-cultured with the resistance test vector host cells or with the resistance test vector virus particles obtained from the filtered supernatant of the resistance test vector host cells. Infect. Each antiviral agent or combination thereof is added to the target host cells prior to or at the time of infection to achieve the same final concentration of the given agent, or agents, present during the transfection. .

Measurement of the expression or inhibition of the indicator gene in target host cells infected by co-culture or with the filtered viral supernatant can be performed, for example, if the indicator gene is firefly luc
In the case of a gene, it is determined by measuring luciferase activity by expression of an indicator gene. Observed in target host cells infected with a given preparation of resistance test vector viral particles in the presence of a given antiviral agent or agents, as compared to a control run in the absence of the antiviral agent. The decrease in luciferase activity generally relates to the log of the concentration of the antiviral agent as a sigmoid curve. The inhibition curve is used to calculate the apparent inhibitory concentration (IC) of the agent or agent combination for the viral target product encoded by the patient-derived segment present in the resistance test vector.

In one case of cell sensitivity and resistance, the host cell is transfected with the resistance test vector and the appropriate packaging expression vector (s) to generate a resistance test vector host cell. Individual antiviral agents, or a combination thereof,
At an appropriate range of concentrations, add to individual plates of transfected cells at the time of their transfer. At appropriate times after the transfection, cells are harvested and assayed for firefly luciferase activity. Transfected cells in culture, as well as hyperinfected cells in culture, can serve as target host cells for indicator gene expression because transfected cells in culture do not express the indicator gene effectively. it can. The decrease in luciferase activity observed in cells transfected in the presence of a given antiviral agent or agents as compared to a control run in the absence of the antiviral agent (s) is generally indicated by a sigmoidal curve. As log of the concentration of antiviral agent. Using this inhibition curve, the apparent inhibitory concentration (IC) of the agent or agent combination is calculated for the viral target product encoded by the patient-derived segment present in the resistance test vector.

Antiviral Drug / Drug Candidate The antiviral drug to be added to the test system is added at a time selected depending on the target of the antiviral drug. For example, in the case of HCV protease inhibitors, they are added at an appropriate range of concentrations to individual plates of packaging host cells at the time of their transfer in a resistance test. HCV protease inhibitors can also be added to target host cells at the time of infection to achieve the same final concentration added during the transfection. In HCMV, phosphotransferase, DNA polymerase and protease inhibitors (including GCV, Sidhobil, Foscarnet) are added at test concentrations to individual target host cells at the time of transfer / infection with the resistance test vector virus particles. Add to plate. In addition, antiviral agents can be present throughout the assay. The test concentrations are selected from a range of concentrations designed to give a satisfactory inhibition profile for resistant and sensitive isolates.

In another embodiment of the invention, candidate antiviral compounds are tested in the drug susceptibility and resistance tests of the invention. Candidate antiviral compounds at appropriate concentrations
Add to the test system at selected times depending on the protein target of the candidate antiviral agent. Alternatively, can one test more than one candidate antiviral compound?
Alternatively, candidate antiviral compounds can be tested in combination with an approved antiviral drug such as GCV for HCMV or a compound undergoing clinical trials. The efficacy of a candidate antiviral agent will be assessed by measuring the suppression or inhibition of the indicator gene. In another aspect of this embodiment, drug mutants can be screened using drug sensitivity and resistance tests. Following the identification of resistant mutants to either a known or candidate antiviral agent, the resistant mutants are isolated and the DNA analyzed. Thus,
A library of virus resistant mutants can be assembled and screened for candidate antiviral agents, alone or in combination. This allows those skilled in the art to identify effective antiviral agents and design effective treatments.

General Materials and Methods Most of the techniques used to construct vectors, transfect and infect cells are widely practiced in the art, and most practitioners describe specific conditions and techniques. Be familiar with standard resource materials. However, for convenience, the following paragraphs can serve as guidelines.

“Plasmids” and “vectors” are denoted by a lower case p followed by letters and / or numbers. Here, the starting plasmids are commercially available, publicly available on a non-restrictive basis, or can be constructed from available plasmids according to published procedures. In addition, plasmids equivalent to the described are known in the art and will be apparent to those skilled in the art.

Construction of the vectors of the present invention uses standard ligation and restriction techniques that are well understood in the art (Ausubel et al., (1987) Current Protocols in Molecular Biology).
, Wiley-Interscience or Maniatis et al., (1992) in Molecular Cloning: A laborat
ory Manual, Cold Spring Harbor Laboraroty, NY). The isolated plasmid, DNA sequence, or synthesized oligonucleotide is cut, tailored, and religated into the desired form. The sequence of all DNA constructs incorporating synthetic DNA is D
Confirmed by NA sequence analysis (Sanger et al. (1977) Proc. Natl. Accd. Sci. 74,5463-
5467).

“Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences and restriction sites in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements are known to those skilled in the art. For analytical purposes, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. Alternatively, an excess of restriction enzyme is used to ensure complete digestion of the DNA substrate.
Variations can be tolerated, but an incubation time of about 1 to 2 hours at about 37 ° C. can be used. After each incubation, proteins are removed by extraction with phenol / chloroform, followed by ether extraction, and nucleic acids are recovered from the aqueous fraction by precipitation with ethanol. If desired, size separation of the cleaved fragments can be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separation can be found in Methods of Enzymology 65: 499-560 (1980).

The restriction fragments were 50 mM Tris (pH 7.6), 50 mM NaCl, 6 mM MgCl ₂ , 6 mM DTT and
Large amounts of E. coli DNA polymerase I (Klenow) in 5-10 micromolar dNTPs in the presence of four deoxynucleotide triphosphates (dNTPs) using an incubation time of about 15 to 25 minutes at 200 ° C. Blunt ends can be obtained by treating with fragments. The Klenow fragment fills the 5 'sticky ends, but even in the presence of four dNTPs, reversely chews the protruding 3' single strand. If desired, selective repair can be performed by providing only one dNTP, or with a selected dNTP, within the limits dictated by the nature of the sticky end. After treatment with Klenow, the mixture is extracted with phenol / chloroform and ethanol precipitated. Treatment under appropriate conditions with S1 nuclease or Bal-31 results in the hydrolysis of any single-stranded portion.

The ligation is performed in a volume of 15-50 μl under the following standard conditions and temperatures:
20 mM Tris-Cl pH7.5, 10 mM MgCl ₂ , 10 mM DTT, 33 mg / ml BSA, 10 mM-50 mM NaCl, and 40 μM ATP at 0 ° C, 0.01-0.02 (Weiss) unit T4 DNA ligase (for “sticky end” ligation ) Or 1 mM ATP at 14 ° C, 0.3-0.6 (Weiss) unit T4
DNA ligase ("blunt end" ligation). Intermolecular “sticky end” ligation is usually performed at 33-100 μg / ml total DNA concentration (5-100 mM total final concentration).
Intermolecular blunt end ligation (usually using a 10-30 fold molar excess of linker)
Is performed at a total final concentration of 1 μM.

“Transient expression” refers to unamplified expression within about one to two weeks after transfection. The optimal time for transient expression of a particular desired heterologous protein will vary depending on several factors including, for example, trans-acting factors, translation control mechanisms and host cells that can be used. Can be. Transient expression is
Occurs when the particular transfected plasmid functions, ie, is transcribed and translated. During this time, the plasmid DNA that has entered the cell is translocated to the nucleus. The DNA is unincorporated and free in the nucleus. Transcription of the plasmid taken up by the cells occurs during this period. Following the transfer, the plasmid DNA is degraded or diluted by cell division. Random integration within the cellular domain occurs.

In general, vectors containing promoter and control sequences from a species compatible with the host cell are used in the particular host cell. Promoters suitable for use in prokaryotic hosts illustratively include hybrid promoters such as the beta-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter system, and the tac promoter. However, other functional bacterial promoters are suitable. In addition to prokaryotes, eukaryotic microbes such as yeast cultures can also be used. Saccharomyces cerevisiae, or ordinary Baker's yeast, is the most commonly used eukaryotic microorganism, but many other strains are routinely available.

[0079] Promoter-controlled transcription from a vector in a mammalian host cell can be accomplished by a variety of sources, including polyoma, simian virus 40 (SV40), adenovirus,
It can be obtained from the genome of a virus such as a retrovirus, hepatitis B virus and preferably cytomegalovirus, or from a heterologous mammalian promoter, for example the β-actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. Of course, promoters from the host cell or related species are also useful herein.

[0080] The vectors used herein can contain a selection gene, also called a selectable marker. The selection gene encodes a protein required for survival or growth of the host cell transformed with the vector. Examples of suitable selectable markers for mammalian cells include the dihydrofolate reductase gene (DHFR), ornithine decarboxylase gene, multidrug resistance gene (mdr), adenosine deaminase gene, and glutamine synthase gene. When such a selectable marker is successfully introduced into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selection methods. The first category is based on the metabolism of cells and the use of mutant cell lines that lack the ability to grow independently of the supplemented media. The second category is the selection scheme used in any cell type, the dominant selection that does not require the use of mutant cell lines. These schemes typically use an agent to block the growth of the host cell. Cells with the new gene will express the protein that carries drug resistance and will survive the selection. Examples of such dominant selections include the drug neomycin (Southern and Berg (1982) J. Molec. Appl. Genet. 1,327), mycophenolic acid (Mulligan and Berg (1980) Science 209,1422), or hygromycin (Sugden et al. (1985)). Mol. Cell. Biol. 5,410-413). The three examples given above illustrate the use of bacterial genes under eukaryotic control, respectively, with the drug neomycin (G41
8 or gentisin), xgpt (mycophenolic acid) or hygromycin.

“Transfection” means introducing DNA into a host cell so that it is expressed, whether or not it is functionally expressed;
The DNA can be replicated as an extrachromosomal element or by chromosomal integration. Unless otherwise noted, the method used herein for the transformation of host cells is the calcium phosphate co-precipitation method of Graham and van der Eb (1973) Virology 52, 456-457. Another method of transfer is electroporation,
DEAE-dextran method, lipofection and biolistics (Krigler (1990) Gene Transfer and Expression: A Laboratory Manual, Stoc).
kton Press).

[0082] Host cells are transfected with the expression vectors of the present invention and suitably cultured in conventional nutrient media modified to induce promoters, selective transformants or amplified genes. Host cells are cultured in F12: DMEM (Gibco) 50:50, supplemented with glutamine and without antibiotics. Culture conditions, such as temperature, pH, etc., have been previously used in the host cell selected for expression and will be apparent to those skilled in the art.

The following examples now merely illustrate the best mode known for practicing the invention, but should not be construed as limiting the invention. All documents throughout this application are incorporated herein by reference.

Example 1 HCV Drug Susceptibility and Resistance Tests Using a Resistance Test Vector Containing a Patient-Derived Segment (s) and a Functional Indicator Gene Fused to an HCV Polypeptide Indicator Gene Virus Vector-Construction Indicator Gene Virus Vector (IGVV) pXHCV-luc was designed using an HCV genomic virus vector containing a functional indicator gene fused to an HCV polyprotein. IGVV uses an open reading frame for the indicator gene as a cD containing the entire HCV genome producing in-frame fusion proteins.
Constructed by inserting into NA construct. IGVV is HCV RN
A contains all cis-acting regulatory elements in the 5 'and 3' untranslated regions (UTRs) required for replication, transcription and translation. In one embodiment, the luciferase open reading frame provides a spacer region containing the recognition sequence for the NS3 / 4A protease and is located immediately downstream of the NS5 coding region (FIG. 3A). An example of such a cleavage site is TEDVVCC-SMSYTWT, which represents the junction between NS5A and NS5B (Grakoui et al. 1993, J. Virol. 67: 2832; S
teinkuehler et al., 1996, J. Virol. 79: 6694). Expected cleavage products are HCV NS5B containing a C-terminal extension (eg, TEDVVCC), and luciferase containing an N-terminal extension (eg, SMSYTWT). In a second embodiment, the luciferase open reading frame is located between the NS5A and NS5B open reading frames, and the NS5A-5B cleavage sequence is at both the N-terminal NS5A-luc and C-terminal luc-NS5B junctions. . The luciferase protein produced from this construct was N-terminal SMSYT
Contains WT and C-terminal TEDVCC VCC extensions. In a third embodiment,
The luciferase open reading frame is placed between the NS4B and NS5A open reading frames, and the NS4B-5A cleavage sequence (SECT
TPC-SGSWLRD) has N-terminal NS4B-luc and C-terminal luc-
At both NS5A junctions. The luciferase protein produced from this construct contains an N-terminal SGSWLRD and a C-terminal SECTTPC extension. In a fourth embodiment, the luciferase open reading frame is NS
NS4A-4 placed between the 4A and NS4B open reading frames.
The B cleavage sequence (FDEMEEC-SQHLPYI) is at both the N-terminal NS4A-luc and C-terminal luc-NS4B junctions. The luciferase protein produced from this construct has N-terminal SQHLPYI and C-terminal FDE
Contains MEEC extension. At the N and C termini of the luciferase or at the N
Short extensions at the C-terminus of S5B do not dramatically affect activity.

The viral vector is assembled from a full-length HCV cDNA construct consisting of a 5 ′ UTR (in the 5 ′ to 3 ′ orientation), an open reading frame for a 3010 amino acid polyprotein, and a 3 ′ UTR. The polyprotein contains therein a capsid (C) open reading frame, envelope glycoprotein genes (E1 and E2), NS2 (a cis-acting autoprotease that cleaves the polyprotein at specific sites at the NS2-NS3 junction), NS3 (Helicase and serine protease), NS4A (required as a cofactor for NS3 activity), NS4B, NS5A, and NS5B (RNA-dependent RNA polymerase) open reading frame. In addition, the open reading frame of luciferase contains open reading frames located variously in the polyprotein as described above.

[0086] In one embodiment, IGVV is present in the transfected cells in RNV.
It contains a eukaryotic promoter at the 5 'end of the HCV sequence for the production of A, and a transcription terminator at the 3' end. Examples of transcription promoters include, but are not limited to, the CMV immediate-early promoter, or the SV40 promoter; examples of transcription terminators include, but are not limited to, the transcription terminators found in the SV40 or human β-globin genes. / Polyadenylation signal (see FIG. 3B). In a second embodiment, the promoter is a promoter for a bacteriophage RNA polymerase such as T7, T3 or SP6;
Terminators are sequence signaling terminations of transcription recognized by polymerases, or self-cleaving ribozymes (see, for example, Chowrira et al., 1994; J. Biol. Chem.
269: 25864). IGVV is transfected as DNA into cells that express RNA polymerase in the cytoplasm. Such expression can be achieved by several methods, including cotransfection with a polymerase expression vector, infection with a recombinant vaccinia virus expressing the polymerase (Fuerst et al., 1986, PNAS 83: 8122), or permanently by polymerase. Is achieved by pre-establishing a cell line that expresses (see FIG. 3C). IGVV additionally shows that the transcribed RNA has a poly-
It contains a poly-A or poly-U sequence immediately following the 3 'end of HCV, such as containing an A or poly-U tail. In a third embodiment, IGVV having a bacteriophage RNA polymerase promoter at the 5 'end and a terminator sequence at the 3' end is transcribed in vitro and the nucleic acid representing the IGVV is R
Transfected as NA. A terminator can be a specific sequence recognized by bacteriophage RNA polymerase as a terminator site or a self-cleaving ribozyme (Chowrira et al., (1994) J. Biol. Chem. 269, 25856-2).
5864). Alternatively, the terminator is a restriction endonuclease site that allows for linearization of the DNA template prior to transcription (see FIG. 3D). In this case, the vector contains a poly-A or poly-U sequence at the 3 'end.

In transfected cells, an internal ribosome entry sequence (IR
The RNA is translated using an internal initiation mechanism via ES) to yield the HCV polyprotein-luc fusion protein. The release of active luciferase from the HCV polyprotein fusion depends on the action of NS3 / 4A itself expressed from genomic RNA. When the genomic RNA is replicated and amplified in transfected cells, high levels of expression occur, depending on the action of viral polymerase NS5B and viral polymerases NS2 and NS3 / 4A. If luciferase is inactive as part of a large HCV polyprotein, activity can be measured directly in the transfected cells. This is because the release of active luciferase is dependent on HCV RNA replication (one-cell assay). If luciferase has significant activity as a fusion protein, progeny virions are collected and used to infect new target cells (two-cell assay). The transfer of IGVV RNA from transfected cells to infected target cells depends on replication and encapsidation in the transfected cells, which, in turn, correct expression, processing of HCV viral structural and nonstructural proteins. And activity. Move (ie, I
To increase the efficiency of GVV RNA packaging into new virions), target cells can be co-infected with wild-type HCV virus or wild-type HCV R
It can be transfected with a NA or cDNA expression construct. IGV
To further increase V RNA replication and packaging, the input RNA (FIG. 3D) is co-transfected with purified NS5B protein so that transcription can be started immediately upon uptake into cells ( Ie, as an RNP complex). This strategy, or a variant thereof, is used for influenza virus (Enami and Palese 1991, J. Virol. 65; 2711-2713), rabies virus (Sch
nell et al., 1994, EMBO J. 13: 4195-4203), and vesicular stomatitis virus (Lawson et al.,
1995, PNAS 92: 4477-4481).

Resistance Test Vector—Construction The resistance test vector (RTV) is an antiviral drug target (eg, NS3 / 4A)
, NS5B, or IRES), the region of the HCV genome corresponding to the virus and / or RN present in the blood and / or cells of the infected patient.
The indicator gene is constructed from the viral vector by replacing it with the corresponding region from A (patient-derived segment, or PDS). In one embodiment, for an NS3 / 4A protease inhibitor, IGVV is NS3 / 4A
It is modified by inserting a unique restriction site in or near the gene (nucleotides 3418-5473 of the H strain of HCV), called the patient sequence acceptor site (PSAS). The patient-derived segment obtained from the patient-derived virus is then introduced into the PSAS in IGVV (FIG. 3A). The wild-type NS3 / 4A region is removed from IGVV by digestion with a restriction endonuclease that recognizes a specific sequence acceptor site: generated by RT / PCR from plasma or serum or patient-derived viral RNA obtained from cells These sequences are replaced with the DNA fragment. PCR using primers containing the restriction endonuclease sites necessary to generate compatible sticky ends for cloning into digested IGVV
Produce the product. The RT and PCR primer binding sites are selected and the primer sequences are designed to allow amplification of as many different subtypes of HCV as possible. In a second embodiment, in the case of an inhibitor of NS5B RDRP,
Sequence spanning the B open reading frame (nucleotide 7 of HCV strain H)
601-9373) is removed from the IGVV at the unique patient sequence acceptor site; then, plasma or serum or patient viral RNA obtained from cells
Replace these sequences with the corresponding PDS generated by RT / PCR from. In a third embodiment, in the case of an IRES inhibitor, the sequence spanning the IRES (nucleotides 1-709 of the H strain of HCV) is removed from IGVV at the unique patient sequence acceptor site; obtained from plasma or serum or cells Replace these sequences with the corresponding PDS generated by RT / PCR from the generated patient viral RNA. Previous methods have identified a gene encoding the target of the drug in IGVV; introducing a unique patient sequence acceptor site into IGVV; and then replacing the target gene with PDS to provide another target for the anti-HCV drug. Applied to

Drug Sensitivity and Resistance Tests Drug resistance and sensitivity tests are performed using a one-cell or two-cell assay with a resistance test vector prepared as described above by transfection (as DNA or RNA). Transfection of the host cells with the resistance test vector generates HCV virions containing the encapsidated indicator gene RNA.

Replication transfection can be performed in the absence of antiviral drugs or at increasing concentrations of anti-HCV drugs (eg, HCV NS3 / 4A protease inhibitor, N
S5B polymerase inhibitor, or IRES inhibitor). After maintaining the packaging house cells for several days in the presence or absence of anti-HCV agents, isolated HCV obtained in host packaging cell lysates or by harvesting the host packaging cell culture medium By measuring indicator gene expression anywhere in the particles, the level of drug sensitivity or resistance can be assessed. Another approach can be used to assess drug sensitivity and resistance in cell lysates and isolated HCV particles.

In one embodiment of a one-cell assay, drug sensitivity or resistance is determined by measuring luciferase expression or activity in a transfected packaging host cell in the presence or absence of an antiviral drug. Be evaluated. The agent is used to reduce the luciferase activity observed in cells transfected in the presence of a given antiviral agent or agent combination, as compared to a control run in the absence of the antiviral agent (s). Is calculated or an S-shaped curve is generated for the log of the concentration of antiviral agent against luciferase activity.

In a second embodiment, a two-cell assay, drug sensitivity or resistance is luciferase gene expression and / or activity in target host cells following infection with HCV particles obtained by transfecting the host cells. Is evaluated by measuring At the time of transfection or infection, depending on the drug target, the appropriate concentration of the antiviral drug is added to the host or target cell culture. Several days after infection, the target host cells are lysed and luciferase expression is measured.
A decrease in luciferase expression can be caused by a drug that inhibits HCV replication, for example, by inhibiting either the protease (NS3 / 4A) or RDRP (NS5B) activity of HCV, as compared to a control run in the absence of the drug. Will be observed in cells infected in the presence of

In a third embodiment, where changes in RNA structure are used as indicators, drug sensitivity and resistance are assessed by measuring the level of HCV RNA replication that has taken place in the transfected host cells. HCV virus vector DNA
RNA is transcribed as a positive (mRNA) sense RNA that can serve as a template for the production of negative sense cDNA by the action of NS5B polymerase in a host cell transfected with Transcribed in vitro and transfected directly). Alternatively, the positive sense RNA is transfected, translated and serves as a template for negative sense cRNA synthesis. To measure HCV RNA replication, RN
A is isolated from the transfected cells and treated with DNAse to remove residual input DNA. (The DNAse treatment is not absolutely necessary if the cells are transfected with positive sense RNA. ). RT primers are designed to specifically hybridize to negative sense HCV RNA and serve as the starting point for the synthesis of positive sense cDNA; RNAse digestion prevents reverse transcription of positive sense RNA using PCR primers. Thereafter, the cDNA is amplified by PCR.

The formation of a positive sense amplified target cDNA in transfected cells follows the onset of HCV RNA replication resulting in the formation of a negative sense RNA. HCV RN
Production of an antiviral agent that inhibits A replication (RNA-dependent RNA polymerase activity), or the active form of the polymerase (by the NS3 / 4A protease)
Limits the formation of NA target sequences, which is measured as a reduction in amplified DNA product using one of a number of quantitative amplification assays.

[0095] In another embodiment using a change in RNA structure as an indicator, the amplified D
The 5 'exonuclease activity of the amplification enzyme (eg, Taq polymerase) is measured rather than the production of NA (Heid et al., 1996, Genome Research 6: 986-994). 5 'exonuclease activity is measured by monitoring the nucleolytic cleavage of a fluorescently tagged oligonucleotide probe capable of binding to an amplified DNA template region flanked by PCR primer binding sites. . The efficiency of this assay depends on the close proximity of the 3 'end of the upstream primer to the 5' end of the oligonucleotide probe. When the primer is extended, it replaces the 5 'end of the oligonucleotide probe such that the 5' exonuclease activity of the polymerase cleaves the oligonucleotide probe.

Drug Screening Drug screening is performed using an indicator gene viral vector containing a functional indicator gene fused to the HCV polyprotein. In the transfected host cell, the indicator gene viral vector contains the R containing the indicator gene.
Produce an NA transcript (alternatively, the RNA is transcribed in vitro and directly transfected). Translation of this RNA or of the produced mRNA as a result of replication and transcription by viral RDRP (NSSB) results in the necessary structural and enzymatic viral functions for viral RNA replication and particle formation.
The transfected cells are encapsidated indicator gene viral vector RNA that also contains a functional indicator gene fused to the HCV polyprotein gene.
To produce HCV virions containing

The drug screening is performed as follows: The host cell is transfected with the indicator gene viral vector DNA or RNA. Replication transfection is performed using potential antiviral compounds (eg, candidate HCV NS3 / 4A).
(A protease or NS5B polymerase inhibitor) in the absence or presence of a series of packaging host cell cultures. After maintaining the transfected host cells in the presence or absence of the candidate antiviral agent for up to several days, the level of inhibition of RNA replication is determined in the transfected host cell lysate or in the transfected host cells. By assessing the expression of the indicator gene directly in the isolated HCV particles obtained by harvesting the cultured cell culture medium or in target cells infected with the isolated HCV particles. Using the RNA detection or indicator gene activity methods described above, potential anti-HCV
Drug candidates can be evaluated.

Example 2 HCV Drug Susceptibility and Resistance Tests Using Functional Indicator Genes Expressed from Patient-Derived Segment (s) and Internal Ribosome Initiation Sequence Indicator Gene Viral Vector-Construction Occurs through an independent internal initiation mechanism. The 5 'end of the viral RNA, including the untranslated region (UTR) and the first 369 nucleotides of the C open reading frame, has a cap-
Contains sequences and / or structures that direct independent translation initiation (Tsukiyama-Ko
Hara et al., J. Virol. 66: 1476, 1992; Wang et al., J. Virol. 67: 3338, 1993; Lu and Wimmer,
PNAS 93: 1412, 1996). Poliovirus (PV) (Pelletier and Sonenberg (19
86), Nature, 334, 320-325), encephalomyocarditis virus (EMCV) (Jang et al. (1989), JV
irol. 63,1651-1660), rhinovirus (RV) (Rohll et al. (1994), J. Virol. 68, 43).
84-4391), hepatitis A virus (HAV) (Brown et al. (1994), J. Virol. 68, 1066-107).
4; Glass et al., (1993) Virol. 193, 842-852), and the pestivirus, bovine virus diarrhea virus (BVDV), in which HCV is closely related (Poole et al., (1995) Virology).
189, 285-292) use a similar mechanism for translation initiation, but the sequence that acts as an internal ribosome entry site (IRES) varies from virus to virus. Some cellular mRNAs are known to initiate translation internally via the IRES (Macejak and Sarnow (1991), Nature, 353, 90-94). These RNA elements are located between the two open reading frames, as well as the RNA
If it is located at the 5 'end of, translation initiation can be commanded. These bicistronic RNAs can be used to obtain expression of two proteins from the same RNA by independently directing the translation of both open reading frames.

An indicator gene viral vector containing a functional IG expressed from an internal ribosome initiation sequence contains an entire H as a second cistronic element preceded by an IRES.
It is constructed by inserting an open reading frame for an indicator gene, eg, luciferase, into a cDNA construct containing the CV genome. HC
Insertion of the IRES (either the native HCV 5'UTR or that of another virus) and luciferase downstream of the V polyprotein provides luciferase gene expression independent of that of the HCV protein (Figure 4). HCV IR
When testing for resistance to drugs that inhibit ES function, it was noted that the IRES used for luciferase expression must be derived from a virus other than HCV that is not affected by the drug. Thus, IGVV contains the following elements in the 5 'to 3' direction: promoter sequence, HCV 5 'UTR,
Contains the complete HCV polyprotein coding sequence, IRES, luciferase coding region, HCV 3'UTR, and transcription terminator.

[0100] In one embodiment, the IGVV is the R in the transfected cells.
It contains a eukaryotic promoter at the 5 'end of the HCV sequence for production of NA, and a transcription terminator at the 3' end. Examples of transcription promoters include, but are not limited to, the CMV immediate-early promoter, or the SV40 promoter: Examples of transcription terminators include, but are not limited to, the transcription terminators found in the SV40 or human β-globin genes. It contains a polyadenylation signal (FIG. 3B). In a second embodiment, the promoter is T7, T3 or SP6
The terminator is the sequence signaling termination of transcription recognized by the polymerase, or a self-cleaving ribozyme. IGVV is transfected as DNA into cells that express RNA polymerase in the cytoplasm. Such expression may be achieved by several methods, including co-transfection with a polymerase expression vector, infection with a recombinant vaccinia virus expressing the polymerase, or by pre-establishing a permanent cell line expressing the polymerase. Achieved (
(FIG. 3C). IGVV additionally contains a poly-A or poly-U sequence immediately after the HCV 3 'end, such that the transcribed RNA contains a poly-A or poly-U tail at the 3' end. In a third embodiment, an I with a bacteriophage RNA polymerase promoter at the 5 'end and a terminator sequence at the 3' end
GVV is transcribed in vitro and nucleic acids representing IGVV are transfected as RNA. The terminator can be a termination site or a specific sequence recognized by a bacteriophage RNA polymerase as a self-cleaving ribozyme. Alternatively, the terminator is a restriction endonuclease site that allows for linearization of the DNA template prior to transcription (FIG. 3D). In this case, the vector contains a poly-A or poly-U sequence at the 3 'end.

Resistance Test Vector-Construction A resistance test vector containing a functional indicator gene expressed from an internal ribosome initiation sequence is constructed from the IGVV described above and the patient-derived HCV sequence described in Example 1. The IGVV was obtained from PDS (described in Example 1 (see FIG. 3A)).
Modified to include PSAS for insertion of NS3 / 4A, NS5B or IRES containing

Drug Sensitivity and Resistance Tests Drug resistance and sensitivity tests are performed using either one-cell or two-cell assays,
Transfection (as either DNA or RNA) is performed with the resistance test vector prepared as described above. Transfection of host cells with a resistance test vector results in HCV virions containing the encapsidation indicator gene RNA. Drug resistance and sensitivity tests are performed as described in Example 1.

Drug Screening IGVV containing a functional indicator gene expressed from an internal ribosome initiation sequence
Is performed substantially as described in Example 1.

Example 3 HCV Drug Susceptibility and Resistance Tests Using a Resistance Test Vector Containing a Patient-Derived Segment (s) and a Functional Indicator Gene Expressed from a Replication-Defective Minigenome Indicator Gene Virus Vector-Construction Dependence of the Indicator Gene on HCV Replication Expression is determined by the HCV 5'UTR and, if desired (
Artificial H consisting of part of the amino terminus of the C open reading frame (needed as part of the IRES), IG, eg luciferase, and HCV 3′UTR
Achieved by constructing a CV subgenomic viral vector, or "minigenome" (FIG. 5). Luciferase is produced responsible for the N-terminal extension from the C open reading frame; alternatively, the ATG at the start of the C open reading frame is mutated so that translation starts at the luciferase ATG. The N-terminus of the 5'UTR plus C and the 3'UTR contain all the cis-acting signals required for RNA translation, replication and packaging. The luciferase minigenome is co-transfected with the full-length helper HCV genomic construct as either DNA or RNA (see Example 1, FIGS. 3B-3D); replication and packaging of the minigenomic RNA into progeny virus is performed by It depends on the HCV replication machine, including the NS3 / 4A protease and NS5B RDRP produced from HCV genomic RNA, and the cis-acting regulatory elements of the minigenome.

An indicator gene viral vector containing a functional indicator gene expressed from a replication defective mini-genome and a helper HCV genomic construct are constructed as follows.
The minigenome contains the following elements in the 5 'to 3' orientation: promoter sequence, HCV 5 'UTR, the first 24 or 3 of the C open reading frame.
69 nucleotides, luciferase open reading frame, HCV 3 '
UTR, and transcription terminator. The helper HCV genomic construct contains a promoter, the complete HCV cDNA, and a terminator.

[0106] In one embodiment, the IGVV is the R in the transfected cells.
It contains a eukaryotic promoter at the 5 'end of the HCV sequence for production of NA, and a transcription terminator at the 3' end. Examples of transcription promoters include but are not limited to the CMV immediate-terminator, or the SV40 promoter; examples of transcription terminators include, but are not limited to, the transcription terminator / polyadenyl found in the SV40 or human β-globin genes. (See FIG. 3B). In a second embodiment, the promoter is T7, T3 or S
A promoter for a bacteriophage RNA polymerase such as P6, the terminator being the sequence signaling termination of transcription recognized by the polymerase or a self-cleaving ribozyme. IGVV is transfected as DNA into cells that express RNA polymerase in the cytoplasm. Such expression may be achieved by several methods, including co-transfection with a polymerase expression vector, infection with a recombinant vaccinia virus expressing the polymerase, or by pre-establishing a permanent cell line expressing the polymerase. Achieved (see FIG. 3C). IGVV additionally includes a poly- immediately following the HCV 3 'end, such that the transcribed RNA contains a poly-A or poly-U tail at the 3' end.
Contains the A or poly-U sequence. In a third embodiment, IGVV having a bacteriophage RNA polymerase promoter at the 5 'end and a terminator sequence at the 3' end is transcribed in vitro, and the nucleic acid representing IGVV is transfected as RNA. The terminator can be a termination site or a specific sequence recognized by a bacteriophage RNA polymerase as a self-cleaving ribozyme. Alternatively, the terminator is a DNA
Restriction endonuclease sites allowing template linearization (FIG. 3D
reference). In this case, the vector contains a poly-A or poly-U sequence at the 3 'end.

Resistance Test Vector-Construction A resistance test vector comprising a functional indicator gene and a helper HCV genomic construct expressed from a replication defective minigenome was constructed as described in Example 1 using a helper HCV genomic construct and a patient-derived HCV. Constructed from an array. Helper HCV
The genomic construct was NS3 / 4A or NS5B-containing PDS (described in Example 1,
3A) is modified to include a PSAS for insertion. In the case of an agent targeting the function of the IRES, the PSAS is also inserted into the minigenome construct.

Drug Sensitivity and Resistance Tests [0108] Resistance and sensitivity tests using the IGVV system containing a functional indicator gene expressed from the minigenome and a helper HCV genomic construct are performed by transfection using either one-cell or two-cell assays. Performed with the resistance test vector prepared as described above (either as DNA or RNA). Transfection of host cells with a resistance test vector (helper HCV genomic construct containing luciferase mini-genome + PDS) comprises transfection of HCV containing encapsidated luciferase gene RNA and / or encapsidated HCV genomic RNA.
This produces virus particles. The drug resistance and sensitivity tests are then performed as described in Example 1.

Drug Screening Drug screening using an IGVV system containing a functional indicator gene expressed from the minigenome and a helper HCV genomic construct is performed substantially as described in Example 1 above.

Example 4 HCV Drug Susceptibility and Resistance Tests Using a Resistance Test Vector Containing a Patient-Derived Segment (s) and a Functional Indicator Gene Expressed from an Antisense Replication-Defective Minigenome Index Gene Virus Vector-Construction Expression from a Replication-Defective Minigenome Non-functional indicator gene and helper HCV
The indicator gene viral vector containing the genomic construct is expressed as follows.
The minigenome contains the following elements in the 5 'to 3' orientation: promoter sequence, HCV 3 'UTR (antisense orientation), luciferase open reading frame (antisense), the first 24 or 369 nucleotides of the C open reading frame ( Antisense), HCV 5'UTR (antisense)
, And transcription terminator. The helper HCV genomic construct contains a promoter, the complete HCV cDNA (sense orientation), and a terminator. In this example, the minigenome is introduced into cells as negative-strand RNA, ie, a replicative intermediate RNA copy of the minigenome described above (see FIG. 6).
. Expression in transfected cells is dependent on the activity of NS3B, whose production depends on the action of NS3 / 4A and, in the case of IRES, cis-acting regulatory elements. Thus, the indicator gene is non-functional until acted upon by the viral replication machine.

[0111] In one embodiment, the IGVV is the R in the transfected cells.
A eukaryotic promoter at the 5 'end of the HCV sequence for production of NA, and 3'
Contains a terminal transcription terminator. Examples of transcription promoters include, but are not limited to, the CMV intermediate-early promoter, or the SV40 promoter; examples of transcription terminators include, but are not limited to, the SV40 or human β-globin genes. Includes transcription terminator / polyadenylation signal (see FIG. 3B). In a second embodiment, the promoter is T7,
A promoter for a bacteriophage RNA polymerase, such as T3 or SP6, where the terminator is the sequence signaling termination of transcription recognized by the polymerase or a self-cleaving ribozyme. IGVV is transfected as DNA into cells that express RNA polymerase in the cytoplasm. Such expression may be achieved by several methods, including co-transfection with a polymerase expression vector, infection with a recombinant vaccinia virus expressing the polymerase, or by pre-establishing a permanent cell line expressing the polymerase. Achieved (see FIG. 3C). IGVV additionally contains a poly-A or poly-U sequence immediately after the HCV 3 'end, such that the transcribed RNA contains a poly-A or poly-U tail at the 3' end. In a third embodiment, 5 ′
IGVV with a terminal bacteriophage RNA polymerase promoter and a 3 'terminator sequence is transcribed in vitro, and the nucleic acid representing IGVV is transfected as RNA. The terminator can be a termination site or a specific sequence recognized by a bacteriophage RNA polymerase as a self-cleaving ribozyme. Alternatively, the terminator is a restriction endonuclease site that allows for linearization of the DNA template prior to transcription (see FIG. 3D). In this case, the vector contains poly-A or poly-
Contains U sequence.

Tolerance test vector-construction Non-functional indicator gene and helper HCV expressed from a replication defective minigenome
A resistance test vector comprising a genomic construct is constructed from a helper HCV genomic construct and a patient-derived HCV sequence as described in Example 1. Helper HC
The V genomic construct was NS3 / 4A or NS5B containing PDS (described in Example 1,
3A) is modified to include a PSAS for insertion. In the case of an agent targeting the function of the IRES, the PSAS is also inserted into the minigenome construct.

Drug Susceptibility and Resistance Testing [0113] Material resistance and susceptibility testing using a resistance test vector containing a non-functional indicator gene expressed from the minigenome and a helper HCV genomic construct include transfection using either one-cell or two-cell assays. (As either DNA or RNA) with the resistance test vector prepared as described above.
Tolerance test vector (helper HC containing luciferase mini genome + PDS)
Transfection of the host cell with the V genome construct) results in HCV viral particles containing the encapsidated luciferase gene RNA and / or the encapsidated HCV genomic RNA. Then, drug resistance and susceptibility tests were performed in Example 1.
This is performed as described in.

Drug Screening Drug screening using a resistance test vector containing a non-functional indicator gene expressed from the minigenome and a helper HCV genomic construct is performed substantially as described in Example 1 above.

Example 5 HCV Drug Susceptibility and Resistance Test Vectors Using a Resistance Test Vector Containing a Patient-Derived Segment (s) and a Functional Indicator Gene Expressed as a Replication-Defective Genome Indicator Gene Virus Vector-Construction Expressed from a Replication-Defective Minigenome The indicator gene virus vector containing the functional indicator gene and the packaging vector construct is expressed as follows.
IGVV contains the following elements in the 5 'to 3' direction: promoter sequence, H
CV5'UTR, first 24 or 369 nucleotides of C open reading frame, indicator gene open reading frame, NS2 of HCV genome
The NS5B portion (nucleotides 2768-9373 of the H strain of HCV), HC
V3'UTR, and transcription terminator. The packaging vector contains the promoter, HCV C, E1 and E2 open reading frames (HCV H
Strain nucleotides 342-2578) and a terminator. When the indicator gene is luciferase or another cytoplasmic protein, with some modifications, host signal peptidases in the endoplasmic reticulum cause IG-NS2
Ensures proper processing of the joint. In the case of a secreted indicator gene, for example secreted alkaline phosphatase, such a modification is not necessary.

Infectious recombinant virions consist of two vectors: an IGVV containing IG and viral non-functional proteins, and a packing vector containing a second vector, a viral structural protein (C / E1 / E2; see FIG. 7). ) Produced from the transfected cells. To produce infectious particles, IGVV DNA
(Or its corresponding RNA, see Example 1, FIGS. 3A-3D) are co-transfected with a packaging vector. Alternatively, it is pseudotyped with an envelope glycoprotein gene from a related flavivirus such as BVDV or classic swine fever virus (CSFV). The pseudotyped virus is used to establish a cell culture system for single cycle infection assays. The virus produced in this manner is then used to infect target cells and luciferase expression is subsequently measured. This approach adds the advantage of minimizing the amount of manipulation performed with replication competent infectious agents.

[0117] In one embodiment, the IGVV is the R in the transfected cell.
A eukaryotic promoter at the 5 'end of the HCV sequence for production of NA, and 3'
Contains a terminal transcription terminator. Examples of transcription promoters include, but are not limited to, the CMV intermediate-early promoter, or the SV40 promoter; examples of transcription terminators include, but are not limited to, the SV40 or human β-globin genes. Includes transcription terminator / polyadenylation signal (see FIG. 3B). In a second embodiment, the promoter is T7,
A promoter for a bacteriophage RNA polymerase, such as T3 or SP6, where the terminator is the sequence signaling termination of transcription recognized by the polymerase or a self-cleaving ribozyme. IGVV is transfected as DNA into cells that express RNA polymerase in the cytoplasm. Such expression may be achieved by several methods, including co-transfection with a polymerase expression vector, infection with a recombinant vaccinia virus expressing the polymerase, or by pre-establishing a permanent cell line expressing the polymerase. Achieved (see FIG. 3C). IGVV additionally contains a poly-A or poly-U sequence immediately after the HCV 3 'end, such that the transcribed RNA contains a poly-A or poly-U tail at the 3' end. In a third embodiment, 5 ′
IGVV with a terminal bacteriophage RNA polymerase promoter and a 3 'terminator sequence is transcribed in vitro, and the nucleic acid representing IGVV is transfected as RNA. The terminator can be a termination site or a specific sequence recognized by a bacteriophage RNA polymerase as a self-cleaving ribozyme. Alternatively, the terminator is a restriction endonuclease site that allows for linearization of the DNA template prior to transcription (see FIG. 3D). In this case, the vector contains poly-A or poly-
Contains U sequence.

Resistance Test Vector-Construction A resistance test vector containing a functional indicator gene and a packaging vector construct expressed from a replication defective minigenome was prepared as described in Example 1 above.
It is constructed from GVV and HCV sequences from patients. IGVV is a PSA for insertion of NS3 / 4A or IRES containing PDS (described in Example 1, see FIG. 3A).
Modified to include S.

Drug Sensitivity and Resistance Tests Drug resistance and sensitivity tests are performed on resistance test vectors prepared as described above (either as DNA or RNA) by transfection using either one-cell or two-cell assays. Transfection of host cells with a resistance test vector results in HCV virions containing the encapsidation indicator gene RNA. Drug resistance and sensitivity tests are performed as described in Example 1.

Drug Screening Drug screening using an IGVV containing a functional indicator gene expressed from a replication defective genome and a known gas for a packaging vector is performed substantially as described in Example 1 above. Example 6 Sensitivity and Resistance Tests of HCV Protease Inhibitors Using Patient-Derived Segment (s) and Resistance Test Vectors Containing NS3 / 4A BVDV Chimeric Virus Vector Indicator Gene Virus Vector-Construction Functional Indicator Gene and Related Parts of HCV (Class ) (Eg, the NS3 / 4A protease domain) were designed to have a relevant viral backbone that replicates well in culture. An example of such a virus is BVDV. B
The complete cDNA for the VDV genome has been assembled and shown to generate infectious RNA by in vitro transcription (Vassiler et al., 1997, J. Virol.
71: 471-478). BVDV polyprotein is processed in a manner very similar to that of HCV using both host (signal peptidase) and virally encoded proteases. Chimeric IGVV based on the BVDV backbone contains the entire NS3 / 4A open reading frame replacing the NS3 protease domain or the corresponding region of BVDV (FIG. 8). By mutating the cleavage site normally recognized by the BVDV NS3 protease to that recognized by HCV NS3 / 4A, BVDV chimeric RNA replication and IG expression are dependent on HCV NS3 / 4A activity Will.

A chimeric indicator gene virus vector containing a functional indicator gene in the NS3 / 4A BVDV chimeric virus vector is constructed as follows. IGVV
Contains the following elements in the 5 'to 3' direction: promoter sequence, BVDV5
'UTR, C to NS2 region of BVDV (NADL strain), NS3 / 4 of HCV
A region, HCV NS4A / 4B cleavage site, BVDV NS4B open reading frame, HCV NS4B / 5A cleavage site, BVDV NS5A open reading frame, HCV NS5A / 5B cleavage site, BVDV NS5
B open reading frame, luciferase open reading frame, BVDV 3'UTR and transcription terminator. In a second embodiment, the IGVV contains an open reading frame of luciferase preceded by an IRES in a conformation similar to that described in Example 2. In a third embodiment, the luciferase gene is expressed from a mini-genome similar to those described in Examples 3 or 4.

[0122] In one embodiment, IGVV is the R in transfected cells.
A eukaryotic promoter at the 5 'end of the HCV sequence for production of NA, and 3'
Contains a terminal transcription terminator. Examples of transcription promoters include, but are not limited to, the CMV intermediate-early promoter, or the SV40 promoter; examples of transcription terminators include, but are not limited to, the SV40 or human β-globin genes. Includes transcription terminator / polyadenylation signal (see FIG. 3B). In a second embodiment, the promoter is T7,
A promoter for a bacteriophage RNA polymerase, such as T3 or SP6, where the terminator is the sequence signaling termination of transcription recognized by the polymerase or a self-cleaving ribozyme. IGVV is transfected as DNA into cells that express RNA polymerase in the cytoplasm. Such expression may be achieved by several methods, including co-transfection with a polymerase expression vector, infection with a recombinant vaccinia virus expressing the polymerase, or by pre-establishing a permanent cell line expressing the polymerase. Achieved (see FIG. 3C). IGVV additionally contains a poly-A or poly-U sequence immediately after the HCV 3 'end, such that the transcribed RNA contains a poly-A or poly-U tail at the 3' end. In a third embodiment, 5 ′
IGVV with a terminal bacteriophage RNA polymerase promoter and a 3 'terminator sequence is transcribed in vitro, and the nucleic acid representing IGVV is transfected as RNA. The terminator can be a termination site or a specific sequence recognized by a bacteriophage RNA polymerase as a self-cleaving ribozyme. Alternatively, the terminator is a restriction endonuclease site that allows for linearization of the DNA template prior to transcription (see FIG. 3D). In this case, the vector contains poly-A or poly-
Contains U sequence.

Resistance Test Vector-Construction A resistance test vector containing a functional indicator gene in the NS3 / 4A BVDV chimeric virus vector was constructed from the IGVV and patient-derived HCV NS3 / 4A sequences as described in Example 1. Is done. IGVV is NS3 / 4A-
Modified to include PSAS for insertion of contained PDS (described in Example 1, see FIG. 3A).

Drug Sensitivity and Resistance Tests Drug resistance and susceptibility tests are performed by transfection using either one-cell or two-cell assays on resistance test vectors prepared as described above (either as DNA or RNA). Transfection of host cells with a resistance test vector results in HCV virions containing the encapsidation indicator gene RNA. Drug resistance and sensitivity tests are performed as described in Example 1.

Drug Screening IGVV containing a functional indicator gene expressed from an internal ribosome initiation sequence
Is performed substantially as described in Example 1 above.

Example 7 Sensitivity and Resistance Testing of HCV Drugs Using a Patient-Derived Segment (s) and Resistance Test Vectors Containing NS5B BVDV Chimeric Virus Vector Indicator Gene Virus Vector-Construction With the exception of NS5B derived from HCV, BVDV structures and non- A chimeric IGVV containing a functional protease is designed to have a BVDV backbone. In addition, the 5 ′ and 3 ′ UTRs of BVDV are replaced with the corresponding regions from HCV to ensure recognition by the homologous polymerase (FIG. 9).

An indicator gene virus vector containing a functional indicator gene in the NS5B BVDV chimeric virus vector is constructed as follows. IGVV contains the following elements in the 5 'to 3' orientation: promoter sequence, HCV 5 'UTR, I
Sequence from N-terminus of HCV C open reading frame required for RES function, Npro to NS5A region of BVDV (NADL strain), NA5 of HCV
B region, luciferase open reading frame, HCV 3′UTR,
And a transcription terminator. In a second embodiment, the IGVV contains an open reading frame of luciferase preceded by an IRES in a conformation similar to that described in Example 2. In a third embodiment, the luciferase gene is expressed from a mini-genome similar to those described in Examples 3 or 4.

[0128] In one embodiment, IGVV is the R in transfected cells.
A eukaryotic promoter at the 5 'end of the HCV sequence for production of NA, and 3'
Contains a terminal transcription terminator. Examples of transcription promoters include, but are not limited to, the CMV intermediate-early promoter, or the SV40 promoter; examples of transcription terminators include, but are not limited to, the SV40 or human β-globin genes. Includes transcription terminator / polyadenylation signal (see FIG. 3B). In a second embodiment, the promoter is T7,
A promoter for a bacteriophage RNA polymerase, such as T3 or SP6, where the terminator is the sequence signaling termination of transcription recognized by the polymerase or a self-cleaving ribozyme. IGVV is transfected as DNA into cells that express RNA polymerase in the cytoplasm. Such expression may be achieved by several methods, including co-transfection with a polymerase expression vector, infection with a recombinant vaccinia virus expressing the polymerase, or by pre-establishing a permanent cell line expressing the polymerase. Achieved (see FIG. 3C). IGVV additionally contains a poly-A or poly-U sequence immediately after the HCV 3 'end, such that the transcribed RNA contains a poly-A or poly-U tail at the 3' end. In a third embodiment, 5 ′
IGVV with a terminal bacteriophage RNA polymerase promoter and a 3 'terminator sequence is transcribed in vitro, and the nucleic acid representing IGVV is transfected as RNA. The terminator can be a termination site or a specific sequence recognized by a bacteriophage RNA polymerase as a self-cleaving ribozyme. Alternatively, the terminator is a restriction endonuclease site that allows for linearization of the DNA template prior to transcription (see FIG. 3D). In this case, the vector contains poly-A or poly-
Contains U sequence.

Resistance Test Vector-Construction A resistance test vector containing a functional indicator gene in a NA5B BVDV chimeric virus vector is constructed from the IGVV and patient-derived HCV NS5B sequences as described in Example 1. IGVV is modified to include PSAS for insertion of NS5B containing PDS (described in Example 1, see FIG. 3A).

Drug Sensitivity and Resistance Tests Drug resistance and susceptibility tests are performed on resistance test vectors prepared as described above (either as DNA or RNA) by transfection using either one-cell or two-cell assays. Transfection of host cells with a resistance test vector results in HCV virions containing the encapsidation indicator gene RNA. Drug resistance and sensitivity tests are performed as described in Example 1.

Drug Screening IGVV containing a functional indicator gene expressed from an internal ribosome initiation sequence
Is performed substantially as described in Example 1 above.

Example 8 Sensitivity and Resistance Testing of HCV Drugs Using a Resistance Test Vector System Containing Patient-Derived Segment (s), Transcriptional Transactivator, and Functional Indicator Gene Indicator Gene Virus Vector-Construction , Were designed to include transcriptional transactivators that activate HCV-dependent expression and expression of indicator genes. An indicator gene, eg, luciferase, is introduced into the host cell as an expression vector by transient or stable transfection. The gene encoding the transactivator protein, eg, that of HIV-1, tat (ie, at the C-terminus or elsewhere) is the same as that described for the luciferase fusion described in Example 1. Similarly, it is fused to the HCV polyprotein via the NS3 / 4A cleavage site linker. Upon expression of the polyprotein, N
Depending on the activity of the S3 / 4A protease, tat is cleaved from a polyprotein that activates transcription of a reporter gene such as luciferase under the control of HIV-1 long terminal repeat (LTR).

An indicator gene viral vector system containing a functional indicator gene comprising segment (s) from the patient, a transcriptional transactivator, and a functional indicator gene, comprises:
It is constructed as follows. The viral vector contains the following elements in the 5 'to 3' orientation: a promoter, an HCV 5 'UTR, and an open reading frame for tat, variously positioned as described in Example 1, containing 3010 therein. Open reading frame, 3′UTR and transcription terminator for amino acid HCV polyprotein. The indicator gene construct is HIV-1
Contains LTR, luciferase open reading frame, and transcription terminator. The indicator gene construct can be co-transfected with a viral vector or, preferably, the stable integrated DNA in host cell DNA.
Exists as a segment.

In one embodiment, the viral vector contains a eukaryotic promoter at the 5 ′ end of the HCV sequence for transcription of RNA in the transfected cells, and a transcription terminator at the 3 ′ end. Examples of transcription promoters include, but are not limited to, the CMV intermediate-early promoter, or the SV40 promoter; examples of transcription terminators include, but are not limited to, SV4
0 or contains the transcription terminator / polyadenylation signal found in the human β-globin gene (see FIG. 3B). In a second embodiment, the promoter is a promoter for a bacteriophage RNA polymerase such as T7, T3 or SP6, and the terminator is the sequence signaling termination of transcription recognized by the polymerase or a self-cleaving ribozyme. The viral vector is transfected as DNA into cells that express RNA polymerase in the cytoplasm. Such expression may be achieved by several methods, including co-transfection with a polymerase expression vector, infection with a recombinant vaccinia virus expressing the polymerase, or by pre-establishing a permanent cell line expressing the polymerase. Achieved (see FIG. 3C). Viral vectors additionally contain a poly-A or poly-U sequence immediately after the HCV 3 'end, such that the transcribed RNA contains a poly-A or poly-U tail at the 3' end. Third
In an embodiment, a viral vector having a bacteriophage RNA polymerase promoter at the 5 'end and a terminator sequence at the 3' end is transcribed in vitro, and the nucleic acid representing the viral vector is transfected as RNA. The terminator can be a termination site or a specific sequence recognized by a bacteriophage RNA polymerase as a self-cleaving ribozyme. Alternatively, the terminator is a restriction endonuclease site that allows for linearization of the DNA template prior to transcription (see FIG. 3D). In this case, the vector contains a poly-A or poly-U sequence at the 3 'end.

Resistance Test Vector-Construction A functional indicator gene, including the patient-derived segment (s), a transcriptional transactivator, and a resistance test vector containing a functional indicator gene, were prepared as described in Example 1 using the virus Constructed from vector and patient-derived HCV sequences. The viral vector is modified to include a PSAS for insertion of a PDS containing a relative portion of the HCV genome (described in Example 1, see FIG. 3A).

Drug Sensitivity and Resistance Tests Transfection into host cells containing the indicator construct is performed (as either DNA or RNA) with the resistance test vectors prepared as described above. Drug resistance and sensitivity tests are performed as described in Example 1.

Drug Screening Drug screening using a functional indicator gene comprising a patient-derived segment (s), a transcriptional transactivator, and an indicator gene viral vector containing a functional indicator gene is substantially as described in Example 1 above. Performed as described.

Example 9 Cytomegalovirus Drug Susceptibility and Resistance Test Using a Resistance Test Vector Containing a Patient-Derived Segment (s) Embedded in a Defective Helper Virus and a Functional Indicator Gene Indicator Gene Virus Vector-Construction In Wild-Type Virus , RNA, e.g., indicator gene comprising TR _L or "b" functional indicator gene inserted into the ORF of HCMV under the control of the endogenous viral promoters controlling expression of the positions the beta _{2, 7} transcript repeat A viral vector (FIG. 12) was designed. Indicator gene viral vectors _{(HCMV-β 2, 7 F} -IG / Δ gene X) was further modified, it lacks a segment of the genome containing the viral genes which are the target of the anti-viral drug (s) (s) Due to duplication there are defects. The gene X product has a patient sequence acceptor site (P
SAS) and an indicator gene provided on an ampicillon plasmid (pA-CMMV-VS-geneX) containing the cis-acting functions required for trans-supplementation of the viral vector (specifically, these are the HCMV origin of replication and HCMV "a" sequence directing HCMV genome cleavage and packaging) (Figure 13). PS
AS is designed to allow PDS to a cassette containing the appropriate regulatory signals for individual viral gene / drug targets, and is derived from the context of the viral gene / drug target in wild-type HCMV. The defect indicator gene virus vector and the ampicillon / gene X plasmid constitute a resistance test vector system. The defect indicator gene viral vector grows as a virus stock in a packaging cell / target host cell system in which a functional copy of the viral gene X is trans. Viral stocks from such packaging host / target host cell lines are prepared, and then these viral stocks are used to infect the isolated cells and / or DNA and into cell types that are permissive for HCMV infection. Used to transfect packaging host cells / target host cells as part of a resistance test vector system in combination with the introduction of the ampicillon / gene X plasmid by transfection. Trans-supplementation of the deleted gene by the ampicillon / gene X plasmid results in a self-perpetuating viral population, which results in a decrease in the activity of the viral gene encoded by the patient-derived segment inserted into the ampicillon / gene X plasmid. The expression of the dependent reporter gene is increased.

In another embodiment, the amplicon / gene X plasmid (pA-C
MV-CS-gene X) is a PS that allows PDS to cleave gene X sequences from patients under the control of a heterologous promoter and polyadenylation signal.
Contains AS. In one embodiment of this example, the expression cassette containing the CMV IE enhancer promoter region, the PSAS, and the SV40 polyadenylation (pA) signal comprises, in addition to the cis-acting functions required for trans-supplementation of the indicator gene viral vector, Amplicon / gene X plasmid (pA-CMv
-CS-gene X) (specifically, these include the HCMV origin of replication and the HCMV "a" sequence that directs HCMV genome cleavage and packaging).

In another embodiment, the helper virus vector can be supplied as a series of overlapping cosmids that undergo recombination upon transfection into cells, resulting in the expression of a full array of helper functions. This modification can be used to provide helper virus sequences in all further examples as well.

In various embodiments of the invention, the viral gene / drug target (gene X) comprises: 1) HCMV DNA polymerase (UL54), 2) phosphotransferase (UL97), 3) viral serine protease (UL80), ) It can be any viral gene that encodes a true or possible target for a drug susceptibility test or a drug screening test. Such viral genes are not limited, but include UL44, UL57, UL105, UL102, UL70,
L114, UL98, or UL84.

[0142] The plasmids described for the CMV resistance test vector are named using the following convention: indicates that it is a plasmid DNA molecule that can replicate in a laboratory strain of E. coli, and "A" indicates that the plasmid is an amplicon and thus carries the cis-acting function required for propagation by helper virus; Typically, these ampicicon plasmids direct the viral origin of replication and genomic mutation, cleavage and packaging, enable the genome to be inverted,
CMV indicates that the amplicon sequence is specific for HCMV (alternatively, H
SV-1 indicates that the homology signal from HSV-1 is present in the amplicon), V indicates that the regulatory region controlling the expression of the viral gene / drug target is derived from the HCMV genome, and that the viral gene C indicates that the regulatory region is used for the expression of the drug target, and C indicates that the regulatory region used to control the expression of the viral gene / drug target is heterologous; in this example, CMV Includes IE promoter / enhancer and SV40 polyadenylation signal, S indicates that the construct is a subgenomic construct, and gene X indicates the antiviral agent (s)
Confirming the viral gene (s) that are the targets of, the examples given herein are:
It may be UL54, UL80 or UL97, indicating DNA polymerase, serine protease, or phosphotransferase. The helper virus or indicator gene helper virus vector is named as follows: HCMV is HC
MV strain, β _2,7 F-IG indicates a functional indicator gene inserted into the β _2,7 ORF in an appropriate reading frame under the control of the β _2,7 regulatory region;
Gene X indicates that the viral gene / drug target has been deleted from the virus, and in the examples provided herein, ΔUL54 indicates the deletion of DNA polymerase, serine protease, or phosphotransferase, respectively. , ΔUL
80 or ΔUL97.

Resistance Test Vector—Construction The resistance test vector consists of 1) an amplicon / gene X plasmid (pA-CMV-VS-gene) by introducing a unique site called the patient sequence acceptor site (PSAS) in the gene X coding region. X or pA-CMV-
CS-gene X), 2) amplify the patient-derived segment (PDS) corresponding to the CMV drug target (gene X) by amplification of viral DNA present in the blood or tissue of the infected patient, and then 3) amplification The prepared segment is prepared by correctly inserting the segment into the ampicillon / gene X plasmid in PSAS. Further embodiments include the isolation of viral RNA from tissue and the use of reverse transcription to convert the RNA to a DNA copy prior to amplification of PDS.

Testing for Drug Sensitivity and Resistance Testing for drug sensitivity and resistance was performed using the ampicillon / gene X plasmid (pA
-CMV-VS-gene X or pA-CMV-CS-gene X) and HCM
The test is performed in a two-part resistance test vector system containing the corresponding indicator gene virus vector such as V-β _2,7 F-IG / Δgene X. In one embodiment, the ampicillon / gene X plasmid is transfected into a packaging host cell / target host cell, and the cells are then infected with a defective indicator gene viral vector. In another embodiment, the ampicillon / gene X plasmid and the defect indicator gene viral vector DNA are co-transfected into a packaging host / target host cell simultaneously. The packaging host cell / target host cell can be any cell that is permissive for wild-type HCMV infection. Trans-supplementation of the deleted gene by the ampicillon / gene X plasmid results in a self-perpetuating viral population, resulting in the activity of the viral gene encoded by the patient-derived segment introduced into the ampicillon / gene X plasmid. -Dependent reporter gene expression is increased. Some reporter gene expression will be observed due to basal levels of expression from the defective indicator gene viral vector, but the replication of the genome of the defective indicator gene viral vector and thus its replication by trans-supplementation with the ampicillon / gene X plasmid Amplification results in increased reporter gene expression in target cells. Antiviral agents that inhibit HCMV replication via inhibition of viral gene / drug targets will limit amplification and expansion of the defective indicator gene viral vector, which is measured as a decrease in expression of the reporter gene product Can be.

Drug Screening Drug screening was performed on the ampicillon / gene X plasmid (pA-CMV-
VS- gene X or pA-CMV-CS- performed using a Gene X) and HCMV-β _2, 7 consisting of the indicator gene viral vector, such as F-IG / delta gene X resistance test vector system. The PDS may be derived from the genome of a laboratory strain of HCMV or from a patient-derived sample, may be of the wild-type sequence, or may have a viral gene / drug target against a known antiviral drug. It may also contain sequences that make it resistant.

The drug screening is performed as follows: Amplicon / gene X plasmid (pA-CMV-VS-gene X or pA-CMV-CS-gene X)
And HCMV-β _2,7 F-IG / Δgene X are introduced into cells in the absence or presence of potential antiviral compounds. After maintaining the culture for a period of time suitable to allow expansion of the defect indicator gene viral vector through the culture, the level of ampicillon expression of the reporter gene is measured and the degree of inhibition in the presence of the drug is calculated.

Example 10 Cytomegalovirus Drug Sensitivity and Resistance Tests Using a Resistance Test Vector Containing a Patient-Derived Segment (s) Under the Control of a Viral Promoter and a Functional Indicator Gene Indicator Gene Virus Vector-Construction Virus Replication for Activity A target host cell is constructed that expresses a functional indicator gene under the control of a dependent viral promoter. The defective helper virus vector (HCMV / Δgene X) was constructed to be defective for replication due to the fact that a segment of the genome containing the viral gene / drug target (gene X) has been deleted from the virus. You. The viral gene / drug target (gene X) is located on an ampicillon plasmid (pA-CMV-VS-gene X) containing the patient sequence acceptor site (PSAS) and the cis-acting functions required for trans-supplementation of the indicator gene viral vector (Specifically, these are HC
HCM directing MV origin of replication and HCMV genome cleavage and packaging
V "a" sequence). The PSAS in pA-CMV-VS-gene X was designed to allow PDS to a cassette containing the appropriate regulatory signals for the individual viral gene / drug target, and the viral gene / drug target in wild-type HCMV was From the context of Defective packaging / helper virus vector (HCMV / Δgene X) and ampicillon / gene X plasmid (pA
-CMV-VS-gene X) and the indicator cell line constitute a resistance test vector system. Defective packaging / helper virus vectors can only propagate as viral stocks in packaging host cells / target host cell systems where the deleted viral gene / drug target is trans. Virus stocks from such packaging host cells / target host cell systems are prepared, and the packaging host cells / target host cells or DNs from which these virus stocks were isolated.
A in combination with transfection of an ampicillon / gene X plasmid by transfection into a cell type that is permissive for HCMV infection.
Used to transfect packaging / target host cells as part of a resistance test vector system. Trans-supplementation of the deleted gene by the ampicillon / gene X plasmid results in a self-perpetuating viral population, so that the viral gene / drug target encoded by the patient-derived segment inserted into the ampicillon / gene X plasmid Increases the expression of a reporter gene that depends on the activity of

In another embodiment, the Amplicon / Gene X plasmid (pA-C
MV-CS-gene X) contains a PSAS that allows PDS to express a viral gene drug target (gene X) from a patient under the control of a heterologous promoter and polyadenylation signal. In one embodiment of this example, C
An expression cassette containing the MV IE enhancer promoter region, PSAS, and the SV40 polyadenylation (pA) signal provides, in addition to the cis-acting functions required for transsupplementation of the indicator gene viral vector, an ampicillon / gene X plasmid (pA-CMV). -CS-gene X) (specifically, these include the HCMV origin of replication and the HCMV "a" sequence that directs HCMV genome cleavage and packaging).

In various embodiments of the invention, the viral gene / drug target is 1) HCM
V DNA polymerase (UL54), 2) phosphotransferase (UL9
7) 3) Viral serine protease (UL80), 4) Any viral gene encoding a true or potential target for drug susceptibility or drug screening tests. Such viral genes include, but are not limited to, UL44, UL57, UL105, UL102, UL70, UL114,
L98 or UL84.

Resistance Test Vector-Construction The resistance test vector consists of 1) introducing an unique site called the patient sequence acceptor site (PSAS) in the viral gene / drug target (gene X) coding region, by combining the amplicon / gene X plasmid ( pA-CMV-VS-
Modifies gene X or pA-CMV-CS-gene X) and 2) amplifies patient-derived segment (PDS) corresponding to CMV drug target (gene X) from viral DNA present in blood or tissue of infected patient And then 3) prepared by correctly inserting the amplified segment into the ampicicon / gene X plasmid in the PSAS. Further embodiments include the isolation of viral RNA from tissue and the use of reverse transcription to convert the RNA to a DNA copy prior to amplification of PDS.

Testing for Drug Sensitivity and Resistance Testing for drug sensitivity and resistance was performed using the ampicillon / gene X plasmid (pA
-CMV-VS-gene X or pA-CMV-CS-gene X), HCMV /
The test is performed in a resistance test vector system that includes a defective viral vector such as the Δgene X and a target cell line that contains an indicator gene under the control of the HCMV promoter that depends on viral replication for activity. In one embodiment, the ampicillon / gene X plasmid is transfected into a packaging host cell / target host cell, and the cells are then infected with a defective helper virus vector. In another embodiment, the ampicillon / gene X plasmid and the defective helper virus vector DNA are co-transfected into a packaging host cell / target host cell. Trans-supplementation of the deleted gene by the ampicillon / gene X plasmid results in a self-perpetuating viral population, so that the viral gene / drug encoded by the patient-derived segment introduced into the ampicillon / gene X plasmid Increased reporter gene expression is dependent on the activity of the target. Antiviral agents that inhibit HCMV replication via inhibition of the viral gene / drug target will limit amplification and expansion of the defective helper virus vector, which is measured as a decrease in expression of the reporter gene product.

Drug Screening Drug screening was performed using the ampicillon / gene X plasmid (pA-CMV-
(VS-gene X or pA-CMV-CS-gene X) and a HCV / Δgene X, using a resistance test vector system including a helper virus vector. The PDS may be derived from the genome of a laboratory strain of HCMV or from a sample from a patient, may be of the wild-type sequence, or may be of the viral gene /
It may also contain sequences that render the drug target resistant to known antiviral drugs.

The drug screening is performed as follows: Amplicon / gene X plasmid (pA-CMV-VS-gene X or pA-CMV-CS-gene X)
And a helper virus vector, such as HCMV / Δgene X, is introduced into the cells in the absence or presence of a potential antiviral compound. After maintaining the culture for a time suitable to allow expansion of the defective helper virus vector through the culture, the level of reporter gene expression is measured and the degree of inhibition in the presence of the drug is calculated. Example 11 Cytomegalovirus drug susceptibility and resistance testing using a resistance test vector containing a non-functional indicator gene with a patient-derived segment (s) and an exchanged promoter Indicator gene virus vector-construction Non-functional with exchanged promoter The indicator gene viral vector containing the target indicator gene is designed using an HCMV ampicillon plasmid containing the viral gene (gene X) which is the target of the antiviral agent (s). Includes a non-functional indicator gene with an exchanged promoter, all of the cis-acting regulatory elements required for HCMV replication (ie, the "a" sequence and the HCMV origin of replication), and a viral gene / drug target expression cassette with a PSAS Indicator gene virus vector (
pA-CMV-VS-gene X- (NF-IG) PP). PSASs are designed to allow PDS into cassettes containing regulatory signals appropriate for individual viral gene / drug targets and are derived from the context of the viral gene / drug target in wild-type HCMV. The promoter region is incorrectly oriented with respect to the indicator gene ORF,
That is, a non-functional marker gene cassette is assembled so as to be antisense or at the wrong position of the marker gene ORF, that is, downstream of the marker gene ORF (FIG. 14). The promoter and indicator gene ORFs are segregated and located within regions of the genome that undergo reversal to each other during genome replication (FIG. 11). This reversal, if it occurs, carries the two parts of the exchanged promoter indicator gene cassette in the proper orientation, allowing expression of the indicator gene product. Defective helper virus vector (
HCMV / Δgene X) is defective for replication. This is because the segment of the genome containing the viral gene / drug target (gene X) is deleted from the virus. The indicator gene virus vector and the defective helper / packaging virus vector constitute a resistance test vector system. Defective helper virus vectors (HCMV / Δgene X) can only propagate in cell lines where the deleted viral gene / drug target is trans. Virus stock packaging host cells / target host cell lines can be prepared from which these viral stocks are used to infect the isolated packaging host cells / target host cells or DNA, and cells that are permissive for HCMV infection. Used to transfect packaging / target host cells as part of a resistance test vector system in combination with the introduction of the indicator gene viral vector by transfection into the type. Trans-supplementation of the deleted gene by the indicator gene viral vector results in a self-perpetuating viral population. During replication of the indicator gene viral vector, a concatamer of the indicator gene indicator is formed and reversal occurs as part of the normal replication cycle of HCMV (see FIG. 11), so that two segments of the replaced promoter cassette are now available. The rearrangement is effected to direct transcription of the RNA, which will be in the proper orientation and will allow expression of the reporter gene.

In another embodiment, the indicator gene viral vector contains a PSAS that allows PDS to express a gene X sequence from a patient under the control of a heterologous promoter and polyadenylation signal. In one embodiment of this example, the CMV IE enhancer promoter region, PSAS, and SV
Expression cassettes containing the 40 polyadenylation (pA) signal provide the cis-acting functions required for trans-recruitment of the indicator gene viral vector (specifically, they direct the HCMV origin of replication and HCMV genome cleavage and packaging). In addition to the HCMV "a" sequence) and the exchanged promoter cassette segment, the indicator gene viral vector (pA-CMV-CS-gene X- (N
F-IG) Included on PP) In various embodiments of the invention, the viral gene / drug target is 1) HCM
V DNA polymerase (UL54), 2) phosphotransferase (UL9
7) 3) Viral serine protease (UL80), 4) Any viral gene encoding a true or potential target for drug susceptibility or drug screening tests. Such viral genes include, but are not limited to, UL44, UL57, UL105, UL102, UL70, UL114,
L98 or UL84.

Resistance Test Vectors—Construction The resistance test vectors consist of 1) the introduction of a unique restriction site called the patient sequence acceptor site (PSAS) in the viral gene / drug target (gene X) coding region by introducing an indicator gene viral vector ( pA-CMV-VS-gene X- (NF-IG) PP or pA-CMV-CS-gene X- (NF-I
G) virus D which modifies PP) and 2) is present in the blood or tissue of infected patients
Patient-derived segment (PDS) corresponding to CMV drug target from NA (gene X)
) And then 3) insert the amplified segment exactly into the Amplicon / Gene X plasmid in the PSAS. A further embodiment is the isolation of viral RNA from tissue and the isolation of DDS prior to amplification of PDS.
Includes the use of reverse transcription to convert RNA to NA copies.

Testing for Drug Sensitivity and Resistance Testing for drug sensitivity and resistance was performed using the indicator gene viral vector (pA-CM
V-VS-gene X- (NF-IG) PP or pA-CMV-CS-gene X
-8NF-IG) PP), and a resistance test vector system containing defective helper virus vectors such as HCMV / Δgene X. In one embodiment,
Indicator gene virus vector (pA-CMV-VS-gene X- (NF-IG)
PP or pA-CMV-CS-gene X- (NF-IG) PP) is transfected into appropriate cells, which are then transfected into a defective helper virus vector (H
CMV / Δgene X). In another embodiment, the indicator gene virus vector and the defective helper / packaging virus vector DNA
Is co-transfected into a packaging host cell / target host cell. Trans-supplementation of the deleted gene by the indicator gene viral vector results in a self-perpetuating viral population. During replication of the indicator gene viral vector, a concatamer of the indicator gene viral vector is formed and reversal occurs as part of the normal replication cycle of HCMV, resulting in the proper orientation and expression of the indicator gene. The two segments of the exchanged promoter cassette are rearranged so as to direct the transcription of the RNA. The expression of the indicator gene depends on the activity of the viral gene / drug target encoded by the patient-derived segment introduced into the indicator gene viral vector. Antiviral drugs that inhibit HCMV replication via inhibition of viral gene / drug targets limit the replication of the indicator gene viral vector, which in turn can reduce the expression of the reporter gene product, where reversal can occur Limits the number of genomes that can be measured as

Drug Screening Drug screening was performed using the indicator gene viral vector (pA-CMV-VS-
Gene X- (NF-IG) PP or pA-CMV-CS-Gene X- (NF-
IG) PP) and a resistance test vector system containing defective packaging / helper virus vectors such as HCMV / Δgene X. The PDS is HC
It can be derived from the genome of a laboratory strain of MV, or from a sample from a patient, may be of the wild-type sequence, or may render the viral gene / drug target resistant to known antiviral drugs It can also contain sequences.

The drug screening is performed as follows: indicator gene viral vector (pA-CMV-VS-geneX- (NF-IG) PP) or pA-CMV-
Defective helper virus vectors such as CS-gene X- (NF-IG) PP) and HCMV / Δgene X are introduced into cells in the absence or presence of potential antiviral compounds. After maintaining the culture for a time suitable to allow replication of the indicator gene viral vector, the level of reporter gene expression is measured and the degree of inhibition in the presence of the drug is calculated.

Example 12 Cytomegalovirus Drug Susceptibility and Resistance Tests Using a Resistance Test Vector Containing a Non-Functional Indicator Gene with Patient-Derived Segment (s) and an Exchanged Coding Region Indicator Gene Virus Vector-Constructed Exchanged Coding An indicator gene viral vector containing a non-functional indicator gene having a region is designed using the HCMV ampicillon plasmid, which is the target (gene X) of the antiviral agent (s). Indicator gene virus vector (pA-
CMMV-VS-gene X- (NF-IG) PCR) is a non-functional indicator gene with an exchanged coding region, all of the cis-acting regulatory elements required for HCMV replication (ie, the "a" sequence and HCMV). Origin of replication), and a viral gene / drug target (gene X) expression cassette with PSAS. The PSASs are packaged with PDS in cassettes containing the appropriate regulatory signals for individual viral gene / drug targets.
And derived from the context of the viral gene / drug target in wild-type HCMV. The non-functional indicator gene cassette is such that the promoter region and the 5 'portion of the coding region are misoriented with respect to the remaining 3' portion of the coding region, ie, are antisense, or the remaining 3 'portion of the coding region. Assembled so as to be located at the wrong position, i.e., located downstream thereof (FIG. 15). The promoter and 5 'portion of the coding region are separated from the 3' portion of the coding region and are located within regions of the genome that undergo reversal with respect to each other during replication of the genome (Figure 11). This reversal, when it occurs, carries the two parts of the exchanged coding region indicator gene cassette in the proper orientation, allowing expression of the indicator gene product (FIG. 16). The defective helper virus vector (HCMV / Δgene X) is a viral gene / drug target (gene X)
Is defective in replication by the fact that is deleted from the virus. The indicator gene virus vector and the defective helper / packaging virus vector constitute a resistance test vector system. Defective helper / packaging virus vector (H
CMV / Δgene X) grows only in packaging host cell / target host cell systems in which the deleted viral gene is provided in trans. Viral stocks from such packaging host / target host cell lines can be propagated and used to infect cells, or DNA from these viral stocks
Can be isolated and used to transfect packaging / target host cells as part of a resistance test vector system in combination with the introduction of the indicator gene viral vector by transfection into a cell type permissive for HCMV infection. Can be used. Trans-supplementation of the deleted gene by the indicator gene viral vector results in a self-perpetuating viral population. During replication of the indicator gene viral vector, a concatamer of the indicator gene viral vector is formed and HC
Reversal occurs as part of the normal replication cycle of the MV, so that the two segments of the exchanged coding region cassette are now in the proper orientation to direct the transcription of RNA that would allow reporter gene expression. The relocation is effected as directed (FIG. 16).

In another embodiment, the indicator gene viral vector comprises a PSAS that permits PDS to express a viral gene sequence (gene X) from a patient under the control of a heterologous promoter and polyadenylation signal. . In one embodiment of this example, the CMV IE enhancer promoter region, PSA
S, and the expression cassette containing the SV40 polyadenylation (pA) signal provide the cis-acting functions required for trans-recruitment of the indicator gene viral vector (specifically, they provide the HCMV origin of replication and HCMV genome cleavage and packaging). In addition to the directing HCMV "a" sequence) and the exchanged coding region cassette segment, the indicator gene viral vector (pA-CMV-CS-
Gene X- (NF-IG) PCR) In various embodiments of the invention, the viral gene / drug target is 1) HCM
V DNA polymerase (UL54), 2) phosphotransferase (UL9
7) 3) Viral serine protease (UL80), 4) Any viral gene encoding a true or potential target for drug susceptibility or drug screening tests. Such viral genes include, but are not limited to, UL44, UL57, UL105, UL102, UL70, UL114,
L98 or UL84.

Resistance Test Vectors—Construction The resistance test vectors consist of 1) introducing a unique restriction site, called the patient sequence acceptor site (PSAS), into the viral gene / drug target (gene X) coding region, which results in an indicator gene viral vector ( pA-CMV-VS-gene X- (NF-IG) PCR or pA-CMV-CS-gene X- (NF-
IG) PCR) and 2) a patient-derived segment (P) corresponding to the CMV drug target (gene X) from viral DNA present in the blood or tissue of the infected patient
DS), and then 3) prepared by inserting the amplified segment exactly into the ampicillon / gene X plasmid in the PSAS. Further embodiments include the isolation of viral RNA from tissue and the use of reverse transcription to convert the RNA to a DNA copy prior to amplification of PDS.

Testing for Drug Sensitivity and Resistance Testing for drug sensitivity and resistance was performed using the indicator gene viral vector (pA-CM
V-VS-gene X- (NF-IG) PCR or pA-CMV-CS-gene X- (NF-IG) PCR) and a resistance test vector system containing defective helper virus vectors such as HCMV / Δgene X Done. In one embodiment, the indicator gene viral vector (pA-CMV-VS-geneX- (NF-I
G) PCR or pA-CMV-CS-geneX- (NF-IG) PCR) is transfected into an appropriate target host cell and then infected with the defective helper virus vector (HCMV / ΔgeneX). . In another embodiment, the indicator gene virus vector and the defective helper virus vector DNA
At the same time into cells. Trans-supplementation of the deleted gene by the indicator gene viral vector results in a self-perpetuating viral population. During replication of the indicator gene viral vector, a concatamer of the indicator gene viral vector is formed and reversal occurs as part of the normal replication cycle of HCMV, resulting in the proper orientation of the reporter gene for expression. R
Two segments of the exchanged coding region cassette are rearranged to direct transcription of the NA (FIG. 16). Reporter gene expression is dependent on the activity of the viral gene / drug target encoded by the patient-derived segment introduced into the indicator gene viral vector. HC via inhibition of viral gene / drug targets
Antiviral agents that inhibit MV replication limit the replication of the indicator gene viral vector, which in turn allows the reversal to occur and the number of genomes that can be measured as a decrease in expression of the reporter gene product. Restrict.

Drug Screening Drug screening was performed using the indicator gene viral vector (pA-CMV-VS-
Gene X- (NF-IG) PCR or pA-CMV-CS-Gene X- (NF
-IG) PCR) and a resistance test vector system containing defective helper virus vectors such as HCMV / Δgene X. PDS can be derived from the genome of a laboratory strain of HCMV, or from a sample from a patient, may be of the wild-type sequence, or may have a viral gene / drug target resistant to known antiviral drugs. May be included.

Drug screening is performed as follows: indicator gene viral vector (pA-CMV-VS-gene X- (NF-IG) PCR or pA-CMV-
Defective helper virus vectors such as CS-gene X- (NF-IG) PCR) and HCMV / Δgene X are introduced into cells in the absence or presence of potential antiviral compounds. After maintaining the culture for a time suitable to allow replication of the indicator gene viral vector, the level of reporter gene expression is measured and the degree of inhibition in the presence of the drug is calculated.

Example 13 Cytomegalovirus Drug Susceptibility and Resistance Tests Using a Resistance Test Vector Containing a Patient-Derived Segment (s) and a Functional Indicator Gene Indicator Gene Virus Vector-Construction Functional Indicator Under the Control of an Endogenous Virus Promoter The indicator gene viral vector containing the gene is based on the HCMV ampicillon plasmid containing the viral gene which is the target (gene X) of the antiviral agent (s). The indicator gene virus vector (pA-CMV-VS-gene X-F-IG) is a functional indicator gene under the control of a viral promoter that depends on viral replication, HCMV.
All of the cis-acting regulatory elements required for replication (ie, the “a” sequence and HCMV
Origin of replication), and a viral gene / drug target (gene X) expression cassette with PSAS (FIG. 17). The PSAS is designed to allow PDS in a cassette containing regulatory signals appropriate for individual viral genes and is derived from the context of the viral gene / drug target (gene X) in wild-type HCMV. The defective helper virus vector (HCMV / Δgene X) is defective in replication due to the fact that a segment of the genome containing the viral gene / drug target has been deleted from the virus. The indicator gene virus vector and the defective helper virus vector constitute a resistance test vector system. Defective helper virus vector (
HCMV / Δgene X) can only be propagated in packaging / target host cell systems in which the deleted viral gene is provided in trans. Virus stocks from such cell lines are prepared and used to infect packaging / target host cells, or DNA from these virus stocks is isolated and transferred to a cell type that is permissive for HCMV infection. Used to transfect packaging host cells / target host cells as part of a resistance test vector system in combination with the introduction of the indicator gene viral vector by transfection. As a result of trans-supplementation of the deleted gene by the indicator gene viral vector,
A self-persistent virus population is provided. Replication of the indicator gene virus vector depends on trans-recruitment between the indicator gene virus vector pA-CMV-VS-gene X-F-IG and the defective helper virus vector HCMV / Δgene X.

In another embodiment, the indicator gene viral vector comprises a PSAS that permits PDS to express a patient-derived gene / drug target (gene X) under the control of a heterologous promoter and polyadenylation signal. Including. 1 of this example
In one embodiment, the CMV IE enhancer promoter region, PSAS
And an expression cassette containing the SV40 polyadenylation (pA) signal
The cis-acting functions required for trans-supplementation of the indicator gene viral vector (specifically, these include the HCMV origin of replication and the HCMV "a" sequence that directs HCMV genome cleavage and packaging) and those replaced In addition to the region cassette segment, it is contained on the indicator gene viral vector (pA-CMV-CS-gene X-F-IG).

In various embodiments of the invention, the viral gene / drug target is: 1) HCM
V DNA polymerase (UL54), 2) phosphotransferase (UL9
7) 3) Viral serine protease (UL80), 4) Any viral gene encoding a true or potential target for drug susceptibility or drug screening tests. Such viral genes include, but are not limited to, UL44, UL57, UL105, UL102, UL70, UL114,
L98 or UL84.

Resistance Test Vectors—Construction The resistance test vectors consist of 1) the introduction of a unique restriction site called the patient sequence acceptor site (PSAS) into the coding region of the viral gene / drug target (gene X), which results in a marker gene viral vector ( pA-CMV-VS-gene X-F-IG or pA-CMV-CS-gene X-F-IG),
2) amplify the patient-derived segment (PDS) corresponding to the CMV drug target (gene X) from the viral DNA present in the blood or tissue of the infected patient, and then 3) transcribe the amplified segment in the PSAS into an amplicon / gene X Prepared by inserting correctly into the plasmid. Further embodiments include the isolation of viral RNA from tissue and the use of reverse transcription to convert the RNA to a DNA copy prior to amplification of PDS.

Testing for Drug Sensitivity and Resistance Testing for drug sensitivity and resistance was performed using the indicator gene viral vector (pA-CM
V-VS-gene X-F-IG or pA-CMV-CS-gene X-F-IG
), And a resistance test vector system containing a defective helper virus vector such as HCMV / Δgene X. In one embodiment, the indicator gene viral vector (pA-CMV-VS-gene X-F-IG or pA-CMV-CS-
The gene X-F-IG) is transfected into a suitable packaging host cell / target host cell and the cells are then transferred to a defective helper virus vector (HCMV /
Superinfection with Δgene X). In another embodiment, the indicator gene viral vector and the defective helper virus vector DNA are co-transfected into a packaging host cell / target host cell. Trans-supplementation of the deleted gene by the indicator gene viral vector results in a self-perpetuating viral population, resulting in the activity of the viral gene / drug target encoded by the patient-derived segment introduced into the indicator gene viral vector. Expression of a reporter gene that is directly dependent on HCM through inhibition of viral gene / drug targets
Antiviral agents that inhibit V replication limit the growth and expansion of defective helper virus vectors, which in turn can be measured as a reduction in the expansion of the reporter gene product.

Drug Screening Drug screening was performed using the indicator gene viral vector (pA-CMV-VS-
Gene X-F-IG or pA-CMV-CS-gene X-F-IG) and H
This is performed using a resistance test vector system containing a defective helper virus vector such as the CMV / Δgene X. PDS can be derived from the genome of a laboratory strain of HCMV, or from a sample from a patient, may be of the wild-type sequence, or may have a viral gene / drug target resistant to known antiviral drugs. May be included.

Drug screening is performed as follows: indicator gene viral vector (pA-CMV-VS-gene X-F-IG or pA-CMV-CS-gene X-F-IG) and HCMV / Δgene X Defective helper virus vectors, such as, are introduced into cells in the absence or presence of potential antiviral compounds. After maintaining the culture for a time suitable to allow replication of the indicator gene viral vector, the level of reporter gene expression is measured and the degree of inhibition in the presence of the drug is calculated.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of HCV replication cycle. Virions bind to the cell surface via specific interactions between viral surface glycoproteins and cell surface receptors (1).
Following receptor-mediated endocytosis (2) and low pH-dependent membrane fusion (3), the nucleocapsid core is released into the cytoplasm (4). Virion RNA is translated in close association with the endoplasmic reticulum, and polyproteins are processed by specific endoproteolytic cleavage mediated by host signal peptidases in the ER, or one of two viral proteases ( 5). After sufficient nonstructural protein has been produced, viral RNA is replicated via a negative strand intermediate to produce a more positive sense RNA for translation and packaging into new virions.
(6). Assemble structural proteins and RNA and extend to ER (7)
Form new viral particles that are secreted via the cellular pathway (8, 9) and release progeny virions.

FIG. 2. Schematic diagram of HCV genomic structure -9.5 kb HCV RNA. The order of the individual HCV proteins is shown in the HCV polyprotein with the relevant putative functions shown below. Cleavage sites for protein processing are indicated by triangles (open triangles are for host signal peptidase, black triangles are for NS2 / 3, and gray triangles are NS
3 / 4A). Internal ribosome entry site (IRES)
Is located at the 5 'end of the RNA and contains the entire untranslated region (UTR) and some sequences at the start of the C ORF. The 3 ′ end of the RNA depends on the type of HCV,
Contains poly (A) or poly (U) tail.

FIG. 3. Resistance test (luciferase fusion protein) Diagram representation of the resistance test vector (pXHCV-luc, where X is CMV or T7), with the patient sequence acceptor site (PSAS) for the introduction of a segment from the patient indicated by the arrow below the polyprotein. Promoter and terminator sequences are shown generally in this and subsequent figures so that several different types of regulatory elements can be used (as described below). The luciferase reporter gene is expressed as a fusion protein with the HCV polyprotein and then cleaved by the action of NS3 / 4A. B. A method of transfection using DNA transfection of a resistance test vector (pCMVHCV-luc) containing a CMV IE promoter and an SV40 polyadenylation signal. The RNA is transcribed by the cellular RNA polymerase in the nucleus of the transfected cell and then transported to the cytoplasm, where translation and replication can occur. C. DN of resistance test vector (pT7HCV-luc1) containing T7 RNA polymerase promoter and T7 RNA polymerase terminator
Transfection method using A transfection. The DNA is T7
Cells that express RNA polymerase are transfected (eg, after recombinant vaccinia virus or by co-transfection with a T7 RNA polymerase expression plasmid); RNA is transcribed cytoplasmically by T7 polymerase. D. RNA transfection of RNA derived from a resistance test vector (pT7HCV-luc2) containing a T7 RNA polymerase promoter and a restriction site located at the 3 'end for DNA linearization prior to transcription in vitro was performed. The transfection method used. The synthetic RNA can then be directly transfected into cells, where translation and replication can occur.

FIG. 4 shows a resistance test vector (bicistronic luciferase expression). A resistance test vector (pX) containing an IRES element for luciferase translation.
HCV-IRESluc). The IRES may be a native HCV IRES or may be derived from other viruses that use such elements for internal initiation of their mRNA. Luciferase expression occurs by internal initiation of translation from bicistronic RNA in the cytoplasm of the transfected cells.

FIG. 5: Resistance test vector (positive sense minigenome) Resistance test vector (pXH containing positive sense luciferase RNA minigenome)
Diagram representation of CV and pXIRESluc). The two constructs are co-transfected into cells; HCV non-structural proteins expressed from pXHCV act on both RNAs to replicate and package them. The replicated RNA is packaged in progeny virions, which are then used for infection of fresh target cells; the target cells are infected with HCV or transfected with pXHCV, and the luciferase minigenome is expressed. You.

FIG. 6: Resistance test vector (negative sense minigenome) Resistance test vector (pXHCV-ASIR) containing a negative sense RNA minigenome
ESluc) diagram display. The two constructs are co-transfected into cells; HCV nonstructural proteins expressed from pXHCV act on both RNAs, leading to their replication. The replicated RNA is packaged into progeny virions, which can then be used for infection of fresh target cells;
Infected with CV or transfected with pXHCV, the luciferase minigenome (now positive sense RNA) is expressed.

FIG. 7 is a diagrammatic representation of resistance test vectors (pXluc-NSHCV and pXSGCV) that express defective genomic RNA. The two constructs are co-transfected into cells; non-structural proteins expressed from pXluc-NSHCV act to
uc-NSHCV RNA; newly replicated RNA is packaged into virions from pXSHCV using structural proteins (C. E1 and E.
2). The progeny virions are then used to infect new cells.

FIG. 8: Resistance test vector (BVDV NS3 / 4A chimera, luc fusion protein) A diagrammatic representation of the genome of BVDV is shown at the top. HCV protease cleavage sites are indicated by gray triangles, and BVDV protease cleavage sites are indicated by cross-hatched diamonds (signal peptidase and NS2
/ 3 protease cleavage site is not shown). The resistance test vector pXBVDV (H
CVNS3) luc is a BVDV structural protease gene, BVDV NS2, H
Contains the CV NS3 / 4A protease, and BVDV NS4B and NSS; the cleavage sites in the non-structural protein regions indicate that they are HCV NS3 /
Modified to be recognized by the 4A protease. The luciferase reporter gene is expressed as a fusion with the chimeric polyprotein and contains the HCV NS3 /
Released by cleavage by 4A.

FIG. 9: Resistance test vector (BVDV NS5B chimera, luc fusion protein) Resistance test vector pXBVDV (HCVNS5B) luc; containing BVDV structural protein gene, BVDV NS2, NS3 / 4A protease, NS4B and NSSA, and HCV NS5B; Cis-acting regulatory elements recognized by NS5B polymerase and C
The amino-terminal region of the ORF is from HCV. The luciferase reporter gene is expressed in fusion with the chimeric polyprotein and released by cleavage with EVDV NS3 / 4A.

FIG. Diagram display of HCMV genome. The genome has terminal direct repeats labeled "a" present at 1-10 copies per genome. The “a” sequence is LS
It exists in the opposite direction at the junction (a '). The inverse repeats “b” and “c” are displayed as blocks, and “b” and “c ′” are used to indicate “b” in the antisense orientation.
And "c" iteration. U _L and U _S indicates a unique region of the L and S components of the genome. The blocks of the ORF are shown below the genome. The three genes encoding for the targets of the antiviral drugs UL54, UL80 and UL97 are indicated by arrows. OriLYT refers to HCMV soluble origin of replication. B. Cyclization of the monomeric HCMV genome following infection

FIG. Diagram display of HCMV genome. B. Cyclization of the HCMV genome following infection. C. The four isomers of the HCMV genome following genome inversion during replication. U _L and U _S under segment arrow highlights the reversal of L and S segments of genomic relative to each other.

FIG. 12 is a diagrammatic representation of the HCMV genome. The β _2,7 transcript present in the “b” region of the genome is present in wild-type HCMV (A), which is HCMV-β _2,7 −
Shown as modified by F-IG (B).

FIG. 13 is a diagrammatic representation of an ampicillon plasmid lacking a functional indicator gene. ORF
Gene X indicates an antiviral target (eg, UL54, UL80, UL97).
Large black arrows represent promoters, circles (pA +) indicate polyadenylation signals. The promoter and polyadenylation signal can be derived from the HCMV genome and can be, as appropriate, for a viral gene / drug target (gene X) or as an exogenous regulatory element described herein. PSAS indicates the patient's sequence acceptor site.

FIG. 14 is a diagrammatic representation of an amplicon plasmid containing a non-functional indicator gene including an exchanged promoter. ORF gene X is an antiviral target (eg, UL54
, UL80, UL97). Large black arrows represent promoters and round circles (pA +) represent polyadenylation signals. The promoter and the polyadenylation signal can be derived from the HCMV genome and, suitably,
It can be a drug target (gene X) or an exogenous regulatory element as described herein. PSAS indicates patient sequence acceptor site. The amplicon contains the exchanged promoter cassette as described herein.

FIG. 15 is a diagrammatic representation of an amplicon plasmid containing a non-functional indicator gene with an exchanged coding region. ORF gene X is an antiviral target (eg, UL
54, UL80, UL97). Large black arrows represent promoters, circles (pA +) indicate polyadenylation signals. The promoter and the polyadenylation signal can be derived from the HCMV genome and can be derived from the viral gene / drug target (gene X
) Or may be an exogenous regulatory element as described herein. This amplicon contains the exchanged coding region cassette described herein.

FIG. 16 is a diagrammatic representation of the four isomers of the HCMV genome present after viral replication and the relative positions of the exchanged coding region cassettes after rearrangement. Panel C
In, the two halves of the cassette are now in the proper orientation and direct the expression of the reporter gene. The arrangement shown in panel B results in proper juxtaposition of the two halves of the cassette following contacammerization of the rearranged genome.

FIG. 17 is a diagrammatic representation of an amplicon plasmid containing a functional indicator gene. ORF
Gene X indicates an antiviral target (eg, UL54, UL80, UL97).
Large black arrows represent promoters and circles (pA +) represent polyadenylation signals. The promoter and polyadenylation signal can be derived from the HCMV genome and can be suitable for a viral gene / drug target (gene X) or can be an exogenous regulatory element as described herein. PSAS indicates patient sequence acceptor site. This amplicon contains the functional indicator gene cassette described herein.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP , KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (72) Inventor Perkin, Nail Tea United States, California 94401 Belmont, Farnwood Way 1815F Terms (reference) 2G045 AA40 4B024 AA11 AA13 BA07 BA14 CA04 DA02 EA02 FA10 FA17 GA11 HA01 4B063 QA01 QA18 QQ10 QQ20 QQ95 QR60 QR72 QR79 QR80 QS05 QX02 4B065 AA90X AA92Y AA93X AA95BA ABA28 AB25 CAABA28

Claims (111)

[Claims] 1. A method for determining susceptibility to an HCV antiviral drug, comprising: (a) introducing into a host cell a resistance test vector containing a segment from a patient containing a hepatitis C virus gene and an indicator gene. (B) culturing the host cell obtained in step (a); (c) measuring the expression of the indicator gene in a target host cell; and (d) obtaining the expression obtained in step (c). Comparing the expression of the indicator gene with the expression of the indicator gene measured when performing steps (a) to (c) in the absence of the antiviral agent, comprising the steps of: A method in which a test concentration of the HCV antiviral agent is present in step (c), step (b) to step (c), or step (c). 2. The method according to claim 1, wherein the resistance test vector comprises a genomic virus vector DNA. 3. The method according to claim 1, wherein the resistance test vector comprises:
A method comprising RNA of a genomic virus vector. 4. The method according to claim 1, wherein the resistance test vector comprises:
A method comprising a gene encoding C, E1, E2, NS2, NS3, NS4, or NS5. 5. The method of claim 1, wherein the patient-derived segment comprises a functional viral sequence. 6. The method of claim 1, wherein the patient-derived segment encodes a protein that is a target of an antiviral drug. 7. The method of claim 1, wherein the patient-derived segment encodes two or more proteins that are targets for antiviral agents. 8. The method of claim 5, wherein said functional viral sequence comprises an IRES sequence. 9. The method according to claim 1, wherein the indicator gene is a functional indicator gene, the host cell is a resistance test vector host cell, and the target host cell is a resistance test vector virus particle. A method comprising the additional step of infecting with. 10. The method according to claim 1, wherein the indicator gene is a specific functional indicator gene. 11. The method according to claim 10, wherein the host cell is a packaging host cell / resistance test vector host cell. 12. The method according to claim 11, wherein the culture is a co-culture. 13. The method of claim 11, wherein said target host cell comprises:
A method of infecting with a resistance test vector virus particle using the filtered supernatant from the packaging host cell / resistance test vector host cell. 14. The method according to claim 1, wherein the indicator gene is a luciferase gene. 15. The method according to claim 1, wherein the indicator gene is a β-lactamase gene. 16. The method according to claim 11, wherein the packaging host cell / resistance test gene / resistance test vector host cell is a human cell. 17. The method of claim 11, wherein said packaging host cell / resistance test vector host cell is a human hepatocyte. 18. The method of claim 11, wherein said packaging host cell / resistance test vector host cell is a Huh7 cell. 19. The method of claim 1, wherein said target host cell is He.
A method which is a pG2 cell. 20. It comprises a flaviviridae gene and an indicator gene,
A resistance test vector containing a patient-derived segment. 21. The resistance test vector according to claim 20, wherein the patient-derived segment comprises a flavivirus gene. 22. The resistance test vector according to claim 20, wherein the segment derived from the patient is one gene. 23. The resistance test vector according to claim 20, wherein the segment derived from the patient is two or more genes. 24. The resistance test vector according to claim 20, wherein the patient-derived segment comprises an HCV gene. 25. The resistance test vector according to claim 20, wherein the patient-derived segment comprises an NS3 / 4A protease gene. 26. The resistance test vector according to claim 20, wherein the patient-derived segment comprises the NS5B RDRP gene. 27. The resistance test vector according to claim 20, wherein the patient-derived segment comprises an IRES. 28. The resistance test vector according to claim 20, wherein the indicator gene is a functional indicator gene. 29. The resistance test vector according to claim 20, wherein the indicator gene is a non-functional indicator gene. 30. The resistance test vector according to claim 20, wherein the indicator gene is a luciferase gene. 31. A packaging host cell transfected with the resistance test vector according to claim 20. 32. The packaging host cell of claim 31, which is a mammalian host cell. 33. The packaging host cell of claim 31, which is a human host cell. 34. The packaging host cell according to claim 31, which is a human hepatocyte. 35. The packaging host cell according to claim 31, which is HepG2. 36. The packaging host cell of claim 31, which is Huh7. 37. A method for determining susceptibility to an HCV antiviral agent, comprising: (a) providing a resistance test vector comprising a segment from a patient containing a hepatitis C virus gene and a non-functional indicator gene to a host. (B) culturing the host cell obtained in step (a); (c) measuring the expression of the indicator gene in a target host cell; and (d) step (c). Comparing the expression of the indicator gene obtained in the step with the expression of the indicator gene measured when performing the steps (a) to (c) in the absence of the high-viral drug, In (a) to (c), (b) to (c), or (c), a test concentration of the HCV antiviral agent is present. 38. The method according to claim 37, wherein the resistance test vector comprises a genomic virus vector DNA. 39. The method of claim 37, wherein said resistance test vector comprises a genomic virus vector RNA. 40. The method of claim 37, wherein said resistance test vector comprises a gene encoding C, E1, E2, NS2, NS3, NS4, or NS5. 41. The method of claim 37, wherein the patient-derived segment encodes one protein. 42. The method of claim 37, wherein the patient-derived segment encodes more than one protein. 43. The method of claim 37, wherein the patient-derived segment comprises a functional viral sequence. 44. The method according to claim 37, wherein the indicator gene is a luciferase gene. 45. The method of claim 37, wherein said host cell is a packaging host cell. 46. The method of claim 37, wherein said packaging host cell is a human cell. 47. The method of claim 37, wherein said packaging host cell is a human hepatocyte. 48. The method of claim 37, wherein said packaging host cell is a Huh7 cell. 49. The method of claim 37, wherein said packaging host cell is a HepG2 cell. 50. The method of claim 37, wherein said non-functional indicator gene comprises a negative sense sequence. 51. The method of claim 37, wherein said host cell and target cell are the same cell. 52. The method of claim 37, wherein said target cells are human cells. 53. The method of claim 37, wherein the target host cell is
A method of infecting with a resistance test vector virus particle using the filtered supernatant derived from the packaging cell / resistance test vector host cell. 54. The method of claim 37, wherein said culturing is by co-culturing. 55. A method for determining HCV antiviral drug resistance in a patient, comprising: (a) generating a standard curve of drug susceptibility for the HCV antiviral drug; and (b) according to the method of claim 1, Measuring HCV antiviral drug susceptibility in the patient; and (c) comparing the HCV antiviral drug susceptibility in step (b) to the standard curve determined in step (a). The method wherein the reduced susceptibility indicates the development of HCV antiviral drug resistance in the patient. 56. A method for determining HCV antiviral drug resistance in a patient, comprising: (a) creating a standard curve of drug sensitivity for the HCV antiviral drug; and (b) according to the method of claim 37, Measuring HCV antiviral drug susceptibility in the patient; and (c) comparing the HCV antiviral drug susceptibility in step (b) to the standard curve determined in step (a). The method wherein the reduced susceptibility indicates the development of HCV antiviral drug resistance in the patient. 57. A method for determining HCV antiviral drug resistance in a patient, comprising: (a) initially determining the HCV antiviral drug susceptibility in the patient according to the method of claim 1, wherein the method comprises the steps of: Obtaining the segment from the patient at about the time; (b) measuring the HCV antiviral drug susceptibility in the same patient at a later time; and (c) HCV measured in steps (a) and (b). Comparing the antiviral drug susceptibility at the later time point with the susceptibility at the first time point, wherein the decrease in HCV antiviral susceptibility at the first time point is the occurrence of HCV antiviral drug resistance in the patient or How to indicate progress. 58. A method for determining HCV antiviral drug resistance in a patient, comprising: (a) initially determining the HCV antiviral drug susceptibility in the patient according to the method of claim 37; Obtaining the segment from the patient at about the time; (b) at a later time measuring HCV antiviral drug susceptibility in the same patient; and (c) HCV measured in steps (a) and (b). Comparing the antiviral drug susceptibility at the later time point with the susceptibility at the first time point, wherein the decrease in HCV antiviral susceptibility at the later time point is the occurrence or progression of HCV antiviral drug resistance in the patient. How to show. 59. A method for determining susceptibility to an HCMV antiviral agent, comprising: (a) introducing into a host cell a resistance test vector comprising a patient-derived segment containing an HCMV gene and an indicator gene; (B) culturing the host cell from step (a); (c) measuring the expression of the indicator gene in a target host cell; and (d) measuring the expression of the indicator gene obtained in step (c). Comparing the expression with the expression of the indicator gene measured when the steps (a) to (c) are performed in the absence of the antiviral agent, and The method wherein the viral agent is present in steps (a)-(c); steps (b)-(c); or step (c). 60. The method of claim 59, wherein said resistance test vector comprises a genomic virus vector DNA. 61. The method of claim 59, wherein said resistance test vector comprises a subgenomic virus vector DNA. 62. The method of claim 59, wherein said resistance test vector is a phosphotransferase (UL97), a DNA polymerase (UL54),
Protease (UL80), UL54, UL44, UL57, UL105, UL
DNA encoding 102, UL70, UL114, UL98, or UL84
The method that contains. 63. The method of claim 59, wherein said patient-derived segment comprises a functional viral sequence. 64. The method of claim 59, wherein the method encodes a protein that is a target of an antiviral drug. 65. The method of claim 59, wherein the method encodes two or more proteins that are targets of the antiviral agent. 66. The method of claim 59, wherein said indicator gene is a functional indicator gene, said host cell is a resistance test vector host cell, and said target host cell is a resistance test vector virus particle. A method comprising the additional step of infecting. 67. The method of claim 59, wherein said indicator gene is a non-functional indicator gene. 68. The method of claim 59, wherein said host cell is a packaging host cell / resistance test vector host cell. 69. The method according to claim 68, wherein the culture is a co-culture (co-culture).
cultivation). 70. The method of claim 69, wherein said target host cell comprises:
A method of infecting with a resistance test vector virus particle derived from the packaging host cell / resistance test vector host cell. 71. The method according to claim 59, wherein said indicator gene is a luciferase gene. 72. The method according to claim 59, wherein the indicator gene is β-
A method that is a lactamase gene. 73. The method of claim 68, wherein said packaging host cell / resistance test vector host cell is a human cell. 74. The method of claim 68, wherein said packaging host cell / resistance test vector host cell is a human foreskin fibroblast. 75. The method of claim 68, wherein said packaging host cell / resistance test vector host cell is an MRC5 cell. 76. The method of claim 59, wherein said target host cell is a human fetal lung cell. 77. A resistance test vector comprising a patient-derived segment containing a herpesviridae gene and an indicator gene. 78. The resistance test vector of claim 77, wherein the patient-derived segment is an α-herpesviridae. 79. The resistance test vector according to claim 77, wherein the patient-derived segment is one gene. 80. The resistance test vector according to claim 77, wherein said patient-derived segment is two or more genes. 81. The resistance test vector of claim 77, wherein said patient-derived segment comprises an HCMV gene. 82. The resistance test vector according to claim 77, wherein the indicator gene is a functional indicator gene. 83. The resistance test vector according to claim 77, wherein the indicator gene is a non-functional indicator gene. 84. The resistance test vector according to claim 77, wherein the indicator gene is a luciferase gene. 85. A packaging host cell infected with the resistance test vector of claim 77. 86. The packaging host cell of claim 85, which is a mammalian host cell. 87. The packaging host cell of claim 85, which is a human host cell. 88. The packaging host cell of claim 85, which is a human fetal lung cell. 89. The packaging host cell of claim 85, which is an MRC5 cell. 90. The packaging host cell of claim 85, which is a human foreskin fibroblast cell line. 91. A method for determining susceptibility to an HCMV antiviral agent, comprising: (a) providing a resistance test vector comprising a segment from a patient containing the HCMV gene and a non-functional indicator gene to a host cell. (B) culturing the host cell obtained in step (a); (c) measuring the expression of the indicator gene in target host cells; and (d) obtaining in step (c). Comparing the expression of the indicator gene with the expression of the indicator gene measured when performing the steps (a) to (c) in the absence of the antiviral agent. ) To (c), (b) to (c), or (c), wherein a test concentration of the HCV antiviral agent is present. 92. The method of claim 91, wherein said resistance test vector comprises a genomic virus vector DNA. 93. The method of claim 91, wherein said resistance test vector comprises DNA of a subgenomic virus vector. 94. The method of claim 91, wherein said resistance test vector is a phosphotransferase (UL97), a DNA polymerase (UL54),
Protease (UL80), UL54, UL44, UL57, UL105, UL
DNA encoding 102, UL70, UL114, UL98, or UL84
The method that contains. 95. The method of claim 91, wherein said patient-derived segment encodes one protein. 96. The method of claim 91, wherein the method encodes two or more proteins that are targets for antiviral agents. 97. The method according to claim 91, wherein said indicator gene is a luciferase gene. 98. The method of claim 91, wherein said host cell is a packaging host cell. 99. The method of claim 91, wherein said packaging host cell is a human cell. 100. The method of claim 91, wherein said packaging host cell is a human fetal lung cell. 101. The method of claim 91, wherein said packaging host cell is a foreskin fibroblast. 102. The method of claim 91, wherein said non-functional indicator gene is an altered promoter. 103. The method of claim 91, wherein said non-functional indicator gene is an altered coding region. 104. The method of claim 91, wherein the finger host cell and the target cell are the same cell. 105. The method of claim 91, wherein said target cells are human cells. 106. The method of claim 91, wherein said target host cell is infected with a resistance test vector virus particle from said packaging host cell / resistance test vector host cell. 107. The method of claim 91, wherein said culturing is a co-culture (c
o-cultivation). 108. A method for determining HCMV antiviral drug resistance in a patient, comprising: (a) generating a standard curve of drug sensitivity to the HCMV antiviral drug; and (b) according to the method of claim 59. Measuring the HCMV antiviral drug susceptibility in the patient; (c) measuring the expression of the indicator gene in a target host cell; and (d) measuring the HCMV antiviral drug susceptibility in step (b). B) comparing to a standard curve generated in the above step, wherein a decrease in HCMV antiviral drug sensitivity is indicative of the development of HCMV antiviral drug resistance in said patient. 109. A method for determining HCMV antiviral drug resistance in a patient, comprising: (a) generating a standard curve of drug sensitivity to the HCMV antiviral drug; and (b) according to the method of claim 91. Measuring the HCMV antiviral drug susceptibility in the patient; (c) measuring the expression of the indicator gene in a target host cell; and (d) measuring the HCMV antiviral drug susceptibility in step (b). B) comparing to a standard curve generated in the above step, wherein a decrease in HCMV antiviral drug sensitivity is indicative of the development of HCMV antiviral drug resistance in said patient. 110. A method for determining HCMV antiviral drug resistance in a patient, comprising: (a) initially measuring HCV antiviral drug susceptibility in the patient according to the method of claim 59, wherein said segment is derived from said patient. (B) measuring HCV antiviral drug susceptibility in the same patient at a later point in time; and (c) HCV measured in steps (a) and (b). Comparing the antiviral drug susceptibility at the later time point with the susceptibility at the first time point, wherein the decrease in HCV antiviral susceptibility at the later time point is the occurrence of HCV antiviral drug resistance in the patient or How to indicate progress. 111. A method for determining HCMV antiviral drug resistance in a patient, comprising: (a) initially determining HCV antiviral drug susceptibility in the patient according to the method of claim 91, wherein said segment is derived from said patient. (B) measuring HCV antiviral drug susceptibility in the same patient at a later point in time; and (c) HCV measured in steps (a) and (b). Comparing the antiviral drug susceptibility at the later time point with the susceptibility at the first time point, wherein the decrease in HCV antiviral susceptibility at the later time point is the occurrence of HCV antiviral drug resistance in the patient or How to indicate progress.