JP2003510033A - Compositions and methods for inhibiting human immunodeficiency virus infection by down-regulating human cell genes - Google Patents

Abstract

  (57) [Summary] The present invention relates to nucleic acid molecules involved in HIV infection, proteins encoded by the nucleic acid molecules, and protective compounds comprising inhibitors of the nucleic acid molecules, proteins, and products encoded by the nucleic acid molecules. In addition, the present invention also provides the use of the cellular gene and its encoded product in screening assays to select additional gene repressing elements, cellular genes corresponding to the GSE, and further inhibitors of HIV. On how to do.

Description

Detailed Description of the Invention

CROSS REFERENCE TO RELATED APPLICATIONS This application describes PCT International Patent Application No. PCT / U filed June 2, 1998
S98 / 11452 and U.S. Pat. No. 0 filed May 29, 1998
It is a continuation-in-part application of 9 / 087,609, both of which were issued on June 2, 1997.
This is a continuation-in-part application of U.S. patent application Ser. No. 08 / 867,314 filed dated. Each patent application referenced in this chapter is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION The present invention relates to the identification of specific human genes as cellular targets for designing therapeutic agents to suppress human immunodeficiency virus (HIV) infection. These genes encode products required for HIV infection. This is because HIV infection is blocked when the expression of these genes is downregulated. Therefore, compounds that inhibit the expression of these genes or the function of the encoded gene products can be used as therapeutic agents for the treatment and / or prevention of HIV infection. Further, the present invention provides H
Method for identifying additional cellular genes as therapeutic targets to suppress IV infection and HI
It relates to the use of such cellular genes and their encoded products in screening assays to select protective compounds that block V infection.

BACKGROUND OF THE INVENTION HIV has been shown to be the major cause of acquired immunodeficiency syndrome (AIDS) (Barre-Sinoussi et al., 1983, Science 220 :).
868-870; Gallo et al., 1984, Science 224: 500-.
503). HIV causes an immune deficiency in an individual by infecting and depleting important cell types of the immune system. This then leads to opportunistic infections, neoplastic growth and death.

HIV is a member of the lentivirus family of retroviruses (T
eich et al., 1984, RNA tumor virus, edited by Weiss et al., CSH-Pre.
ss, 949-956). A retrovirus is a virus with a small envelope that contains a diploid single-stranded RNA genome and that replicates through a DNA intermediate produced by RNA-dependent DNA polymerase, a reverse transcriptase encoded by the virus. (Varmus, 1988, Science 240: 1427.
~ 1439). HIV has at least two distinct subtypes: HIV-
1 (Barre-Sinoussi et al., Ibid .: Gallo et al., Ibid.) And HI.
V-2 (Clavel et al., 1986, Science 233: 343-346.
Guyader et al., 1987, Nature 326: 662-669). Genetic heterogeneity exists within each of these HIV subtypes.

CD4 ⁺ T cells are the main target of HIV infection. This is because the CD4 cell surface protein acts as a cell receptor to which HIV attaches (Dal
gleish et al., 1984, Nature 312: 763-767; Klat.
Zmann et al., 1984, Nature 312: 767-768; Maddo.
n et al., 1986, Cell 47: 333-348). Viral entry into cells depends on binding of the viral protein gp120 to cellular CD4 receptor molecules (McDougal et al., 1986, Science 231: 382-385;
Maddon et al., 1986, Cell 47: 333-348).

HIV infection is pervasive, and HIV-related diseases have become a global health problem. Despite considerable efforts in the design of anti-HIV modalities, to date there have been no successful preventive or curative measures against AIDS. However, several stages of the HIV life cycle are considered as potential targets for therapeutic intervention (Mi
tsuya et al., 1991, FASEB J. et al. 5: 2369-2381). For example, the virus-encoded reverse transcriptase is the main focus of drug development. AZT, d
Many reverse transcriptase-targeted drugs have been shown to be active against HIV, including 2 ', 3'-dideoxynucleotide analogs such as dI, ddC and ddT (Mitsuya et al., 1990). , Science 249: 153
3-1544). These nucleotide analogues, while beneficial, are not curative, presumably due to the rapid emergence of drug resistant HIV variants (La
Nder et al., 1989, Science 243: 1731-1734). Moreover, these drugs often exhibit adverse side effects such as myelosuppression, vomiting and liver abnormalities.

Another stage of the HIV life cycle that has been targeted is the early stage of HIV infection,
It is the entry of a virus into a cell. This approach is mainly based on recombinant soluble CD4
The protein has been used to block infection of CD4 ⁺ T cells by several HIV-1 strains (Smith et al., 1987, Science 238: 1704-).
1707). However, certain primary HIV-1 isolates are relatively insensitive to inhibition by recombinant CD4 (Daar et al., 1990, Proc. Natl. Acad.
． Sci. USA 87: 6574-6579). So far, recombinant soluble CD
4 clinical trials yielded inconclusive results (Schooley et al.,
1990, Ann. Int. Med. 112: 247-253; Kahn et al., 1
990, Ann. Int. Med. 112: 254-261; Yarchoan.
, 1989, Proc. Vth Int. Conf. on AIDS, Item 564, MCP 137).

Moreover, later stages of HIV replication, which also include important virus-specific processes of specific viral proteins and enzymes, have been targeted for anti-HIV drug development. The late stage process depends on the activity of the virus-encoded protease,
Drugs including saquinavir, ritonavir, and indinavir were developed to inhibit this protease (Pettit et al., 1993, Persp. Dru).
g Discov. Design 1: 69-83). In this class of drugs,
The emergence of drug-resistant HIV variants is also a problem; resistance to one inhibitor often confers cross resistance to other protease inhibitors (Condra et al.,
1995, Nature 374: 569-571). Also, these drugs
It often shows adverse side effects such as nausea, change in taste, paresthesia, fat accumulation, diarrhea and nephrolithiasis.

Antiviral therapy of HIV using different combinations of nucleotide analogs and protease inhibitors has recently been shown to be more effective than using only a single drug (Torres et al., 1997, INF. Med. 14:14.
2-160). However, despite being able to significantly reduce viral load, to date there is no evidence that AIDS is curatively treated with the available drug combinations.

[0010] Another potential approach to developing treatments for AIDS involves delivering foreign genes to infected cells. One such gene therapy approach involves the use of genetically engineered viral vectors to introduce toxic gene products and kill HIV infected cells. Another form of gene therapy is designed to protect virus-infected cells from cytolysis by specifically disrupting viral replication. Stable expression of RNA-based (decoy, antisense and ribozymes) or protein-based (transdominant mutants) HIV-1 antiviral agents can block certain stages of the viral life cycle. Decoy RNA of TAR or RRE (Sullenger et al., 1990, Cell 63: 6).
01-608; Sullenger et al., 1991, J. Am. Virol. 65:68
11-6816; Lisziewicz et al., 1993, New Biol. 3:
82-89; Lee et al., 1994, J. Am. Virol. 68: 8254-8264
), Ribozyme (Sarver et al., 1990, Science 247: 122).
2-1225; Wecrasinghe et al., 1991, J. Am. Virol. 65:
5531-5534; Dropulic et al., 1992, J. Am. Virol. 66:
1432-1441; Ojwang et al., 1992, Proc. Natl. Aca
d. Sci. USA 89: 10802-10806; Yu et al., 1993, Pr.
oc. Natl. Acad. Sci. USA 90: 6340-6344; Yu.
Et al., 1995, Proc. Natl. Acad. Sci. USA 92: 699.
~ 703; Yamada et al., 1994, Gene Therapy 1: 38-.
45), an antisense RNA complementary to the mRNA of the viral gag, tat, rev or env gene (Sezakiel et al., 1991, J. Virol. 6).
5: 468-472; Chatterjee et al., 1992, Science 2.
58: 1485-1488; Rhodes et al., 1990, J. Am. gen. Viro
l. 71: 1965, Rhodes et al., 1991, AIDS 5: 145-15.
1; Sezakiel et al., 1992, J. Am. Virol. 66: 5576-558
1; Joshi et al., 1991, J. Am. Virol. 65: 5524-5530),
And Rev (Bevec et al., 1992, Proc. Natl. Acad.S.
ci. USA 89: 9870-9874), Tat (Pearson et al., 19).
90, Proc. Natl. Acad. Sci. USA 87: 5079-50
83; Modesti et al., 1991, New Biol. 3: 759-768)
, Gag (Trono et al., 1989, Cell 59: 113-120), En.
v (Bushschacher et al., 1995, J. Virol. 69: 1344.
~ 1348) and proteases (Junker et al., 1996, J. Virol).
． 70: 7765-7772), and many anti-HIV suppressors have been reported, including transdominant mutants.

Antisense polynucleotides are designed to complex and sequester HIV-1 transcripts (Holmes et al., WO 93/11230; Lipp.
s et al., WO 94/10302; Kretschmer et al., EP 594,881;
And Chatterjee et al., 1992, Science 258: 1485.
). In addition, an enzymatically active RNA called a ribozyme was used to cleave the viral transcript. The use of ribozymes in hematopoietic cell lines has been reported to produce resistance to HIV-1 (Ojwang et al., 1992, Proc. Na.
tl. Acad. Sci. USA 89: 10802-06; Yamada et al.,
1994, Gene Therapy 1: 38-45; Ho et al., WO 94/2.
6877; and Cech and Sullenger, WO 95/13379).
. In a preclinical study, RevM10, a transdominant Rev protein, was
In vitro, transfection of CD4 ⁺ cells of HIV infected individuals was shown to confer survival advantage over cells transfected with vector alone (
Woffendin et al., 1996, Proc. Natl. Acad. Sci. U
SA 93: 2889-2894).

Despite the tremendous efforts in the art, reliable and curative anti-HIV therapeutics and measures have not been developed.

In nature, the evolution of intracellular pathogens such as HIV requires the generation of interactions of multiple cellular components with its genes and gene products. For example, virus-host cell interactions include binding of the virus to specific cell receptor (s) of the virus, translocation through the cell membrane, trans-jacketing, replication of the viral genome, transcription of viral genes, and the like. Each of these events occurs in the cell and involves interactions with cellular components. Thus, the viral life cycle can only be completed if the cells are “permissive” to viral infection. The availability of amino acids and nucleotides for replication of the viral genome and protein synthesis, the energy status of the cells, the presence of cellular transcription factors and enzymes, all contribute to the propagation of the virus in the cell. As a result, cellular components, in part, determine the susceptibility of host cells to infection and can be used as potential targets for the development of new therapeutic interventions. In the case of HIV, one cellular component used towards this goal is CD4, a cell surface molecule for HIV attachment.

Recently, HIV entry into susceptible cells was reported to require expression of a second type of receptor, the chemokine receptor (CCR2, CCR3, CCR5 or CXCR4), in addition to CD4 ( Moore, 1997, Science 276.
: 51-52). Chemokine receptors are usually its natural ligand, RAN.
Binds TES, MIP-1α and MIP-1β. In case of HIV infection, CD
4 first binds to the HIV gp120 protein on the cell surface and then this complex binds to the chemokine receptor, thus it has been proposed that the virus enters the cell (Cohen et al., 1997, Science 275: 1261). ).
Therefore, chemokine receptors may represent additional cellular targets for the design of HIV therapeutics. Inhibitors of the HIV / chemokine receptor interaction are tested as anti-HIV agents. However, there is still a need to find additional cellular targets for the design of anti-HIV therapeutics, especially intracellular targets that disrupt viral replication after the virus has entered the cell.

SUMMARY OF THE INVENTION The present invention prevents HIV infection by down-regulating the expression of certain human cell genes and / or by inhibiting the activity of the products encoded by said genes. To compositions and methods. In particular, it relates to many human cell-derived nucleic acid molecules that prevent HIV infection of susceptible cells. An isolated nucleic acid molecule corresponds to a portion of a cellular gene or its complement and is referred to herein as a gene repression element (GSE). The cellular gene encodes an intracellular product required for productive HIV infection. Furthermore, small molecule inhibitors of the same cellular gene and its encoded product are also within the scope of the invention. The present invention also relates to methods of identifying additional cellular genes as therapeutic targets for suppressing HIV infection, and methods of using the cellular genes and their encoded products to select additional inhibitors of HIV.

The present invention provides, in part, that nucleic acid molecules isolated from human cells prevent both latent HIV-1 activation in CD4 ⁺ cell lines and proliferative HIV infection in said cells. In turn, the nucleic acid molecule is based on Applicants' discovery that it corresponds to a fragment of a particular human cell gene. In this regard, any cell or viral marker associated with HIV infection can be used for selection of the nucleic acid molecule. An example of said marker is CD4, which is conveniently monitored by using specific antibodies.

Based on considerable sequence identity (90% to 100%), many isolated GSEs correspond to a portion of the human cellular genes that encode different subunits of NADH dehydrogenase in the mitochondrial enzyme complex. There is. In addition, inhibitors of this enzyme inhibit HIV infection of susceptible host cells, including newly isolated human CD4 ⁺ T cells. In addition, there is considerable sequence identity (90% -100%) with the following human cell genes:
%), 2-oxoglutarate dehydrogenase, pyruvate kinase type M2 / cytosolic thyroid hormone binding protein, calnexin, ADP-ribosylation factor 3, eukaryotic initiation factor 3, protein tyrosine phosphatase, Herpesvirus-associated ubiquitin-specific protease, eukaryotic initiation factor 4B, CD44, phosphatidyl-inositol 3 kinase, elongation factor 1α, bone morphogenetic protein-1, double-strand break DNA repair gene protein,
Rat guanine nucleotide releasing protein, anti-proliferative factor (BTG-1), lymphocyte-specific protein 1, protein phosphatase 2A, squalene synthetase, eukaryotic releasing factor 1, GTP binding protein, importin β subunit, cell adhesion molecule L1 , U-snRNP associated cyclophilin, resepin, Arg /
Abl interacting protein (ArgBP2A), keratin-related protein, p1
8 protein, p40 protein, glucosidase II, α enolase, macrophage inflammatory protein 1α, translationally regulated oncoprotein 1 (TCTP1), BB
C1, Nef interacting protein, Na ⁺ -D-glucose cotransporter gene protein, hsp90 chaperone protein, FK506 binding protein A
1, Rox, β signal sequence receptor, adult tumor primordium protein and cell surface heparin binding protein.

Among the GSEs selected to block HIV infection, some function in the sense orientation while others function in the antisense orientation. While not wanting to consolidate with any particular theory, it is believed that the GSEs of the present invention downregulate cellular genes by different mechanisms. GSE is expressed in a host cell by encoding an RNA molecule, which may or may not encode a protein product. GSE in the sense orientation can exert its effect as a transdominant mutant or RNA decoy. Transdominant mutants are expressed proteins or peptides that competitively inhibit the normal function of the wild-type protein. RNA decoys are protein binding sites that titrate these proteins. GSE in the antisense orientation can exert its effect as antisense RNA, that is, a nucleic acid molecule complementary to the mRNA of the target gene. These nucleic acid molecules bind to the mRNA and block the translation of the mRNA. Some antisense nucleic acid molecules can act directly at the DNA level to block transcription. Down-regulation of cellular genes by GSE then removes the cellular components required for HIV replication, thus blocking HIV infection.

A wide variety of applications are encompassed by the present invention, including but not limited to the treatment and prevention of HIV by introducing a protective GSE compound as an inhibitory composition into an HIV sensitive cell type. For example, GSE can be introduced into T cells, particularly CD4 ⁺ T cells, which is the major cell population targeted by HIV. Alternatively, GSE can be introduced into hematopoietic stem cells in vitro and then transplanted into autologous or histocompatible or even histoincompatible recipients. In another embodiment, HI
Any cell susceptible to V infection can be directly transduced or transfected with GSE in vivo. In yet another embodiment, NADH dehydrogenase, 2-oxoglutarate dehydrogenase, M2-type pyruvate kinase /
Cytosolic thyroid hormone binding protein, calnexin, ADP-ribosylation factor 3, eukaryotic initiation factor 3, protein tyrosine phosphatase, herpesvirus-associated ubiquitin-specific protease, eukaryotic initiation factor 4B, CD44, phosphatidyl-inositol 3 kinase, elongation factor 1α, bone morphogenetic protein-1,
Double-strand break DNA repair gene protein, rat guanine nucleotide-releasing protein, anti-proliferative factor (BTG-1), lymphocyte-specific protein 1, protein phosphatase 2A, squalene synthetase, eukaryotic releasing factor 1, GTP binding protein, import Tin β subunit, cell adhesion molecule L1, U-snRNP
Associated cyclophilin, recepin, Arg / Abl interacting protein (ArgB
P2A), keratin-related protein, p18 protein, p40 protein, glucosidase II, α enolase, macrophage inflammatory protein 1α, translationally regulated oncoprotein 1 (TCTP1), BBC1, Nef interacting protein, N
a ⁺ -D-glucose cotransport regulator gene protein, hsp90 chaperone protein, FK506 binding protein A1, Rox, β signal sequence receptor,
Inhibitors of adult tumor primordia proteins and cell surface heparin binding proteins can be used in vivo as inhibitory compositions to suppress or prevent HIV infection.

(Detailed Description of Preferred Embodiments) The present invention relates to a novel method for identifying a gene repression element capable of blocking HIV infection, a gene repression element identified by the above method, and a host cell gene involved in HIV infection. And an inhibitory composition that inhibits HIV infection by down-regulating the expression of the cellular gene or inhibits the activity of the gene product. The term "HIV infection" as used herein means that HIV is able to enter and / or replicate in a host cell.

One embodiment of the invention is a method of isolating a gene repression element, referred to herein as GSE, which is 1) randomly fragmenting the cell-derived cDNA into fragments; 2) expressing the fragments in an expression vector. To form a random fragment expression (RFE) library; and 3) introducing the expression library into a population of cells that contain an inducible latent HIV-1 provirus or are susceptible to HIV infection; 4) selecting a subpopulation of cells that comprises a subset of the expression library enriched for GSE by monitoring the expression of cells or viral markers associated with HIV infection;
And 5) collecting GSE from the selected cell population. In a preferred embodiment, cell-derived cDNA is randomly fragmented into 100-700 base pair (bp) fragments. The method further comprises repeating the steps so that a large number of successive selections can be carried out. The method further comprises selecting GSEs by determining continuous expression of cellular markers such as CD4, or reduction of expression of viral markers such as p24 or gp120 using, for example, antibodies.

The present invention is discussed in more detail in the following subsections, which are merely illustrative and not limiting. For clarity of discussion, the specific procedures and methods described herein were applied to OM10.1 cells, CEM-ss cells, tumor necrosis factor-α (TNF-α).
), Anti-CD4 antibody, and anti-p24 antibody, which are merely illustrative for the practice of the invention. Similar procedures and techniques are equally applicable to the isolation of GSEs from other cellular DNA, using any cell line and any marker associated with HIV infection that can be easily assayed.

The cell-derived RFE library can be generated from any mammalian cell nucleic acid molecule, preferably the cDNA of an HIV sensitive cell. In this regard, Example 1 demonstrates that GSE is derived from HL-60 cells that are naturally susceptible to HIV infection, and C
We demonstrate that we can select from HeLa cells that are not naturally susceptible to HIV infection due to the lack of D4 expression. However, expression of CD4 on the surface of HeLa cells using retroviral vectors has been shown to render cells susceptible to HIV infection. Therefore, cell types that are not normally susceptible to HIV infection still have RF
It may be useful as a source of genetic material for the construction of E libraries. It is preferred to prepare a standardized cDNA library (Gudkov and Roninson,
1996, Method of molecular biology 69: 229-231). The DNA is first treated with an enzyme to generate randomly cleaved fragments. This can be conveniently done by DNase I digestion in the presence of Mn ⁺⁺ (Roninson et al., US Pat. No. 5,217,889, column 5, lines 5-20). Then, the randomly cleaved DNA is size-fractionated by gel electrophoresis. The 100-700 bp fragment is the preferred length for making RFE libraries. Single stranded breaks of size selected fragments are repaired by methods known in the art.

Each fragment is ligated with a 5'and 3'adapter selected to have a non-adhesive restriction site so that it can be inserted into the expression vector in an oriented manner. In addition, the 5'adapter contains an initiation (ATG) codon, which allows translation of the fragment containing the open reading frame in the correct phase. The fragment is then inserted into a suitable expression vector. Any expression vector that provides efficient expression in host cells can be used. In a preferred embodiment, the retroviral vector LNCX
(Miller and Rosman, 1989, BioTechniques.
7: 980) and LNGFRM. Alternatively, adenovirus, adeno-associated virus and herpesvirus vectors can be used for this purpose.

If a virus-based vector is used, the ligated vector is first transfected into a packaging cell line to produce viral particles. In the retroviral vector, PA317 (Miller and Butti
more, 1986, Mol. Cell. Biol. 6: 2895-2902:
Any broad host packaging system such as ATCC CRL 9078) can be used for efficient production of the virus. In a preferred embodiment of the invention,
The viral vector also contains a selection gene, such as the neo ^r gene or the partially truncated nerve growth factor receptor (NGFR) gene, which allows the isolation of cells containing the vector.

The number of independent clones present in each RFE expression library can vary.
In a preferred embodiment, a library of cDNA from cells of about 10 ⁶ to 10 ⁸ independent clones can be used.

In the particular embodiment shown, for example, in Example 1, OM10.1 cells were treated with GS
Used for selection of E and maintained in conventional tissue culture medium as described in Butera US Pat. No. 5,256,534. The purpose of using OM10.1 cells for the selection of GSEs is that they are latent HIV-1 inducible by TNF-α.
Including a provirus. Other cell lines can be treated with inducible HIV provirus as well. Examples of cell lines that are infected with latent HIV are U1, U33, 8E5,
Including but not limited to ACH-2, LL58, THP / HIV and UHC4 (Bednarik and Folks, 1992, AIDS 6: 3-16). Various substances have been shown to be capable of inducing latent HIV infected cells, which are
TNF-α, TNF-β, interleukin-1, -2, -3, -4 and -6
, Granulocyte-macrophage colony stimulating factor, macrophage-colony stimulating factor, interferon-γ, transforming growth factor-β, PMA, retinoic acid and vitamin D3 (Poli and Fauci, 1992, AIDS Re.
s. Human Retroviruses 9: 191-197). Separately, G
SEs can be selected based on their ability to directly protect HIV susceptible cells from HIV infection using the methods described herein.

The cell-derived RFE library can be introduced into latent HIV-infected cells or HIV-sensitive cells by any technique known in the art suitable for the vector system used.
In one embodiment of the invention, the viral vector also contains a selectable marker in addition to the random fragments of cellular DNA. A suitable marker is the neo ^r gene, which allows the drug G-418 to be used to select cells containing RFE library members. In a preferred embodiment, the viral vector is anti-N.
It contains a partially truncated low affinity nerve growth factor receptor (NGFR) which allows the selection of cells using a GFR monoclonal antibody. In another embodiment, the multiplicity of infection of the virions of the library is adjusted so that preselection of cells transduced by the vector is not required.

In the case of OM10.1 cells, the transduced cells are treated with 10 U / mL TNF-α for a period of 24 to 72 hours, preferably about 24 hours, according to the method of Butera. Activation of latent HIV-1 provirus at OM10.1 can be detected by suppression of cell surface CD4. (It is believed that the viral protein gp120 binds to CD4 in the cytoplasm, which prevents subsequent expression of CD4 on the cell surface.) HIV replication-resistant clones were identified as cell surface CD4. Continues to develop. Such clones can be selected, for example, by any antibody staining technique for CD4 and cell sorting using a fluorescence activated cell sorter (FACS).

The fraction of CD4 ⁺ cells transduced with the RFE library can be compared to cells transduced with an expression library consisting of vector alone. Increased relative differences between the cell-derived RFE library and the control library can be found with each additional round of TNF-α induction. Therefore, in a preferred embodiment of the present invention, at least two cycles of induction, selection and recloning are performed prior to GSE recovery from cells for further characterization.

After selection, specific nucleic acid molecules corresponding to GSE can be recovered from cells that continue to express CD4 after induction of latent HIV provirus by TNF-α. Specific GS
E is recovered from genomic DNA isolated from FACS-sorted CD4 ⁺ cells after TNF-α induction. GSEs in this population are recovered by PCR amplification, preferably using primers designed from the sequences of the vector.

The recovered GSE can be introduced into an expression vector as discussed in the Examples section herein. The resulting GSE expression library is known as the secondary library. The secondary library can use the same or different vector that was used to create the primary library. The secondary library can be transduced into another population of cells and the resulting population can be selected, recloned and processed as described herein.

Further, each individually recovered GSE can be inserted into a cloning vector to determine its specific nucleotide sequence and its orientation. Then G
The sequence of SE is compared with that of a known gene to determine the portion of the corresponding cellular gene. Alternatively, the PCR product itself can be directly sequenced to determine its nucleotide sequence. At the same time, the isolated GSE can be analyzed to determine its minimal core sequence. A "core sequence" is a consensus sequence found by comparing GSE with overlapping sequences. GSE is further tested for its ability to protect previously uninfected cells from HIV infection.

Another embodiment of the invention includes a method of determining the core sequence of GSE. this is,
It can be carried out by comparing the overlapping sequences of GSE obtained independently. Separately, GSE
Can be altered by additions, substitutions or deletions and assayed for retention of HIV suppressor function. Changes in the GSE sequence can be made using various chemical and enzymatic methods known to those of skill in the art. For example, oligonucleotide-directed mutagenesis can be used to alter the GSE sequence and / or introduce restriction sites at specific regions within the sequence in a defined manner. In addition, deletion mutants were Bal31 or ExoIII
And DNA nucleases such as S1 nuclease. GS
Increasingly large deletions of the E sequence can be made by incubating DNA with nucleases for longer periods of time (for a review of mutagenesis techniques, see Aus.
See ubel et al., ibid.).

The altered sequence can be evaluated for its ability to suppress the expression of HIV proteins such as p24 in a suitable host cell. It is within the scope of the invention that any altered or truncated GSE nucleic acid molecule that retains the ability to suppress HIV infection can be incorporated into a recombinant expression vector for further use.

In order to confirm whether the selected GSE can protect uninfected cells from HIV infection, GSE is introduced into latent HIV-infected host cells or HIV-susceptible host cells,
Then you can be infected with HIV. In this regard, GSEs can also be selected directly from the RFE library for their ability to protect against productive infections by HIV, as shown in the specific embodiments illustrated in the Examples section herein. Protection experiments can be performed on any cell type that has incorporated a potential GSE that is otherwise susceptible to HIV infection. For example, in a preferred embodiment, the CEM-ss cell line is used (Foley et al., 1965, Cancer 18: 522-529). HIV-
The use of CEM-ss cells as a target for quantitative infectivity of 1 was described by Nara & Fi.
chinger (1988, Nature 322: 469-470). Other cell lines that are susceptible to HIV infection include, but are not limited to, HUT-78, H9, Jurkat E6-1, A3.01, U-937, AA-2, HeLaCD4 ⁺ and C8166. In addition, freshly isolated peripheral blood leukocytes can be used.

Testing of potential GSEs can be performed using the same expression vector system used to transduce cells with the RFE library during the initial selection step. In other embodiments, the vector system can be modified to obtain high levels of expression, eg, a linker can be used to introduce a leader sequence that enhances message translation efficiency. One such sequence is Kozak, 1994, Biochemie 7
6: 815-821.

Another way to test potential GSE efficacy against HIV infection is to determine how quickly HIV-1 mutants develop that can counteract the effects of that element. Such tests show that CEM-s at low multiplicity of infection
HIV-sensitive cells such as s.
1 Includes determining if the infection has spread and how rapidly it has spread. The range of useful multiplicity of infection is approximately 10 ⁶ per 10 ⁶ CEM-ss cells.
Tissue culture infectious units (TCID ₅₀ ) of 0 to 1000. The TCID ₅₀ is determined by the endpoint method and is important for determining the input multiplicity of infection (moi).

A development-related parameter in the test broth of the HIV-1 strain that is resistant to the effects of potential GSEs is the percentage of cells infected in the broth. This percentage is
It can be determined by immunofluorescent staining of the fixed permeabilized cells with an antigen specific for the HIV-1 p24 antigen. Commercially available reagents are suitable for carrying out the test (Lee).
Et al., 1994, J. Am. Virol. 68: 8254-8264).

One embodiment of the present invention is an isolated nucleic acid molecule comprising a human cellular gene or at least a portion thereof required for HIV infection. These isolated nucleic acid molecules are referred to herein as "cell-derived nucleic acid molecules." The term "a" or "an" entity means one or more of that entity; for example, a protein (a prot).
It is noted that ein) means one or more proteins or at least one protein. Thus, "a" (or "an"), "one or more" and "
The term "at least one" can be used synonymously herein. "Include (co
It is noted that the terms mprising, "including," and "having" can be used as synonyms. Further, a compound “selected from the group consisting of” means one or more compounds in the list below, including mixtures (ie, combinations) of two or more compounds. According to the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural environment (ie, has been subjected to human engineering). Thus, "isolated" does not reflect the extent to which the nucleic acid molecule has been purified.
Isolated nucleic acid molecule means DNA, RNA, or a derivative or hybrid of DNA or RNA. An isolated nucleic acid molecule of the present invention is derived from its natural source in a productive HI
It can be obtained as a whole (ie complete) gene or part thereof, corresponding to at least part of the gene encoding the product required to cause V infection or to block HIV infection. The term "at least a portion" of an entity as used herein means an amount of the entity that is at least sufficient to have the functional aspects of that entity. For example, at least a portion of a nucleic acid sequence as used herein is the amount of nucleic acid sequence required for HIV infection. The isolated nucleic acid molecule of the present invention also comprises
It can be made using recombinant DNA technology (eg, polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. The isolated nucleic acid molecules of the present invention include naturally occurring nucleic acid molecules and homologues thereof, which by modification promote HIV infection or HI.
It includes naturally occurring allelic variants and modified nucleic acid molecules in which nucleotides are inserted, deleted, substituted and / or inverted in such a manner that the ability of the nucleic acid molecule to prevent V infection is not substantially interfered with. Not limited to.

The nucleic acid sequence complement of any nucleic acid sequence of the present invention is complementary to the strand from which the sequence is referenced (
That is, the nucleic acid sequence of a nucleic acid strand (capable of forming a complete double helix with that strand). It is noted that one strand of the nucleic acid sequence represented by SEQ ID NO: has been determined, the double-stranded nucleic acid molecule of the present invention also includes a complementary strand having a sequence that is the complement of that SEQ ID NO. Thus, a nucleic acid molecule of the present invention, which may be double-stranded or single-stranded, has the sequence number set forth herein and / or the complement of that sequence number (which may or may not be set forth herein). Optional) and nucleic acid molecules that form stable hybrids under stringent hybridization conditions. Methods of deducing complementary sequences are known to those of skill in the art.

One embodiment of a cell-derived nucleic acid molecule is a GSE nucleic acid molecule capable of blocking HIV infection in susceptible cells. Preferred GSE nucleic acid molecules of the present invention are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, sequence. No. 11, sequence number 12, sequence number 13, sequence number 14, sequence number 15, sequence number 16, sequence number 17, sequence number 1
8, sequence number 19, sequence number 20, sequence number 25, sequence number 27, sequence number 29
, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39,
Sequence number 41, sequence number 43, sequence number 45, sequence number 47, sequence number 49, sequence number 51, sequence number 53, sequence number 55, sequence number 57, sequence number 59, sequence number 61, sequence number 63, sequence number 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85; SEQ ID NO: 87; SEQ ID NO: 89; SEQ ID NO: 9
1: SEQ ID NO: 93 and / or SEQ ID NO: 95, and the complements of any of these sequences, homologues thereof, or to these sequences or their complements, under highly or moderately stringent hybridization conditions. It comprises a nucleic acid sequence selected from the group consisting of hybridizable nucleotide sequences. Also included are GSEs with conservative nucleotide substitutions that produce the same protein product. High stringency hybridization conditions were hybridized to the filter bound DNA in 0.5 M NaHPO ₄ , 7% sodium dodecyl sulphate (SDS), 1 mM EDTA at 65 ° C., then 0.1 × SSC / 0. 1% SD
It can be defined as washing in S at 68 ° C. (Ausubel FM et al., Ed., 19
89, Current Protocols in Molecular Biology, Volume 1, Green Publicshi
ng Associates, Inc. And John Wiley & Sons
Company, New York, Section 2.10.3). Medium stringency conditions were performed by hybridizing as above, then in 0.2 x SSC / 0.1% SDS at 42 ° C.
Can be defined as washing (Ausubel et al., 1989, current protocol of molecular biology).

Another embodiment of the cell-derived nucleic acid molecule of the present invention comprises a cellular gene, referred to herein as a “target gene”, that encodes an intracellular product required for proliferative HIV infection.
Preferably, the target gene of the present invention is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, Sequence number 11, sequence number 12, sequence number 13, sequence number 14, sequence number 15, sequence number 16, sequence number 17, sequence number 18, sequence number 19, sequence number 20, sequence number 25, sequence number 27, sequence number 29, sequence number 31, sequence number 33, sequence number 35, sequence number 37, sequence number 39, sequence number 41, sequence number 4
3, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53
, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63,
Sequence number 65, sequence number 67, sequence number 69, sequence number 71, sequence number 73, sequence number 75, sequence number 77, sequence number 79, sequence number 81, sequence number 83, sequence number 85; sequence number 87; sequence number 87 89; SEQ ID NO: 91: SEQ ID NO: 93 and / or
Or G having a nucleic acid sequence selected from the group consisting of SEQ ID NO: 95, and its complement, and any other GSE sequence disclosed herein and its complement.
Includes nucleic acid molecules that correspond to SE.

Particularly preferred GSE nucleic acid molecules of the invention are plasmids CF-315, CF-319, CF-101, CF-117, C, as defined and identified herein.
F-025, CF-128, CF-004, CF-113, CF-204, CF
-001, CF-273, CF-311, CF-313, CF-210, CF-
266, CF-302, CF-317, CF-286, CF-061, CF-2
80, CF-537, CF-320, CF-321, CF-322, CF-33
2, CF-335, CF-42, CG-50, CF-527, CF-528, C
F-529, CF-531, CF-545, CF-547, CF-619, CF
-620, CF-624, CF-630, CF-579, CF-676, CF-
675, CF-653, CF-674, CF-675, CF-673, CF-6
93, CF-287, CF-658, CF-672, CF-679, CF-68
1, CF-622, CF-683, CF-684, CF-685, and CF-
686 is included.

The term “corresponding to” as used herein refers to nucleic acid sequence SEQ ID NO: 1,
Sequence number 2, sequence number 3, sequence number 4, sequence number 5, sequence number 6, sequence number 7,
Sequence number 8, sequence number 9, sequence number 10, sequence number 11, sequence number 12, sequence number 13, sequence number 14, sequence number 15, sequence number 16, sequence number 17, sequence number 18, sequence number 19, sequence number 20, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 2
9, sequence number 31, sequence number 33, sequence number 35, sequence number 37, sequence number 39
, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49,
Sequence number 51, sequence number 53, sequence number 55, sequence number 57, sequence number 59, sequence number 61, sequence number 63, sequence number 65, sequence number 67, sequence number 69, sequence number 71, sequence number 73, sequence number 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85; SEQ ID NO: 87; SEQ ID NO: 89; SEQ ID NO: 91: SEQ ID NO: 93, SEQ ID NO: 95, and at least about the complement thereof. 75%
, More preferably about 80%, more preferably about 85%, more preferably about 90%.
, More preferably about 95% and more preferably about 100% identical. Lack of complete identity between the GSE and the target gene sequence may result from genetic polymorphism between different individuals or mutations introduced during cloning or PCR amplification.

Preferred embodiments of the present invention include NADH dehydrogenase, 2-oxoglutarate dehydrogenase, type M2 pyruvate kinase / cytosolic thyroid hormone binding protein, calnexin, ADP-ribosylation factor 3, eukaryotic initiation factor 3,
Protein tyrosine phosphatase, herpesvirus-associated ubiquitin-specific protease, eukaryotic initiation factor 4B, CD44, phosphatidyl-inositol 3 kinase, elongation factor 1α, bone morphogenetic protein-1, double-strand break DNA repair gene protein, rat guanine nucleotide release Protein, anti-growth factor (BT
G-1), lymphocyte-specific protein 1, protein phosphatase 2A, squalene synthetase, eukaryotic release factor 1, GTP binding protein, importin β subunit, cell adhesion molecule L1, U-snRNP associated cyclophilin, recepin, Arg / Abl interacting protein (ArgBP2A), keratin-related protein, p18 protein, p40 protein, glucosidase II, α enolase, macrophage inflammatory protein 1α, translation control tumor protein 1 (TC
TP1), BBC1, Nef interacting protein, Na ⁺ -D-glucose cotransporter gene protein, hsp90 chaperone protein, FK506 binding protein A1, Rox, β signal sequence receptor, tumor adult primordium protein or cell surface heparin binding. A target gene encoding at least a portion of a protein selected from the group consisting of proteins, wherein said portion of the nucleic acid molecule comprises
Encodes intracellular products required for HIV infection.

The following subsections describe the cellular genes identified herein as important targets for the development of HIV therapeutics.

In aerobic organisms, adenosine triphosphate (ATP) provides the primary source of energy. Due to the production of ATP, energy-rich molecules such as NADH and FADH ₂ are first formed in the glycolysis, fatty acid oxidation and citric acid cycles. When these molecules donate their electrons to molecular oxygen, free energy is released and ATP is produced.

Oxidative phosphorylation is the process by which ATP is formed when electrons are transported from NADH or FADH ₂ to O ₂ by a series of electron carriers (Stryer, 1
988, Biochemistry, Freeman). This process occurs in eukaryotic mitochondria. More specifically, enzymes that catalyze electron-transport chains are present in the inner mitochondrial membrane, where they are found in both nuclear and mitochondrial DNA.
Coded by. These enzymes exist as large protein complexes, the first chain complex known as NADH dehydrogenase or NADH-Q reductase. It has a molecular weight of 850,000 daltons and consists of over 40 polypeptide subunits, seven of which are encoded by the mitochondrial genome. (Anderson et al., 1981, Nature.
290: 457; Chomyn et al., 1985, Nature 314: 592;
Chomyn et al., 1986, Science 234: 619). The nucleotide sequence of the cDNA of the subunit nuclear gene has been described (Walker et al.,
1992, J.I. Mol. Biol. 226: 1051; Fernley et al., 1
989, EMBO J .; 8: 665; Pilkington et al., 1989, Bi.
ochem. 28: 3257). NADH dehydrogenase from NADH
It catalyzes the transport of electrons into electron carriers called ubiquinones.

The 2-oxoglutarate dehydrogenase complex is a rate-limiting enzyme that catalyzes the oxidative decarboxylation of 2-oxoglutarate to succinyl CoA and CO ₂ and regulates substrate influx through the Krebs cycle. (Delvin, TM;
Ed., 1992, Biochemistry textbook, Wiley-Liss). This enzyme complex is located in the inner membrane / matrix compartment of Mycotondria. This complex contains multiple copies of 2-oxoglutarate dehydrogenase (lipoamide) (OGDH).
Or ELO; 2-oxoglutarate: lipoamide 2-oxoreductase (decarboxylation and acceptor succinylation), EC 1.2.4.2), dihydrolipoamide succinyl transferase (designated E20; EC 2.3).
． 1.61) and dihydrolipoamide dehydrogenase (E3; EC1.8.
1.4). The coding sequence for 2-oxoglutarate dehydrogenase has been described (GenBank Accession Nos. D10523 and D90499; Koi).
ke et al., 1992, Proc. Natl. Acad. Sci. USA 89: 1
963-1967; Koike, 1995, Gene 159: 261-266.
).

Pyruvate kinase / thyroid hormone binding protein p58 (TBP) is a pyruvate kinase (ATP pyruvate O ₂ -phosphotransferase, EC2
． 7.1.40) It is a monomer of subtype M2. Its conversion to the tetrameric pyruvate kinase is linked to fructose 1,6, -bisphosphate (Fru-1,6-
P ₂ ). At low glucose concentrations, mammalian cells have low F
Pyruvate kinase is inactive, including ru-1,6-P ₂ . At high glucose concentrations (typical medium contains 5-10 mM glucose), high levels of Fr
u-1,6-P ₂ is found in proliferating and tumor cells, which requires high pyruvate kinase activity for proliferation. Low Fru-1,6-P ₂ favors the formation of p58 and high concentrations have been demonstrated to convert it to a tetrameric enzyme. Increased glucose concentration can lead to multimerization of p58, which in turn activates pyruvate kinase and glycolysis. At the same time, thyroid hormone can be released from the complex with TBP, bind to nuclear and mitochondrial receptors and activate oxidative phosphorylation. The coding sequence for the pyruvate kinase / thyroid hormone binding protein has been described (GenBank Accession No. M26252; Kato et al., 198).
9, Proc. Natl. Acad. Sci. USA 86: 7861-786.
5).

Calnexin, along with two cofactors involved in substrate binding, ATP and Ca ²⁺ , functions in the endoplasmic reticulum (ER) as a molecular chaperone for secreted glycoproteins,
It is a type I membrane protein (Ou et al., 1995, J. Biol. Chem. 270).
: 18051). Folding of gp120 is a newly synthesized gp12
It has been demonstrated to be mediated by calnexin during the 0 translocation to the ER (Li et al., 1996, Proc. Natl. Acad. Sci. USA 93:
9606). The coding sequence for calnexin has been described (GenBank Deposit No. L10284; David et al., 1993, J. Biol. Chem. 268).
: 9585-9592).

ADP-ribosylating factor (ARF) is an in vitro cholera toxin ADP-
A guanine nucleotide binding protein of approximately 20 kDa molecular weight that stimulates ribosyltransferase activity (Tsai et al., 1991, J. Biol. Ch.
em. 266: 23053-23059). Five different ARFs of human cDNA
Was cloned. ARF3 is shown by two mRNAs of 3.7 and 1.2 kb, created by using another polyadenylation signal (Tsai et al., 1991, supra).

Ubiquitin-specific proteases (USPs) play important roles in several cellular processes, including regulation of gene expression, cell cycle regulation, DNA repair and differentiation (H
ochtrasser, 1995, Curr. Opin. Cell. Biol.
7: 215-223; Wilkinson, 1995, Ann. Rev. Nut
r. 15: 161-189). One of the best characterized ubiquitin-dependent pathways involved in the regulation of gene expression is NF-κB activation. NF-
Cleavage of p105, the precursor of the p50 subunit of κB, requires a ubiquitin conjugate (Palombella et al., 1994, Cell 78: 7.
73-785), and second, the destruction of IκB, a process that allows NF-κB to translocate to the nucleus in its activated form, requires phosphorylation-dependent inhibitor ubiquitination ( Scherer et al., 1995, Proc. Natl. Acad. Sci.
USA 92: 11259-11263).

USP has been shown to cleave ubiquitin from model substrates, characterized by the presence of two conserved active site domains (Everett et al., 1997).
, EMBO J .; 16: 556-577). There are two classes in USP. The first involves proteins involved in the production of free ubiquitin, either from precursor fusion proteins or from peptide-linked polyubiquitin after proteosome proteolytic degradation of the substrate (Hochtrasser, 1995, ibid.). The second contains increasing numbers of deubiquitinated proteins that can recognize and stabilize specific substrates by removing ubiquitin adducts. Examples of this class include the Drosophila fat facets protein, whose deubiquitination is required for proper ocular development (Huang et al., 1995, Science 270: 1828-1831). Another example is the immediate early gene, DUB-1 gene, which regulates cell proliferation (Zhu et al., 1
996, Proc. Natl. Acad. Sci. USA 93: 3275-3.
279).

The coding sequence for a herpesvirus-associated ubiquitin-specific protease has been described (GenBank Accession No. Z72499; Everett et al., 1997).
EMBO J.M. 16: 566-577).

CD44 is involved in multiple physiological cell functions such as extracellular matrix-cell adhesion, lymphocyte homing, lymphohematopoiesis, T cell activation, and tumor metastasis.
It is a widely distributed surface receptor glycoprotein (Shimuzu et al., 198).
9, J. Immunol. 143: 2457-2463; Huet et al., 1989.
J. Immunol. 143: 798-801). Some of these functions depend on the ability of CD44 to recognize the extracellular matrix component hyaluronan. CD44 is expressed as several different isoforms that vary from 85 to 200 kDa, depending on the degree of usage and cell-type glycosylation of the 10 exons encoding part of the extracellular domain. Each isoform may have some degree of functional identity (Stamenkovich et al., 1989, Cel.
56: 1057-1062). The most widely expressed molecule is an 85-90 kDa glycoprotein, commonly referred to as CD44H, that has been demonstrated to be the major receptor for hyaluronan (Bartolazzi et al., 199).
6, J. Cell Biol. 132: 1199-1208). CD44H is
Shows the basic isoforms found on hematopoietic cells. It has been shown that CD44, along with other HIV such as CD4, can play a role in viral tropism and affect viral infectivity. HIV causes CD44 down-regulation in monocytes but at post-translational levels (Guo and Children, 19
93, J. Immunol. 151: 2225-2236).

The coding sequence of human CD44 has been described (GenBank Deposit No. X55
150; Stamenkovich et al., 1991, EMBO J. et al. 10: 243
~ 248).

Phosphorylation of serine, threonine and tyrosine residues is one of the key regulatory mechanisms in gene expression and post-translational modification. Tyrosine phosphorylation is important for the control of normal cellular processes such as cell proliferation and differentiation, as well as the control of pathological events such as malignant transformation (Cantley et al., 1991, Cell 64: 281-).
301). The overall level of tyrosine phosphorylation is regulated by the complementary and antagonistic actions of protein tyrosine kinases and protein tyrosine phosphatases. At steady state, less than 1% of total cell phosphorylation is due to tyrosine phosphorylation. However, phosphotyrosine content can dramatically increase during cell transformation (Wa
lton and Dixon, 1993, Ann. Rev. Biochem. 62
: 101-120). There are about 40 known phosphatases (Zolni
erowiez and Hemmings, 1994, Trends Cell
Biol 4: 61-64).

Several viruses are known to contain protein tyrosine kinases and phosphatases. The putative protein tyrosine phosphatase is HIV-1
Found in the 5'LTR (Nandi and Banerjee, 1995, Me.
d. Hypothesis 45: 476-480). Phosphorylation is due to tat-mediated transactivation (Ensoli et al., 1990, Nature 345: 84-8).
6), nef protein function (Balliet et al., 1994, Virology.
200: 623-631; Venkatesh et al., 1990, Virolog.
176: 39-47), and viral matrix association (Camaur et al., 1997, Virology 71: 6834-6841).

The coding sequences for brain-derived phosphatase (BDP1) mRNA and CL100 mRNA-derived protein tyrosine phosphatase have been described (GenBa.
nk deposit no. X79568; Kim et al., 1996, Oncogene 13: 2.
275-2279 and GenBank Deposit No. X68277; Keyse and Emslie, 1992, Nature 359: 644-647).

Phosphatidylinositol 3-kinase (PI3K) has been characterized as an enzyme involved in receptor signaling. There are multiple forms with different substrate specificities. They are associated with a wide variety of cell surface receptors, including receptors for growth factors, thrombin, chemotactic peptides and cytokines. PI3K has been proposed to act as a second messenger, probably through activation of specific protein kinase C isotypes (Liscovitch and Cantry, 1994, Cell 77: 329-334; Toker et al., 1994, J. Biol. Chem. 269: 323598-32367).
PI3K may also play a role in regulating the transport of proteins from the Golgi apparatus to lysosomes (Volinia et al., 1995, EMBO J. 14: 3339-3.
348).

HIV-1 nef expression severely damages PI3K associated with the receptor, which causes Nef to selectively affect the PI3K signaling pathway, thus leading to deleterious effects on host cell function. (Graziani et al., 199)
6, J. Biol. Chem. 271: 6590-659 3). Nef expression was also associated with a decrease in PIK in basal cells, suggesting a role for PI3K in HIV replication (Garcia, 1997, CR Acad. Sci. III.
320: 505-508). PI3K can be activated by gp160, Ta
Involved in t-mediated apoptosis (Mazerolles et al., 1997, Eu.
r. J. Immunol. 27: 2457-2465; Borgatti et al., 1
997, Eur. J. Immunol. 27: 2805-281). Known P
I3K inhibitors include wortmannin and theophylline.

The coding sequence for phosphatidylinositol 3-kinase has been described (G
enBank Deposit No. Z46973; Volinia et al., 1995, EMBO.
J. 14: 3339-3348).

Elongation of the polypeptide chain occurs after translation initiation. The elongation factor uses the energy released by GTP hydrolysis to ensure the proper aminoacyl-tRNA.
To translocate messages and associated tRNAs through the decoding region of the ribosome (Devlin, 1992, Biochemistry textbook, Wiley-Liss).
. EF-1-α is an evolutionarily conserved universal protein synthesis cofactor in all living cells. It carries an aminoacyl-tRNA to the A site of the ribosome in a GTP-dependent manner.

EF-1α expression levels are regulated at various stages of cell life, such as growth arrest, transformation and aging. The level of EF-1α may be an important regulator that regulates the rate of apoptosis. Decreasing EF-1α expression slows apoptosis, while overexpression accelerates the process (Duttaroy et al., 1998,
Exp. Cell Res. 238: 168-176).

The coding sequence of human EF-1α has been described (GenBank Deposit No. J0
4617; Uetshiki et al., 1989, J. Am. Biol. Chem. 264:
5791-5798).

Translation initiation occurs by binding of the 40S ribosomal subunit at or near the cap structure of the mRNA, followed by ribosome scanning of the 5'untranslated region until encountering the initiator AUG. This process is facilitated by a complex group of proteins known as initiation factors. These factors only participate in translation initiation (Devlin, 1992, Biochemistry textbook, Wiley-Liss).
Company).

Eukaryotic initiation factor 3 (eIF3) is the largest multi-subunit complex involved in initiating protein synthesis. It has a mass of 600 kDa and 10 subunits. This factor prevents assembly of ribosomal subunits,
Stabilizes the binding of methionyl-tRNA to the 40S subunit and promotes mRNA binding (Merrick and Hershey, 1996, translational control, He.
Rshey, Mathews and Sonenberg, eds. 31-69, Cold Spring Harbor, Cold Spring Harbor, NY). The sequence of human eukaryotic initiation factor 3 has been described (GenBank Accession No. U78525).

Eukaryotic initiation factor 4B (eIF4B) is an 80 kDa polypeptide that is essential for mRNA binding to ribosomes. eIF4B has RNA binding activity,
Stimulates ATPase and RNA helicase (Mehot et al., 1996, R
NA 2: 38-50; Abramson et al., 1987, J. Am. Biol. Che
m. 262: 3826-3832; Ray et al., 1985, J. Am. Biol. Che
m. 260: 7651-7658; Rozen et al., 1990, Mol. Cell
． Biol. 10: 1134-1144). Furthermore, eIF4B has been reported to have RNA annealing activity and promote base pairing between complementary sequences of RNA strands (Altmann et al., 1995, EMBO J. 14: 3820-382).
7). It also interacts with eIF4A and eIF3 (Methot et al., 1996, Mol. Cell. Biol. 16: 5328-5334). eI
Translation is generally inhibited by overproduction of F4B (Milburn et al., 19
90, EMBO J. et al. 9: 2783-2790).

The sequence of human eIF4B has been described (GenBank Deposit No. X5573).
3; Milburn et al., 1990, EMBO J .; 9: 2783-2790).

Protein extracts obtained from bone may initiate the process of beginning chondrogenesis and ending with new bone formation. This protein extract is a bone morphogenetic protein (BMP
) Is called. It is unclear what are the key components of BMP that direct cartilage and bone formation and the components supplied by the animal during cartilage and bone formation (W
ozney et al., 1988, Science 242: 1528-1534). The amino acid sequence was obtained from a highly purified BMP preparation from bovine bone. B
Human complementary DNA clones corresponding to the three polypeptides present in the MP preparation were isolated and expressed recombinant human proteins. Each of the three expressed human proteins, BMP-1, BMP-2A and BMP-3, appears to be capable of independently inducing cartilage formation in vivo. BMP-2A and BMP-3A are TGF-
A new member of the β supergene family, while the third BMP-1 is a regulatory molecule.

Schizosaccharomyces
human and mouse homologues of the rad21 gene of Pombe (involved in repair of ionizing radiation-induced DNA double-strand breaks) have been described (McKay et al., 1996, Genomics 36: 305-315). mHR12 (sp)
(Mouse homolog of Rad21 of S. pombe) and hHR21 (sp) (S
． The predicted amino acid sequence of the human homolog of Rad21 of pombe) is 96% identical, while human and S. Pombe proteins are 25% identical and 47% similar. RNA blot analysis showed that mHR21sp mRNA was abundant in all adult mouse tissues examined, with highest expression in testis and thymus. In addition to the 3.1 kb constitutive mRNA transcript, a 2.2 kb transcript was present at high levels in post-meiotic spermatids, whereas expression of the 3.1 kb mRNA in testis was reduced. It was limited to the dividing compartment. The cell cycle of hHR21sp mRNA was regulated in human cells, increased in late S phase, and peaked in G2 phase. In situ hybridization showed that mHR21sp resides on chromosome 15D3, while hHR21sp localizes to the synteny 8q24 region. Increased mHR12sp expression in testis and thymus
Ra in the V (D) J immunoglobulin gene and meiotic combination, respectively
The role of the d21 mammalian homologue is shown. S. Cell cycle regulation of rad21, which is conserved in pombe and humans, has been reported in S. Consistent with the conservation of function between the pombe and human rad21 genes.

Small GTP-binding proteins of the ras superfamily are important for exocytosis from eukaryotic cells. These GTP-binding proteins are
Depending on whether it is bound to P or GTP, it can exist in two different conformations and function as a molecular switch that regulates various cellular processes. G
The TP-GDP cycle is regulated by accessory proteins that facilitate the exchange of bound GDP or the hydrolysis of GTP. A cDNA encoding the mammalian GDP-releasing protein mss4 has been cloned (Burton et al., 1993, Nat.
ure 361: 464-467). Mss4 specifically binds to Rab G
A guanine nucleotide exchange factor that facilitates GDP-GTP exchange on a subset of TPases (Burton et al., EMBO J. 13: 5547-555).
8). The Mss4 protein also stimulates GDP release from Ypt1 and from the mammalian protein Rab3a, but not Ras2. Mss4 is a yeast protein with similar biochemical properties, Dss4
Shows a sequence similar to.

The products of B cell translocation gene 1 (BTG1), a member of the antiproliferative protein family, including Tis-21 / PC3 and Tob regulate cell cycle progression (Rodier et al., 1999, Exp Cell Res. .249: 33
7-348). The BTG1 locus has been shown to be involved in the t (8; 12) (q24; q22) chromosomal translocation in certain B-cell chronic lymphocytic leukemias (Rouault et al., 1992, EMBO J. 11: 1663). ~ 1670).
The BTG1 cDNA was isolated (Rouault et al., Supra) and contains an open reading frame of 171 amino acids. BTG1 expression is cell cycle G0 / G1
It is maximal in phase and is downregulated when cells progress through G1. Moreover, transfection experiments of NIH3T3 cells show that BTG1 negatively regulates cell proliferation. The BTG1 open reading frame is 60% homologous to PC3, the immediate early gene induced by nerve growth factor in rat PC12 cells. Sequence and Northern blot analysis indicate that BTG1 and PC3 are not cognate genes. Triiodothyronine (T3) or 8-Br-cAMP was found to bind to B in confluent myoblast cultures.
Increases TG1 nuclear accumulation (Rodier et al., Supra). AP-1 activity, a key target involved in the effects of triiodothyronine myoblasts, suppresses BTG1 expression, thus demonstrating low BTG1 expression levels in proliferating myoblasts. ing. AP-1-like sequences present in the BTG1 promoter have been shown to be involved in negative regulation of BTG-1 expression.

The mouse and human lymphocyte-specific protein 1 (LSP1) gene is Thy
Expressed in normal B and T cells, including 1+ thymocytes, and in normal macrophages and neutrophils (Pulford et al., 1999, Immunology).
96: 262-271). LSP1 is found in all hematopoietic cells and its function is unknown. In intact cells, mitogen-activated protein kinase activator protein (MAPKAP) kinase 2 is rapidly activated by various cytokines, stresses and chemotactic factors. Recently, LSP1 is a MA in human neutrophils.
It has been shown to be a substrate for PKAP2 kinase 2 (Zu et al., 1996, Blo.
od 87: 5287-5296). LSP1 has also been identified as one of the major substrates of B-cell protein kinase C (Carballo et al., 1996, J.
． Immunol. 156: 1709-1713).

Protein phosphatases can regulate the activity of various protein kinases. Protein phosphatase 2A (PP2A) regulates cell growth and division. Researchers have suggested that HIV infection activates protein phosphatase 2A (Han et al., 1992, J. Virol. 66: 4065-).
4072). Proteins from other viruses are known to interact with PP2A. The SV40 small tumor antigen (small-t) was used as a model to identify structural elements involved in the interaction between regulatory proteins and PP2A (Mateer et al., 1998, J. Biol. Chem. 273: 353.
39-35346). NCp7 and Vpr have more PP2A than NCp7 alone.
It forms a strong complex that is a more potent activator of. The ability of NCp7 to activate the protein PP2A is regulated by Vpr. The C-terminal part of Vpr prevents NCp7 from activating the protein PP2A, while the N-terminal part of Vpr
The terminal portion enhances the effect of NCp7 on the activity of PP2A. These findings indicate that Vpr acts on NCp7 as a targeting subunit that directs activation of the protein PP2A (Tung et al., 1997, FEBS.
Lett. 401: 197-201).

The reaction catalyzed by squalene synthetase (SQS) shows many similarities to the reaction carried out by another polyisoprene synthetase, phytoene synthetase (PhS). A 2 kb cDNA (hSQS) encoding human SQS, a 417 amino acid protein with a predicted molecular weight of 48,041, was cloned by identifying a conserved sequence between yeast SQS (ySQS) and PhS. (Summers et al., 1993, Gene 136: 185-1.
92). Two hSs of 2.0 and 1.55 kb differing in their 3'untranslated sequences
A QS mRNA species has been identified. The two mRNAs are present in roughly equal amounts in heart, placenta, lung, liver, kidney, and pancreas, while the 2 kb mRNA is predominantly present in brain and skeletal muscle. In HepG2 cells, both mRNAs were 3-
It is induced 2-fold to 4-fold by lovastatin, a hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor. In contrast, Northern blot analysis of rat tissues revealed a mRNA of only 2.0 kb, which is significantly upregulated by lovastatin in vivo.

Termination of protein synthesis in the ribosome is governed by the termination codon of messenger RNA and by the polypeptide chain releasing factor (RF). eRF
The amino acid sequence of one family member is highly conserved. These RF proteins are directly involved in the termination of translation in eukaryotic cells (Frolova et al.,
1994, Nature 372: 701-703).

Prokaryotic and eukaryotic cells incorporate the amino acid selenocysteine at the UGA codon, which conventionally acts as a termination signal. Eukaryotic selenoprotein mR
Translation of NA requires a nucleotide selenocysteine insert in the 3'untranslated gene region. Erb-1 can recognize a selenocysteine insertion sequence element.

ERF1 has been demonstrated to associate with the catalytic subunit of protein phosphatase 2A (Andjelkovic et al., 1996, EMBO J. 1).
5: 7156-67). It was postulated that eRF1 also functions to recruit PP2A to the polysome, thus bringing phosphatase into contact with a putative target between components of the translation apparatus. By the induction of granulocyte differentiation of HL60 cells by retinoic acid,
Transient and reversible interconversions of phosphatase 2A holoenzyme occur, and
It was demonstrated that the C-terminus of the PP2A catalytic subunit is transiently methylated at S phase in HL-60 cells (Zhu, 1997, Arch Biochem B.
iophys. 339: 210-217).

G-proteins are a family of heterotrimeric guanine nucleotide binding proteins that play important roles in signal transduction, the expression of which is tissue-specifically regulated. G (olf) α is a G protein that was initially thought to mediate signaling exclusively within the olfactory neuroepithelium and was thought to be the major stimulatory G protein of the basal ganglia and several other tissues ( Zigman et al., 1993, Endocrin.
133: 2508-2514).

Nuclear cytoplasmic transport occurs through the nuclear pore. The peripheral pore structure interacts with the transport receptor and its cargo when these receptor complexes first encounter the pore.
Nuclear import of proteins is mediated by the basal nuclear localization signal (NLS), which binds to the importin α (Impα) NLS receptor. Impβ is
It is also required for nuclear imports.

Human immunodeficiency virus type I (HIV-1) Rev protein binds to unspliced HIV-1 pre-mRNA and exports it from the nucleus. Rev itself can be "roundtrip" between the nucleus and the cytoplasm. This bidirectional transport is mediated by two specific Rev sequences: NLS, which overlaps with RNA binding proteins and a distinct nuclear export signal (NES). Impβ
Nuclear import of Tat or Rev (Truant et al., 1999, M.
Ol Cell Biol, 19: 1210-7) through the classical NLS pathway, in which the interaction of Impβ with Tat and Rev results in RanGT.
It is demonstrated by being inhibited by P but not RanGDP.

Importin β also interacts with the HIV-1 protein Vpr (Je
nkins et al., 1998, J Cell Biol, 143: 875-885).
. Vpr contains two distinct nuclear targeting signals that use two different import pathways, both of which are distinct from the classical NLS pathway. Vpr import does not appear to require Ran-mediated GTP hydrolysis and is maintained under low energy conditions. Vpr bypasses many soluble receptors involved in the import of cellular proteins. Vpr directly approaches NPCs, a property that can help HIV replicate in nondividing cellular hosts (Jenkins).
Et al., Ibid.). Overexpression of Vpr or importin β in yeast blocks nuclear export of mRNA. Variants of Vpr, Vpr F34I, which are not localized in the nuclear envelope or which bind to importin alpha and nucleoporins, prevent HIV-1 from efficiently infecting macrophages. However, Vpr F34I still causes G2 arrest, which leads to Vpr
Demonstrate that the dual function of is genetically separable (Vodikka et al.,
1998, Genes Dev, 12: 175-185).

Rodent, avian, and insect L1-like cell adhesion molecules are members of the immunoglobulin superfamily involved in axon growth. The entire coding region of human L1CAM was cloned and found to be highly homologous to mouse L1CAM and 92% identical at the amino acid level (Hlavin et al., 199).
1, Genomics 11: 416-423). Due to this similarity, L1CA
It is suggested that M is a molecule important for normal human nervous system development and nerve regeneration. This molecule is completely unrelated to the HIV-1 life cycle.

Cyclophilin is proposed to act as a chaperone in various cellular processes. The U4 / U6 snRNP-associated cyclophilin has been cloned and sequenced (Horowitz et al., 1997, RNA 3: 1374-13.
87).

The nucleotide sequence of the resepin gene, a novel human liver cDNA encoding a serpin-like molecule, was submitted directly to GenBank.

Arg and c-Abl represent mammalian members of the Abelson family of protein tyrosine kinases. Ar which is an Arg / Abl binding protein
gBP2 was isolated using the segment of the ArgCOOH terminal domain as a bait for the yeast double hybrid system (Wang et al., 1997, J. Biol. Ch.
em. 272: 17542-17550). ArgBP2 contains three COOH-terminal Src homology 3 domains, a serine / threonine-rich domain, and several potential Abl phosphorylation sites. ArgBP2 is Arg and v-Ab
l, its substrate, and is phosphorylated on tyrosine in v-Abl transformed cells. ArgBP2 is widely expressed in human tissues and is highly abundant in the heart. In epithelial cells, ArgBP resembles the reported localization of c-Abl,
Localized to stress fibers and the nucleus. In cardiac muscle cells, ArgBP2 is localized to the sarcomeric Z disk. These observations indicate that ArgBP2 functions as an adapter protein for the association of signaling complexes in stress fibers, and ArgBP2
BP2 is shown to be the link between the Abl family kinases and the actin cytoskeleton.

Interferon (IFN) -γ is involved in the pathogenesis of several autoimmune and inflammatory skin disorders. CDN that detects 1.6 kb mRNA that accumulates in response to IFN-γ but not IFN-α or IFN-β
A was cloned and sequenced (Flohr et al., 1992, Eur J.
Immunol. 22: 975-979). The gene is regulated by IFN-γ in human cell lines of epithelial origin. The mRNA encodes a predicted protein of 432 amino acids, and the primary structure of the protein demonstrates that it is a member of the developmentally regulated keratin class I genes.

The oncoprotein 18 (Op18) gene encodes a proliferation-associated cytosolic protein that is induced in normal lymphocytes after stimulation with mitogens. The cDNA for this gene has been cloned (Zhu et al., 1989, J Biol.
Chem. 264: 14556-14560). It is encoded by two different size full length cDNAs. The two cDNAs differ in their 3'non-coding regions as a result of another polyadenylation. Op18 gene (this is 6.3k
b length) consists of 5 exons and 4 introns and exhibits features common to other genes involved in cell proliferation and reproduction. This gene is highly conserved in several animal species, and low stringency hybridization studies suggest that the p18 gene may be a member of a family of partially homologous genes in the human genome. The increase in Op18 polypeptide in leukemia is
Associated with increased RNA transcription without gene amplification or rearrangement (Melhe
M. et al., 1991, J. Am. Biol. (Chem 266: 17747-17753)
. Treatment of the K562 leukemia cell line with hemin, which induces terminal differentiation, results in Op18
Expression was decreased.

A putative G protein-coupled receptor has recently been cloned (Mayer et al., 19
98, Biochim. Biophys. Acta 1395: 301-308
). The cDNA sequence encodes a protein of 399 amino acids. Northern and RNA dot blot analysis demonstrated that the major 4.8 kb transcript was predominantly expressed in brain. In situ hybridization studies of tissue sections revealed high expression in brain and spinal cord neurons, thymocytes, megakaryocytes and macrophages.

Glucosidase αII is a glycoprotein involved in the processing of N-linked glycans. It resides in the endoplasmic reticulum (ER) and regulates the formation of glycoproteins in the ER. The glucosidase αII gene has been cloned and the encoded protein has not been shown to contain known ER retention signals or hydrophobic regions, which may represent transmembrane domains. However, glucosidase αII has been shown to contain a single N-glycosylation site near the amino terminus. HIV-1 contains two heavily glycosylated envelope proteins, gp120 and gp41, which mediate attachment of virions to the glycosylated cell surface receptor molecule, CD4. Gp120 and gp41 also show syncytia formation and associated HIV
May be involved in the cytopathic effect of. The α-glucosidase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), inhibits HIV replication and HIV in vitro.
It is an inhibitor of induced syncytia formation. NB-DNJ-mediated retention of glycosylation-glycans prevents HIV entry by the combined action of qualitative defects in the remaining gp120 and reduced virion gp120, which causes conformational changes after CD4 binding. Prevent receiving (Fischer et al., 1996, J.V.
irol. 70: 7153-7160).

In mammals, there are at least three isoforms of the glycolytic enzyme enolase, encoded by three similar genes: α, β and γ. The structure of the human gene for the α-enolase locus has been described (Giallongo et al., 199).
0, Eur J Biochem, 190: 567-573). This gene is
It consists of 12 exons, distributed over 18,000 bases. The structure of this gene has a high degree of similarity to that of the human and rat γ-enolase genes, with identical positions for all intron regions. The region of the putative promoter, like that of other housekeeping genes, lacks canonical TATA and CAAT boxes, is highly G + C rich, and contains several potential SP1 binding sites. A 48 kDa protein (p48) that specifically reacts with antisera directed against the 12 carboxyl-terminal amino acids of the c-myc gene product.
Has been demonstrated to be an alpha enolase (Giallongo et al., 198).
6, Proc Natl Acad Sci USA 83: 6741-674.
5).

The murine gene macrophage inflammatory protein 1α (MIP1α) is a cytokine that inhibits proliferation of bone marrow stem cells (Russell et al., 1993, D.
NA Cell Biol. 12: 157-175). MIP1α has been shown to suppress HIV-1 replication in human peripheral blood mononuclear cells. MIP1α
May also repress transcription from the HIV-1 LTR in a transient transfection assay in cells of the Jurkat acute T leukocyte cell line (Han
den et al., 1997, FEBS Lett. 410: 301-302). MIP
1α is a ligand for CCR5 and may prevent M-tropic HIV infection in vitro (Cochi et al., 1995, Science, 270: 1811-1815).
Gong et al., 1998, J .; Biol. Chem. 271: 2599-260
3). Certain individuals with elevated levels of MIP1α expression appear to be resistant to HIV infection (Cho et al., 1997, Biomed. Pharmacother. 51.
: 221-229). However, it is unclear how down-regulation of intracellular expression of MIP1α can affect T cell HIV replication.

Translation-regulated oncoprotein (TCTP) is a growth-related protein that is regulated at the transcriptional level. It is present in mammals, higher plants and Saccharomyces cerevisiae (San
chez et al., 1997, Electrophoresis 18: 150-15.
5). Activation of macrophages by PHA was shown to upregulate TCPP (Walsh et al., 1995, J. Leukoc Bi.
ol. 57: 507-512). TCTP also has the following effects during vitamin D-induced cell death:
Differentially expressed in C6.9 glioma cells (Baudet et al., 1998,
Cell Death Differ, 5: 116-125). TCTP is localized to chromosomes 13q12-q14 (MacDonald et al., 1999, Cy.
Togenet Cell Genet, 84: 128-129). Vitamin D
Both PHA and PHA can induce latent HIV-1. This allows TCTP1
The pathway has been shown to overlap with signaling pathways involved in the HIV-1 life cycle.

The human and simian immunodeficiency virus nef genes are important factors in the pathogenesis and viral replication of acquired immunodeficiency syndrome. Several Nef-induced events have been previously reported, including down-regulation of CD4 in T cells.
Naf1 (Nef-related factor 1) has been cloned (Fukushi, 199).
9, FEBS Lett. 442: 83). The Naf1 gene produces two isoforms, Nafα and β, which contain four coiled-coil configurations. Na
f1 mRNA is ubiquitously present in human tissues and is strongly expressed in peripheral blood lymphocytes and spleen. Naf1 overexpression in T cells increases surface CD4 expression. N
Expression of ef suppresses this increase in CD4 expression induced by Naf1 and provides a novel form of Nef action in CD4 down-regulation.

A human gene for a protein that modifies Na ⁺ -D-glucose cotransport, N
The a ⁺ -D-glucose cotransport regulatory gene (hRS1) has been cloned and sequenced (Lambotte et al., 1996, DNA Cell Bi.
15: 769-777). It is an intronless gene called hRS1 (6,743 bp), which encodes a 617 amino acid protein and is 74% identical to pRS1. By fluorescence in situ hybridization, hRS1 was localized to chromosome 1p36.1. It is homologous to the nucleic acid sequence of pRS1 cloned from porcine kidney cortex and encodes a membrane-associated protein involved in Na ⁺ -conjugated sugar transport. pRS1 is S derived from rabbit intestine
Alters glucose transport by GLT1 or by SMIT from canine kidney homologous to SGLT1. In contrast, pRS1 does not affect transport from other gene families. The function of hRS1 was demonstrated by human gut-derived hRS1 and SGLT1 co-expression experiments in Xenopus-derived oocytes. The hRS1 protein was demonstrated to inhibit Na ⁺ -D-glucose cotransport, which is expressed by human SGL1T, by reducing both transporter V _max and apparent K _m values. Analysis of the 5'non-coding sequence of hRS1 revealed different enhancer consensus sequences not present in the SGLT1 gene, eg several consensus sequences of steroid binding proteins.

Hsp90 is a rich molecular chaperone involved in the folding of a set of signaling molecules, including steroid-hormone receptors and kinases. In vitro experiments suggest that Hsp90 contains two distinct binding sites for non-natural proteins, which can match the properties of predicted folding helper proteins with those of promiscuous chaperones. The heat shock protein Hsp90 is known as an essential component of several signal transduction pathways and has been identified as an important host for hepatitis B virus replication. Hsp90 is
Interacts with viral reverse transcriptase to facilitate the formation of the ribonucleoprotein (RNP) complex between polymerase and RNA ligands (Hu et al., 1996, Pro.
c Natl Acad Sci USA 93: 1060-1064). Hs
p90 is not associated with the HIV life cycle. Specific inhibitors of hsp90, such as the antifungal macrolides geldanamycin and radicicol, can be used against HIV-1 infection.

FK506 binding protein A1 is 100 times more active and potent than cyclosporin A, a cyclic decapeptide used to prevent post-transplant rejection of bone marrow and organs such as kidney, heart and liver. It is an immunosuppressant. FK506 is
It has been shown to bind to cellular proteins distinct from cyclophilin, which is known to bind to cyclosporin A. The cDNA was isolated from human peripheral blood T cells that encode the FK506 binding protein (FKBP) (Maki et al., 19).
90, Proc Natl Acad Sci USA 87: 5440-54.
43). The isolated cDNA contained an open reading frame encoding 108 amino acid residues. The first 40 residues of the deduced amino acid sequence are identical to those of the reported amino-terminal sequence of bovine FKBP, indicating that the isolated DNA sequence represents the gene encoding FKBP. It is expressed in brain, lung, liver, placental cells and leukocytes. Phorbol 12 of Jurkat leukocyte T cells
-Myristate 13-acetate and ionomycin induced FKBP
It did not affect the level of mRNA.

The Myc and Mad family of proteins are involved in transcriptional regulation and mediate cell differentiation and proliferation. These molecules are basic-helix-loop-
The helix-leucine zipper domain (bHLHZip) is shared, and the E-box (CANNTG) consensus sequence forms a heterodimer with Max to form D.
Binds to NA (Meroni et al., 1997, EMBO J. 15-16: 28.
92-906). The Rox transcriptional repressor forms a heterodimer with Max and weakly forms a homodimer. Interestingly, the band-shift assay demonstrates that Rox-Max heterodimers show novel DNA binding specificities and a high affinity for the CACGCG site as compared to the canonical E-box CACGTG site. Transcriptional studies indicate that Rox represses transcription in both human HEK293 cells and yeast. Furthermore, ROX expression was 12-O-.
U9 stimulated to differentiate with tetradecanoylphorbol-13-acetate
It appears to be induced on 37 bone marrow white blood cells. With these data, HIV-
1 is shown to be able to interfere with cell transcription using Rox. Because antisense elements derived from this gene can interfere with HIV-1 replication.

The human signal sequence receptor gene, SSR2, encodes an endoplasmic reticulum (ER) membrane protein associated with protein translocation across the ER membrane. This gene has been cloned (Chinen et al., 1995, Cytogenet Cell).
Genet. 70: 215-217). Northern blot analysis revealed its ubiquitous expression in all organs examined (Chinen et al., Ibid.). The gene is localized to chromosome bands 1q21 to q23. The various GSEs described herein affect the expression of the ER proteins calnexin, glucosidase alpha and glucosyl transferase, which expression has an inhibitory effect on HIV replication. The SSR2 gene inhibits HIV-1 replication in the same manner, gp1
Affects translocation of 60 to ER.

The human tumor imaginal discriminant protein hTid-1, which is a homologue of the Drosophila tumor suppressor protein Tid56, has been cloned (Schill.
Ling, 1998, Virology 247: 74-85). hTid-1
The protein can form a complex with the human papillomavirus E7 oncoprotein. The carboxyl-terminal cysteine-rich metal-binding domain of E7 is hTi
It is the major determinant of interaction with d-1. The hTid-1 protein is a member of the DnaJ family of chaperones. hTid-1 mRNA is HP
It is widely expressed in human tissues, including the normal host cells for V, the HPV-18 positive cervical cancer cell line, HeLa and human reproductive keratinocytes. hT
The id-1 gene has been mapped to the short arm of chromosome 16. The large tumor antigen of polyomavirus encodes a functional J-domain that is important for viral replication and cell transformation. The ability of HPV E7 to interact with the cellular DnaJ protein suggests that these two viral oncoproteins may target common regulatory pathways through the J-domain (Schilling, supra).

Heparin sulphate proteoglycans are expressed on the cell surface and their corresponding binding sites are important during the initial attachment of the murine blastocyst to the uterine epithelium and to the human trophoblast cell line. Have been suggested to play a role. Three major peptide fragments from the heparin-binding protein were isolated and the partial amino-terminal amino acid sequence of each peptide fragment was obtained (Raboudi et al., 1992, J. Bio.
l. Chem. 267, 11930-11939). The full-length cDNA of heparin-binding protein, called HP / HS interacting protein (HIP), is RL9.
Cloned from 5 cells (Liu et al., 1996, J Biol Chem.
271: 11817-11823. ) HIP cDNA encodes a protein of 159 amino acids. Transfection of HIP full-length cDNA into NIH-3T3 cells resulted in cell surface expression of HIP protein with a size similar to HIP expressed by human cells. According to the deduced amino acid sequence,
HIP has been shown to lack a transmembrane region and has no common site for glycosylation. Northern blot analysis revealed total RNA and poly (A ⁺ ) RN
In both A, a single transcript of 1.3 kb was detected. Examination of human cell lines and normal tissues using both Northern and Western blot analysis revealed that HIP was expressed at different levels in various human cell lines and normal tissues.

The minimum size of a nucleic acid molecule of the present invention is the size required for the use of the nucleic acid molecule to block HIV replication. One of ordinary skill in the art will recognize that the length can vary depending on whether the nucleic acid molecule is in the form of RNA or DNA. Nucleic acid molecules of the invention include, but are not limited to, therapeutic probes for identifying additional nucleic acid molecules, as primers for amplifying or extending nucleic acid molecules, or for example, for blocking expression of a target gene of the invention. It can be used for various applications, including but not limited to. Said therapeutic application is eg antisense RNA, which functions to prevent HIV infection.
And DNA molecules, triplex-forming, ribozyme- and / or RNA
It includes the use of said nucleic acid molecules in drug-based technology and is referred to herein as "therapeutic nucleic acid molecules". For example, antisense RNA and DNA molecules can be used to directly block the translation of mRNA encoded by these cellular genes by binding to the targeted mRNA and preventing translation of the protein. Polydeoxyribonucleotides can form sequence-specific triple helices by hydrogen bonding to specific complementary sequences of double-stranded DNA. The formation of a specific triple helix
By inhibiting the specific binding of functional trans-acting factors, replication and / or expression of the targeted gene can be selectively inhibited.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule complementary to the target RNA, followed by nucleotide strand scission. Within the scope of the present invention are engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze nucleotide strand scission of cellular RNA sequences. Antisense RNAs that exhibit high affinity for the target sequence can also be used as ribozymes by the addition of enzymatically active sequences known to those of skill in the art.

The nucleic acid molecule used for triple helix formation should be single-stranded and composed of deoxynucleotides. The base composition of these nucleic acid molecules should be designed by the Hoogsteen base pairing rules to promote triple helix formation, which in general,
A significant size sequence of purine or pyrimidine is required to be present on one strand of the duplex. The nucleic acid molecule sequence may be pyrimidine-based, which
TAT and CGC triplets traverse the three associated strands of the resulting triple helix. A pyrimidine-rich polynucleotide, in an orientation parallel to its strand, provides base complementarity to the purine-rich region of one strand of the duplex. Furthermore, purine-rich nucleic acid molecules can be selected that include, for example, a G residue sequence. These nucleic acid molecules form triple helices with GC pair-rich DNA duplexes, where most of the purine residues are located on one strand of the targeted duplex, and thus the GGC triplet. Crosses the three strands of the triplex.

Alternatively, potential sequences that can be targeted for triple helix formation are so-called "
Can be increased by creating a "switchback" polynucleotide. The switchback polynucleotide is synthesized in an alternating 5'-3 ', 3'-5' fashion,
Thus, they base pair with the first strand of the duplex and then with the others, and a substantial size sequence of purines or pyrimidines is present on one strand of the duplex. There is no need.

Both antisense RNA and DNA molecules of the invention, and ribozymes,
It can be prepared by a method known in the art. These include techniques for chemically synthesizing polydeoxyribonucleotides known in the art, such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules can be made by in vitro and in vivo transcription of DNA sequences encoding antisense RNA. The DNA sequence is T
A wide variety of vectors can be incorporated that incorporate suitable RNA polymerase promoters such as the 7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly can be stably introduced into host cells depending on the promoter used.

Therefore, the present invention provides a method of interfering with the production of a protein encoded by a target gene required for HIV infection according to the present invention, by use of said therapeutic nucleic acid molecule and one or more of the above techniques. Including.

Preferred therapeutic nucleic acid molecules are NADH dehydrogenase, 2-oxoglutarate dehydrogenase, M2 type pyruvate kinase / cytosolic thyroid hormone binding protein, calnexin, ADP-ribosylation factor 3, eukaryotic initiation factor 3, protein tyrosine phosphatase, Herpesvirus-associated ubiquitin-specific protease, eukaryotic initiation factor 4B, CD44, phosphatidyl-inositol 3 kinase, elongation factor 1α, bone morphogenetic protein-1, double-strand break DNA repair gene protein, rat guanine nucleotide releasing protein, antiproliferation Factor (BTG-
1), lymphocyte-specific protein 1, protein phosphatase 2A, squalene synthetase, eukaryotic release factor 1, GTP binding protein, importin β subunit, cell adhesion molecule L1, U-snRNP associated cyclophilin, recepin, Arg / Abl Interacting protein (ArgBP2A), keratin-related protein, p18 protein, p40 protein, glucosidase II, α enolase, macrophage inflammatory protein 1α, translation control tumor protein 1 (TCTP)
1), BBC1, Nef interacting protein, Na ⁺ -D-glucose cotransporter gene protein, hsp90 chaperone protein, FK506 binding protein A1, Rox, β signal sequence receptor, tumor primordium protein or cell surface heparin binding It comprises at least a part of a target gene encoding a protein, or a protein selected from the group consisting of homologues thereof, wherein said part is capable of down-regulating the expression of an associated target gene. More preferred therapeutic nucleic acid molecules are especially SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5.
, Sequence number 6, sequence number 7, sequence number 8, sequence number 9, sequence number 10, sequence number 11, sequence number 12, sequence number 13, sequence number 14, sequence number 15, sequence number 1
6, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 25
, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35,
Sequence number 37, sequence number 39, sequence number 41, sequence number 43, sequence number 45, sequence number 47, sequence number 49, sequence number 51, sequence number 53, sequence number 55, sequence number 57, sequence number 59, sequence number 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85; Sequence number 8
7; SEQ ID NO: 89; SEQ ID NO: 91: SEQ ID NO: 93 and / or SEQ ID NO: 95 or other GSE sequences disclosed herein, as well as the complements of any of these sequences or homologues thereof. GSE nucleic acid molecule of

The invention also includes a recombinant vector, which includes the nucleic acid molecule of the invention inserted into any vector that can deliver the nucleic acid molecule to a host cell. The vector comprises a heterologous nucleic acid sequence, which is a nucleic acid sequence not found naturally adjacent to the cell-derived nucleic acid molecule of the present invention. The vector can be RNA or DNA of prokaryotic or eukaryotic cells, and is typically a virus or plasmid. Recombinant vectors can be used for cloning, sequencing and / or other manipulations of the nucleic acid molecules of the invention. One type of recombinant vector, referred to herein as a recombinant expression molecule and described in more detail below, can be used to express a nucleic acid molecule of the invention. Preferred recombinant vectors are capable of replicating in transformed cells.

Preferred nucleic acid molecules for inclusion in the recombinant vector of the invention are the cell-derived nucleic acid molecules of the invention. Particularly preferred nucleic acid molecules for inclusion in the recombinant vector are NADH dehydrogenase, 2-oxoglutarate dehydrogenase, type M2 pyruvate kinase / cytosolic thyroid hormone binding protein, calnexin, ADP-ribosylation factor 3, eukaryotic initiation factor 3, protein. Tyrosine phosphatase, herpesvirus-associated ubiquitin-specific protease, eukaryotic initiation factor 4B, CD44,
Phosphatidyl-inositol 3 kinase, elongation factor 1α, bone morphogenetic protein-1, double-strand break DNA repair gene protein, rat guanine nucleotide release protein, anti-proliferative factor (BTG-1), lymphocyte-specific protein 1, protein phosphatase 2A, squalene synthetase, eukaryotic release factor 1, GT
P-binding protein, importin β subunit, cell adhesion molecule L1, U-s
nRNP-associated cyclophilin, recepin, Arg / Abl interacting protein (
ArgBP2A), keratin-related protein, p18 protein, p40 protein, glucosidase II, α enolase, macrophage inflammatory protein 1α
, Translationally regulated oncoprotein 1 (TCTP1), BBC1, Nef interacting protein, Na ⁺ -D-glucose cotransport regulator gene protein, hsp90 chaperone protein, FK506 binding protein A1, Rox, β signal sequence receptor, tumor protozoa A protein selected from the group consisting of a basic protein or a cell surface heparin binding protein, or its complement or homologue,
It is at least part of the target gene. Other preferred nucleic acid molecules for inclusion in the recombinant vector are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, sequence No. 10, SEQ ID No. 11
, Sequence number 12, sequence number 13, sequence number 14, sequence number 15, sequence number 16,
Sequence number 17, sequence number 18, sequence number 19, sequence number 20, sequence number 25, sequence number 27, sequence number 29, sequence number 31, sequence number 33, sequence number 35, sequence number 37, sequence number 39, sequence number 41, sequence number 43, sequence number 45, sequence number 47, sequence number 49, sequence number 51, sequence number 53, sequence number 55, sequence number 57, sequence number 59, sequence number 61, sequence number 63, sequence number 65, Sequence number 6
7, sequence number 69, sequence number 71, sequence number 73, sequence number 75, sequence number 77
, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85; SEQ ID NO: 87;
SEQ ID NO: 89; SEQ ID NO: 91: SEQ ID NO: 93, SEQ ID NO: 95, or other GSE sequences disclosed herein, and a nucleic acid selected from the group consisting of the complement or homologue of any of these sequences. A GSE nucleic acid molecule having a sequence.

Recombinant expression molecules of the invention include one or more nucleic acid molecules of the invention operably linked to an expression vector containing one or more regulatory sequences. The term "operably linked" refers to the insertion of a nucleic acid molecule into an expression vector so that the molecule will be expressed when transformed into a host cell. An expression vector as used herein is a DNA or RNA vector capable of transforming a host cell and effecting the expression of a particular nucleic acid molecule. Preferably, the expression vector is also capable of replicating in the host cell. Expression vectors can be prokaryotic or eukaryotic, and are typically viruses or plasmids. Expression vectors of the invention include any vector that functions (ie directs gene expression) in the recombinant cells of the invention, including bacterial, fungal, insect, animal and / or plant cells. Therefore, the nucleic acid molecule of the present invention comprises a promoter,
Operable in expression vectors that include regulatory sequences such as operators, repressors, enhancers, termination sequences, origins of replication, and other regulatory sequences that are compatible with recombinant cells and control the expression of the nucleic acid molecules of the invention. Can be connected to. Regulatory sequences, as used herein, include sequences capable of controlling the initiation, elongation and termination of transcription. Particularly important regulatory sequences are those that control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable regulatory sequences include any transcriptional control sequence that can function in cells susceptible to infection with HIV. A variety of such transcription control sequences are known to those of skill in the art. Additional suitable regulatory sequences include tissue-specific promoters and enhancers, and lymphokine-inducible promoters (eg, promoters inducible by interferons or interleukins).
including. The regulatory sequences of the present invention may also include natural transcriptional control sequences naturally associated with the DNA sequences encoding the nucleic acid molecules of the present invention. If the recombinant molecule of the present invention is expressed as a protein, a specific initiation signal may be required for efficient translation of the inserted nucleic acid molecule. It is necessary to provide exogenous translational control signals, including the ATG initiation codon. In addition, the initiation codon ensures that translation of the entire insert is ensured,
It must be in phase with the reading frame of the inserted nucleic acid molecule. These foreign translation control signals and initiation codons can be of various origins, both natural and synthetic.

Preferred expression vectors of the invention are obtained from viruses such as retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, or papillo-maviruses. Methods known to those of skill in the art can be used to make recombinant molecules of the invention (Sambrook et al., 1989, Molecular Cloning: Experimental Manual, Cold Spring Harbor Laboratory, NY and Ausubel et al., 1989.
Current Protocols in Molecular Biology: Greene Publishing Publishing Ass
associates and Wiley Interscience, N.M. Y. ). When an adenovirus is used as an expression vector, the nucleic acid molecule of the invention can be ligated to an adenovirus transcription-translation control complex, such as the late promoter and ternary leader sequence. This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion into a nonessential region of the viral genome, such as the E1 or E3 region, results in a recombinant virus that is viable and capable of expressing GSE in the infected host (Logan & Shenk,
1984, Proc. Natl. Acad. Sci. USA 81: 3655-
3659).

[0116] The use of recombinant DNA technology can increase, for example, the copy number of the nucleic acid molecule in the host cell, the efficiency with which the nucleic acid molecule is transcribed, the efficiency with which the resulting transcript is translated, and the efficiency of post-translational modification. One of skill in the art will recognize that manipulations can improve the expression of transformed nucleic acid molecules. Recombinant techniques useful for increasing expression of the nucleic acid molecules of the invention include operably linking the nucleic acid molecule to a high copy number plasmid, integrating the nucleic acid molecule into the chromosome of one or more host cells, a vector Addition of stabilizing sequences to the promoter, substitution or modification of transcription control signals (eg promoter,
Operators, enhancers), substitutions or modifications of translational control signals (eg ribosome binding sites, Shine-Dalgarno sequences), modifications of the nucleic acid molecule of the present invention corresponding to the codon usage of the host cell, of transcript destabilizing sequences. Deletion and use of control signals that temporally separate recombinant cell growth from recombinant protein production during fermentation, but are not limited to this. The activity of the expressed recombinant protein of the present invention may be improved by fragmenting, modifying or derivatizing the resulting protein.

Various modifications to nucleic acid molecules can be introduced as a means of increasing intracellular stability and half-life. Possible modifications are addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5'and / or 3'ends of the molecule, or phosphorothioate or 2'O-methyl rather than phosphodiesterase chains within the polydeoxyribonucleotide backbone. Uses include, but are not limited to.

One embodiment of the present invention is an isolated protein required for HIV infection. The protein is referred to as "cell-derived protein". The term "protein" as used herein is meant to include both polypeptides and peptides. Preferably, the present invention provides a peptide or a fragment shorter than the full length of said cell-derived protein of the present invention, wherein expression of said protein in HIV-susceptible cells results from HIV infection, replication, or HIV progeny virus. Inhibit the production of. Also included by this definition is a full-length protein having at least one change in a constitutive amino acid residue, whereby the protein no longer favors HIV infection in said cell, and most preferably it has changed. The protein inhibits HIV infection, replication or production of HIV progeny virus. According to the invention, isolated or
Alternatively, a biologically pure protein is a protein that has been removed from its natural environment. Thus, "isolated" and "biologically pure" do not necessarily reflect the extent to which the protein has been purified. The isolated cell-derived protein of the present invention can be obtained from its natural source, produced using recombinant DNA technology, or produced by chemical synthesis. Preferably, the isolated cell-derived protein of the present invention is NADH dehydrogenase, 2-oxoglutarate dehydrogenase, M2-type pyruvate kinase / cytosolic thyroid hormone binding protein, calnexin, ADP-ribosylation factor 3, eukaryotic initiation factor 3, Protein tyrosine phosphatase, herpesvirus-associated ubiquitin-specific protease, eukaryotic initiation factor 4B
, CD44, phosphatidyl-inositol 3 kinase, elongation factor 1α, bone morphogenetic protein-1, double-strand break DNA repair gene protein, rat guanine nucleotide release protein, antiproliferative factor (BTG-1), lymphocyte-specific protein 1 , Protein phosphatase 2A, squalene synthetase, eukaryotic release factor 1, GTP binding protein, importin β subunit, cell adhesion molecule L1, U-snRNP-associated cyclophilin, lecepin, Arg / Abl interacting protein (ArgBP2A), keratin-related Protein, p18 protein,
p40 protein, glucosidase II, α enolase, macrophage inflammatory protein 1α, translation control tumor protein 1 (TCTP1), BBC1, Nef interacting protein, Na ⁺ -D-glucose cotransport regulator gene protein,
hsp90 chaperone protein, FK506 binding protein A1, Rox, β
It comprises at least part of a protein selected from the group consisting of a signal sequence receptor, an adult tumor primordium protein or a cell surface heparin binding protein or any homologue of said protein. The isolated proteins of the invention (including homologues) can be identified in a straightforward manner by the ability of the proteins to prevent HIV infection. Examples of homologues are those in which the amino acid has been deleted (so that the homologue has at least some ability to block HIV infection.
For example, by partially truncated forms of proteins, such as peptides), insertions, inversions, substitutions and / or derivatizations (eg, glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation, and And / or glycerophosphatidylinositol added), where expression of the wild-type protein is required for HIV infection. A homologue also comprises at least one epitope capable of eliciting an immune response against cell-derived proteins. HIV
Disclosed herein are techniques for measuring infection or its inhibition.

The cell-derived protein homologues of the present invention can be the result of natural allelic variants or natural mutations. The cell-derived protein homologues of the present invention are effective for direct modification to the protein, or for random or targeted mutagenesis, eg, using classical or recombinant nucleic acid technology, to the gene encoding the protein. It can be made using techniques known in the art, including but not limited to modifications.

The minimum size of the cell-derived protein homologue of the present invention is a size sufficient to function as an inhibitor of HIV infection. The minimum size of the homologue is typically at least about 6 to about 10 residues long. Other than practical limits, there are no limits on the maximum size of said homologues, wherein protein homologues include peptides, fragments shorter than the full length of full length proteins, full length proteins, multiple proteins, or parts thereof. Thus, said full-length protein most preferably comprises an altered amino acid sequence whereby the altered protein inhibits HIV infection, replication or infectious virus production.

The present invention also includes mimetopes of the cell-derived proteins of the invention. As used herein, a mimetope of a cell-derived protein of the invention means any compound capable of mimicking the activity of said cell-derived protein (eg, the ability to block HIV infection). This is because the mimetope often has a structure that mimics a cell-derived protein. However, it is noted that the mimetope need not have a chemical structure similar to a cell-derived protein as long as the mimetope functionally mimics the protein. Mimetope is a peptide that has been modified to reduce its susceptibility to degradation; anti-idiotype and / or catalytic antibodies or fragments thereof; non-protein immunogenic portions of isolated proteins (eg, carbohydrate structures); Can be, but is not limited to, synthetic or natural organic or inorganic molecules; and / or any other peptidomimetic compound. The mimetope of the present invention is
It can be designed using the computer-generated structures of the cell-derived proteins of the invention. Mimetopes, such as polynucleotides, peptides or other organic molecules,
Random molecular samples can be generated and obtained by chromatographic techniques using the corresponding binding pairs. Preferred mimetopes are peptidomimetic compounds that are structurally and / or functionally similar to the cell-derived proteins of the invention.

One embodiment of the cell-derived protein of the invention is a fusion protein that comprises a cell-derived protein-containing domain attached to one or more fusion segments.
Suitable fusion segments are known to those of skill in the art, depending on the use of the segment in protein stability or transport of the protein into the cell. Fusion protein
Preferably, it is prepared by culturing a recombinant cell transformed with a fusion nucleic acid molecule, which encodes a cell-derived protein containing a fusion segment.

The isolated cell-derived protein of the invention has the additional feature of being encoded by the cell-derived nucleic acid molecule of the invention. The preferred cell-derived protein of the present invention is
Sequence number 1, sequence number 2, sequence number 3, sequence number 4, sequence number 5, sequence number 6,
Sequence number 7, sequence number 8, sequence number 9, sequence number 10, sequence number 11, sequence number 12, sequence number 13, sequence number 14, sequence number 15, sequence number 16, sequence number 1
7, sequence number 18, sequence number 19, sequence number 20, sequence number 25, sequence number 27
, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37,
Sequence number 39, sequence number 41, sequence number 43, sequence number 45, sequence number 47, sequence number 49, sequence number 51, sequence number 53, sequence number 55, sequence number 57, sequence number 59, sequence number 61, sequence number 63, sequence number 65, sequence number 67, sequence number 69, sequence number 71, sequence number 73, sequence number 75, sequence number 77, sequence number 79, sequence number 81, sequence number 83, sequence number 85; sequence number 87; Sequence number 8
9; SEQ ID NO: 91: having a nucleic acid sequence selected from the group consisting of SEQ ID NO: 93, SEQ ID NO: 95 or other sequences disclosed herein, as well as the complement or homologue of any of these sequences. , Encoded by a nucleic acid molecule.

The isolated GSE nucleic acid molecule of the present invention can suppress HIV activity by encoding a protein or RNA product. The invention includes any of the aforementioned protein products, including fusion proteins, leader peptides and localization signals.

One embodiment of the present invention is an inhibitory composition capable of prophylactically or therapeutically protecting an animal from HIV infection when administered to the animal in an effective manner. The inhibitory composition of the present invention comprises NADH dehydrogenase, 2-oxoglutarate dehydrogenase, M2 type pyruvate kinase / cytosolic thyroid hormone binding protein,
Calnexin, ADP-ribosylation factor 3, eukaryotic initiation factor 3, protein tyrosine phosphatase, herpesvirus-associated ubiquitin-specific protease, eukaryotic initiation factor 4B, CD44, phosphatidyl-inositol 3 kinase, elongation factor 1α, bone morphogenetic protein- 1, double-strand break DNA repair gene protein, rat guanine nucleotide releasing protein, anti-proliferative factor (BTG-1), lymphocyte-specific protein 1, protein phosphatase 2A, squalene synthetase, eukaryotic releasing factor 1, GTP binding protein , Importin β subunit,
Cell adhesion molecule L1, U-snRNP associated cyclophilin, resepin, Arg / A
bl interacting protein (ArgBP2A), keratin-related protein, p18
Protein, p40 protein, glucosidase II, α enolase, macrophage inflammatory protein 1α, translationally regulated oncoprotein 1 (TCTP1), BBC
1, Nef interacting protein, Na ⁺ -D-glucose cotransporter gene protein, hsp90 chaperone protein, FK506 binding protein A1
, Rox, β signal sequence receptor, tumor imaginal discriminant protein or cell surface heparin binding protein, down-regulating the expression of genes, and may include protective compounds. The protection compound comprises an isolated cell-derived nucleic acid molecule of the invention operably linked to regulatory sequences that control expression. The protective compound can be an RNA or DNA molecule as described herein. Especially preferred are the GSE nucleic acid molecules of the invention. Functionally active fragments of GSE, and GSEs containing conservative nucleotide substitutions as functional equivalents of GSE are within the scope of the invention. Preferably, the GSE nucleic acid molecule, or functional equivalent thereof, is operably linked to regulatory sequences controlling its expression.

The inhibitory composition of the present invention comprises NADH dehydrogenase, 2-oxoglutarate dehydrogenase, M2-type pyruvate kinase / cytosolic thyroid hormone binding protein, calnexin, ADP-ribosylation factor 3, eukaryotic initiation factor 3, protein tyrosine. Phosphatase, herpesvirus-associated ubiquitin-specific protease, eukaryotic initiation factor 4B, CD44, phosphatidyl-inositol 3 kinase, elongation factor 1α, bone morphogenetic protein-1, double-strand break DNA repair gene protein, rat guanine nucleotide-releasing protein, Anti-growth factor (BTG-1
), Lymphocyte-specific protein 1, protein phosphatase 2A, squalene synthetase, eukaryotic release factor 1, GTP binding protein, importin β subunit, cell adhesion molecule L1, U-snRNP associated cyclophilin, resepin,
Arg / Abl interacting protein (ArgBP2A), keratin-related protein, p18 protein, p40 protein, glucosidase II, α enolase, macrophage inflammatory protein 1α, translation control tumor protein 1 (TCTP1)
), BBC1, Nef interacting protein, Na ⁺ -D-glucose cotransport regulator gene protein, hsp90 chaperone protein, FK506 binding protein A1, Rox, β signal sequence receptor, tumor imaginal discriminant protein or cell surface heparin binding protein. Protective compounds that inhibit the activity of the gene product, which encode The protective compound is the isolated cell-derived protein of the present invention, particularly a peptide of the cell-derived protein, a mimetope of the cell-derived protein,
And small molecule inhibitors of the activity of the target gene product. Protective compound,
Examples of proteins, mimetopes, nucleic acid molecules, and inhibitors are disclosed herein, for example. It is within the scope of this invention that the inhibitor composition may include one or more protective compounds.

A suitable inhibitor of the activity of a target gene product is usually one that binds to, or otherwise interacts with, or otherwise modifies the active site of the product so that the active site of said product. Includes compounds that interact directly with. Inhibitors of the product can also interact with other regions of the product and inhibit its activity, for example by allosteric interactions. An inhibitor of a product binds or interacts with the product, and thus the product and / or
Alternatively, it is identified by its ability to inhibit HIV infection.

For NADH dehydrogenase, many small molecule inhibitors are available in the art. Suitable inhibitors can be used in the methods of the invention. Establishing an in vitro assay,
DiC6, a pigment that accumulates in mitochondria and cytoplasmic membranes, depending on mitochondrial functional activity and ATP concentration, can be used to screen for additional NADH dehydrogenase complex I inhibitors by measuring the membrane potential of living cells. (Methods in Enzymology, 1995, 260
: 448). Compounds are selected for their ability to reduce membrane potential by methods known in the art. NADH dehydrogenase inhibitors include amital, anonine VI, aurakin A, aurakin B, aureosin, benzimidazole, blactin, capsaicin, ethoxyformic anhydride, ethoxyquin, phenproxymate, mofarotene (Ro40-8757; arotinoid), morbizarin, and mixerxamide PI. , Ochibarin (Anonaseosu acetogenins), pethidine, Feng aramid A _2, phenoxy Sun, piericidin A, p-chloromercuribenzoate, ranolazine (RS-forty-three thousand two hundred eighty-five) Roriniasachin -1, Roriniasachin -2, rotenone, Sukuamoshin, and Chiagazoru (It Singer Et al., 1992, Mol.
． Mechan. in Bioenergetics, Chapter 6, Section 153; De
gli et al., 1994, Biochem. J. 301: 161; Friedric
h et al., 1994, Eur. J. Biochem. 219: 691; Uchida
Et al., 1994, Int. J. Cancer 58: 891; Wyatt et al., 199.
5, Biochem. Pharmacol. 50: 1599; Shimomur
a et al., 1989, Arch. Biochem Biophys. 270: 573
), But is not limited thereto.

Examples of inhibitors of 2-oxoglutarate dehydrogenase are described by Majamaa et al.
1985, Biochem. J. 229: 127-133.

Examples of inhibitors of protein tyrosine phosphatase include, but are not limited to, ortho-vanadate, pervanadate, sodium vanadate, and oxidized phenylarsine. Inhibitors of protein tyrosine phosphatase
fi et al., 1994, Proc. Natl. Acad. Sci USA 91: 1
0868-10872 and Lund-Johansen et al., 1996, Cyt.
Ometry 25: 182-190. Examples of inhibitors of EF-1α include, but are not limited to, quercetin (3,3 ′, 4′5,7-pentahydrodiflavone) and didemnin B.

Once a cellular gene has been identified as a potential important target to support the HIV life cycle, assay systems can be used to select additional compounds such as anti-HIV therapeutics. , Can be established for the screening and selection of additional compounds as anti-HIV therapeutics based on their ability to down regulate the expression of genes or inhibit the activity of their gene products. For example, cell lines naturally expressing or transfected with the gene of interest can be incubated with various compounds. Decreasing the expression of the gene of interest or inhibiting the activity of its encoded product can be used as an indicator that the compound is effective in inhibiting the expression and / or function of said gene. The compound was retested in OM10.1 cells or other assays such as proliferative HIV infection to confirm its activity against HIV infection. These compounds can be screened from known organic compounds, peptide library products and chemical combinatorial library products.

One embodiment of the present invention is a method of identifying compounds that can inhibit HIV infection.
The method comprises contacting, eg, combining or mixing, an isolated cell-derived protein with a putative inhibitory compound under conditions where the protein is active in the absence of the compound (a), and (b) the putative inhibitory compound. Determines whether to inhibit the activity of the cell-derived protein. Putative inhibitory compounds to screen include small organic molecules,
Includes antibodies (including their mimetopes) and substrate analogs. Methods of determining the activity of cell-derived proteins are known to those of skill in the art.

According to the present invention, cell-derived nucleic acid molecules, especially GSE nucleic acid molecules, can be used to design polypeptides or peptides that can block HIV infection. Methods of testing inhibitory polypeptides or peptides for use as therapeutic compounds include (a) delivering a putative inhibitory peptide to cells susceptible to HIV infection; and (b) said peptide being HI.
The step of determining the ability to prevent V infection can be included. Disclosed herein are methods of delivering HIV and determining HIV infection.

One preferred embodiment of the present invention uses the protective compounds of the present invention to
Protecting animals from infection. Disclosed herein are preferred protective compounds of the invention. Additional protection can be obtained by administering additional protective compounds, including other agents known to block HIV infection.

Protective compounds, including cell-derived nucleic acid molecules, were obtained from bone marrow or migrating peripheral blood, CD
Introduced into any HIV sensitive host cell such as 4 ⁺ T cells or hematopoietic progenitor cells such as CD34 ⁺ cells by any DNA transfer technique known in the art such as electroporation, transfection or transduction, The cells can then be transplanted into a recipient. When a progenitor cell containing a cell-derived nucleic acid molecule differentiates in vivo, the progenitor cell expresses the nucleic acid molecule product and becomes resistant to HIV. Preferably, the cell-derived nucleic acid molecule is a GSE nucleic acid molecule of the present invention.

Alternatively, cell-derived nucleic acid molecules can be directly administered in vivo using gene therapy expression vectors. In particular, the molecule has been identified in both HIV infected and uninfected individuals,
It can be delivered or introduced into CD4 ⁺ T cells to protect against the development of HIV infection. Recombinant molecules can also be introduced into stromal cells, including macrophages.

Alternatively, cell-derived nucleic acid molecules can be delivered to target cells by non-viral delivery systems. For example, GSE nucleic acid molecules can be reconstituted into liposomes for delivery to susceptible cells. Liposomes are spherical lipid bilayers with an aqueous interior. All molecules present in aqueous solution at the time of liposome formation (in this case, oligonucleotides)
Are incorporated into this aqueous interior. The liposome contents are protected from the external microenvironment, and because the liposomes fuse with the cell membrane, they are efficiently delivered into the cytoplasm without the need to neutralize the negative charge of the oligonucleotide.

Methods of introducing cell-derived nucleic acid molecules into cells or tissues include insertion of naked nucleic acid molecules, ie by injection into the tissue, introduction of GSE in vitro into cells, ie for use in autologous cell therapy, This includes the use of vectors such as viruses, retroviruses, phages or plasmids, or techniques such as electroporation which can be used in vivo or in vitro.

The protective compounds of the present invention can be formulated and administered by a variety of means, including systemic, localized or topical administration. Formulation and administration technology is "Remington's Pharmaceutical Sciences"
Mack Publishing Co. , Easton, PA. The dosage form can be chosen to maximize delivery to the desired target site in the body.

For systemic administration, injection routes include but are not limited to intramuscular, intravenous, intraperitoneal, and subcutaneous. The cell-derived nucleic acid molecule of the present invention is formulated in an aqueous solution, preferably a physiologically compatible buffer such as Hank's solution, Ringer's solution, or saline buffer. Furthermore, cell-derived nucleic acid molecules can be formulated in solid or lyophilized form and then redissolved or suspended immediately prior to use.

NADH dehydrogenase, 2-oxoglutarate dehydrogenase, M2-type pyruvate kinase / cytosolic thyroid hormone binding protein, calnexin, ADP-ribosylation factor 3, eukaryotic initiation factor 3, protein tyrosine phosphatase, herpesvirus-associated ubiquitin-specific Protease, eukaryotic initiation factor 4B
, CD44, phosphatidyl-inositol 3 kinase, elongation factor 1α, bone morphogenetic protein-1, double-strand break DNA repair gene protein, rat guanine nucleotide release protein, antiproliferative factor (BTG-1), lymphocyte-specific protein 1 , Protein phosphatase 2A, squalene synthetase, eukaryotic release factor 1, GTP binding protein, importin β subunit, cell adhesion molecule L1, U-snRNP-associated cyclophilin, lecepin, Arg / Abl interacting protein (ArgBP2A), keratin-related Protein, p18 protein,
p40 protein, glucosidase II, α enolase, macrophage inflammatory protein 1α, translation control tumor protein 1 (TCTP1), BBC1, Nef interacting protein, Na ⁺ -D-glucose cotransport regulator gene protein,
hsp90 chaperone protein, FK506 binding protein A1, Rox, β
Protective compounds that inhibit the expression of genes encoding signal sequence receptors, adult tumor primordium proteins and cell surface heparin binding proteins, and inhibitors of this encoded product, alone or in human patients in need of such treatment. The inhibitor can be administered in a pharmaceutical composition mixed with a suitable carrier or excipient (s). A therapeutically effective amount means an amount of the compound sufficient to result in inhibition of HIV infection relative to its pre-treatment condition. The technology for formulating and administering the compounds of the present application is described in "Remington's Pharmaceutical Sciences" Mac.
k Publishing Co. You can find it in Easton, PA.

Suitable routes of administration are, for example, oral, rectal, transmucosal, transdermal, or enteral administration; intramuscular, subcutaneous, intramedullary injection, as well as intrathecal, direct intraventricular, intravenous, Parenteral delivery including intraperitoneal, intranasal, or intraocular injection may be included. Alternatively, the compound may be administered locally, rather than systemically, eg, by depot injection of the compound directly into a specific tissue, often in a depot or sustained release formulation.

Furthermore, the compounds can be administered in a targeted drug delivery system, eg, in liposomes and / or conjugated with cell-specific antibodies. The liposomes and cell-specific antibodies target HIV-infected cells and are selectively taken up by HIV-infected cells.

The pharmaceutical compositions of the present invention may be manufactured in a manner known per se, for example by conventional mixing, dissolving, granulating, sugar-coating, gelatinizing, emulsifying, encapsulating, encapsulating or lyophilizing processes. .

The pharmaceutical compositions used according to the invention are therefore one or more physiologically containing excipients and auxiliaries, which facilitate the processing of the active compounds into pharmaceutically usable preparations. Can be formulated by a conventional method using an acceptable carrier. Proper formulation is dependent upon the route of administration chosen.

For injection, the compounds of the invention can be formulated in a suitable aqueous solution, such as a physiologically compatible buffer such as Hank's solution, Ringer's solution, or saline buffer. For transmucosal and transdermal administration, penetrants appropriate to the barrier of penetration are used in the formulation. The penetrants are generally known in the art.

For oral administration, the compounds can be formulated by combining the active compounds with pharmaceutically acceptable carriers well known in the art. The carriers allow the compounds of the invention to be formulated as tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions, etc., for oral ingestion by the patient to be treated. Pharmaceutical preparations for oral use include solid excipients, optionally crushing the resulting mixture, adding suitable auxiliaries, if desired, and then processing the granule mixture, tablets or dragées. It can be obtained by obtaining the core. Suitable excipients are bulking agents such as sugars, including in particular lactose, sucrose, mannitol or sorbitol;
Maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethyl-cellulose,
Cellulose preparations such as sodium carboxymethylcellulose and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or its salts, such as sodium alginate.

Dragee cores are provided with suitable coatings. To this end, a concentrated sugar solution, a lacquer solution and a suitable organic solvent or solvent mixture, which may optionally comprise gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, are provided. Can be used. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Push fit capsules
The active ingredient can be included in admixture with filler such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added. All formulations for oral administration should be in dosages suitable for such administration. For buccal administration, the composition may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds used according to the invention are conventionally provided with suitable propellants, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. Used to be delivered in the form of an aerosol spray from a pressure pack or nebulizer. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mixture of the compound and a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration by injection, eg by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, eg, in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oils or aqueous vehicles and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. further,
Suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use.

The compounds may be formulated in rectal compositions such as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation.
Such long acting formulations may be administered by implant (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymers or hydrophobic materials (eg, as an acceptable emulsion in oil) or ion exchange resins, or as sparingly soluble derivatives, such as sparingly soluble salts.

The hydrophobic compound pharmaceutical carrier of the present invention is a co-solvent system comprising benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous layer. Cosolvent system is VP
It may be a D cosolvent system. VPD is 3% w / volume increased with absolute ethanol
A solution of w benzyl alcohol, 8% w / v apolar surfactant polysorbate 80, and 65% w / v polyethylene glycol 300. VPD co-solvent system (
VPD: 5W) consists of VPD diluted 1: 1 with 5% dextrose in water. This co-solvent system dissolves hydrophobic compounds well and, as such, has low toxicity upon systemic administration. Naturally, the proportion of co-solvent systems can vary considerably without destroying its solubility and toxic characteristics. Furthermore, the identity of the co-solvent component may vary; for example, other less toxic non-polar surfactants may be used in place of polysorbate 80; the fraction size of polyethylene glycol may vary; other biocompatibility The polymer can be replaced with polyethylene glycol, eg polyvinylpyrrolidone; other sugars or polysaccharides can be substituted with dextrose.

Alternatively, other delivery systems for hydrophobic pharmaceutical compounds can be used. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethyl sulfoxide can also be used, but usually at the cost of high toxicity. In addition, the compounds can be delivered using sustained release systems, such as semipermeable matrices of the hydrophobic polymers containing the therapeutic agent. Various sustained release materials have been established and are known to those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Additional strategies for protein and nucleic acid stabilization can be used, depending on the chemical nature and biological stability of the therapeutic agent.

The pharmaceutical compositions may also include suitable solid or gel phase carriers or excipients.
Examples of the carrier or excipient include calcium carbonate, calcium phosphate, various sugars,
Including but not limited to starch, cellulose derivatives, gelatin, and polymers such as polyethylene glycol.

The compounds of the present invention can be provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts can be formed with many acids including, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid and the like. Salts tend to be more soluble in aqueous or other protic solvents than the corresponding free base form.

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredient is contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent or reduce the occurrence of pre-existing symptoms in the subject being treated. Determination of an effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example EC5 as determined in cell culture
Circulating concentration range including 0 (effective dose for 50% increase), ie, half maximal inhibition of HIV replication, retention of CD4 expression, retention of virus p24 or gp120 as assayed by infected cells. The dose can be formulated in animal models to achieve a concentration of the test compound that reduces the concentration and prevents syncytia formation. Such information can be used to more accurately determine useful doses in humans.

The toxicity and therapeutic efficacy of the compounds can be determined, for example, for the determination of LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population), It can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
The dose ratio between toxic and therapeutic effects is the therapeutic index and the LD50 and ED50
It can be expressed as the ratio between. Compounds that exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration used. The specific formulation, administration route, and dose can be selected by the individual physician in view of the patient's condition (eg, Fingl et al., 1975, "Pharmacological basis of therapeutic agents", Chapter 1, paragraph 1).
.

Dosage amount and interval can be adjusted individually to provide plasma levels of the active moiety sufficient to maintain its inhibitory effect. A typical patient dose for systemic administration is 100-2
The range is 000 mg / day. If described in terms of patient body surface areas, usual dosages range from 50~910mg / m ² / day. Normal mean plasma levels are
It should be maintained within 0.1-1000 μM. In the case of local administration or selective uptake, the effective local concentration of the compound may not be related to plasma concentration.

The amount of compound administered will, of course, be dependent on the subject being treated, the subject's body surface area, the severity of the affliction,
It will depend on the mode of administration and the judgment of the prescribing physician.

The following examples are provided for purposes of illustration and are not intended to limit the scope of the invention. The invention is not limited in scope by the illustrated embodiments, which is taken as a description of individual aspects of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications fall within the scope of the appended claims.

[0165]   All publications cited herein are incorporated by reference in their entirety.

EXAMPLES Example 1 This example describes the isolation and identification of human cell-derived GSEs that exhibit HIV suppressive activity.

A. Generation of RFE Library Two RFE libraries were prepared according to the method described by Gudkov et al.
Generated from cDNAs of two human cell lines (1994, Proc. Natl. Ac
ad. Sci. USA 91: 3744). CDNA prepared from HL-60 cells and HeLa cells was partially digested with DNaseI in the presence of Mn ⁺⁺ (S
Ambrook et al., 1989, Molecular Cloning: Experimental Manual, Cold Spring Harbor Laboratory, N. et al. Y. ). Under these conditions, DNase I is known to produce the majority of double-strand breaks.

The resulting fragment was repaired with the Klenow fragment of DNA polymerase I and T4 polymerase and ligated to a synthetic double stranded adapter. The 5'adapter (SEQ ID NOS: 21 and 22) was: 5'-CTCGGAATTCAAGCTTATGGATGGATGGG-3 '3'-CCTTAAGTTCGAATAACTTACACC-5'.

The 3'adapter (SEQ ID NOs: 23 and 24) is 5'TGAGTGAGTGA
ATCGATGGATCCGTCT-3'3'-ACTCACTCACTTAGCTACCTAGGGCAGATCCT-5
'Met.

For libraries made from HL-60 cells, mRNA from uninduced cells
Were first subtracted from the mRNA from TNF-α-induced cells. The subtracted HL-60 library was obtained from Coche et al., 1994, Nucleic Ac.
ids Res. 22: 1322-1323 shows a modification of the procedure. Tracer mRNA was purified from HL-60 cells containing the LNCX plasmid at various time points after induction with TNF-α. The LNCX gene was used as an internal standard to monitor the intensity of sequences present in the tracer after subtraction. MRNA isolated from induced and non-induced cells was isolated separately from oligo dT magnetic beads (
(Obtained from Dyna) and the first cDNA strand was synthesized using reverse transcriptase and oligo dT as primers. The RNA strand was hydrolyzed and the second strand was synthesized on the induced population using a primer containing 3 ATG codons and 10 random nucleotides at the 3'end. The single-stranded cDNA fragment was annealed to excess driver cDNA attached to magnetic beads. This procedure was repeated several times until quite a lot was found in the spiked LNCX sequences. Single chain D
The final population of NA (ssDNA) molecules was amplified using a primer with 3 TGA codons in all 3 reading frames with 10 random nucleotides on the 3'end. The obtained population of cDNA fragments was designated as LNCX.
Cloned into. This step was performed to enhance the mRNA encoded by cellular genes, which may be important in supporting specific steps of the HIV life cycle, in order to compensate for the low retroviral transduction efficiency into OM10.1 cells. This library was represented by 10 ⁶ recombinant clones.

Random fragments of cDNA from HeLa cells were subjected to standardization procedures to provide uniform and abundant different DNA sequences (Gudkov and Roninson, 1
997, Methods in Molecular Biology 69:
221 and N.N. Humana Press, Totowa, J.). This procedure was used to increase the probability of isolating GSE from rare cDNAs. Because all poly A ⁺ R
This is because NA is a mixture of heterogeneously presented sequences. Briefly, this method was denatured by first boiling 20 μg of cDNA for 5 minutes in 25 μl of TE buffer, followed by immediate cooling on ice. Then 25 μL of 2 ×
Hybridization solution was added and the mixture was divided into 4 aliquots, 12.5 μL each, in Eppendorf tubes. One to two drops of mineral oil were added to each sample to avoid evaporation and the tubes were placed in a 68 ° C water bath for annealing. 1
One tube was frozen every 12 hours. After the last time point, each annealing mixture was diluted with water to a final volume of 500 μL and hydroxyapatite (HAP) was added.
Chromatography. The HAP suspension equilibrated with 0.01 M phosphate buffered saline (PBS) was placed in an Eppendorf tube so that the volume of the HAP pellet was about 100 μL. The tubes with HAP and all solutions used below were preheated and kept at 65 ° C. Excess PBS was removed and diluted annealing solution was added. After mixing by shaking in a 65 ° C. water bath, the tubes were left in the water bath until a HAP pellet formed (using a 15 second centrifugation, to avoid damage to HAP crystals, The pellet was collected at 1000 g or less in a centrifuge). Carefully add the supernatant to 1 mL of preheated 0.0
Replaced with 1M PBS and the process was repeated. HAP to elute ssDNA
The pellet is treated with 500 μL of P at the determined single-stranded elution concentration, eg 0.16M.
Suspended in BS, the supernatant was collected and the process was repeated. The supernatants were combined and trace HAP was removed by centrifugation. Concentrate ssDNA by centrifugation, 3 times, 1 mL
Washed with Centricon-100. The isolated ssDNA sequence was amplified by PCR using the sense primer from the adapter, using a minimum number of cycles, yielding 10 μg of product. The size of the PCR product was confirmed to be within the desired range (200-500 bp). Normalization quality was tested by Southern blot or slot-blot hybridization with ³² P-labeled probe for high, medium and low expressed genes using 0.3-1.0 μg of standardized cDNA / lane. . For high expression genes, β-actin and β-tubulin cDNA were used as probes, for medium expression genes, c-myc and topoII cDNA, and for low expression genes, c-fosDNA was used. The cDNAs isolated after various annealing times were compared to the original non-standardized cDNA.
The probes certainly had similar size and specific activity. The best standardized ssDNA fraction was used, which in large scale PCR amplification yielded the most uniform signal intensity with different probes, synthesizing at least 20 μg of product for cloning. More ssDNA template was used to obtain the desired amount by scaling up the number of PCRs or reaction volumes.

After normalization, the mixture of fragments ligated to the adapter was BamHI and EcoR.
I-digested, column-purified, digested with EcoRI and BamHI, the retroviral vector pLNCX (Miller and Rosman, 1989, Bi.
oTechniques 7: 980-990) or pLNGFRM. In the pLNGFRM vector, the neo ^r gene has a partially truncated low affinity N
The same as the pLNCX vector except that it is replaced with the GFR gene. pLN
Cells transduced with GFRM express partially cleaved receptors on their surface, which can be readily selected by anti-NGFR antibody and FACS. The ligate mixture was transformed into E. E.coli. All plasmids were purified from approximately 100,000 recombinant clones. The size distribution of the cloned fragments was tested by PCR amplification using primers derived from vector sequences flanking the adaptor.

B. Cell Lines and Reagents OM10.1 cells are available as CRL 10850 from the American Type Culture Collection, Manassas, Va. (Butera, US Pat. No. 5,256,534). CEM-ss cells are NIH AIDS Re
search and Reference Reagent Program
Available under the serial number 776. HIV-1 _SF2 is NIH AIDS
Research and Reference Reagent Prog
It is available from Ram under the serial number 275. HL-60 cells are American
Available as CCL240 from Type Culture Collection. HIV
-1 _IIIB is available from AIDS Program as serial number 398.

Anti-CD4 (Q4120PF) and anti-p24 (KC-57FITC) antibodies were
Purchased from Sigma and Coulter respectively. TNF-α was obtained from Boehringer Mannheim. G418 was purchased as Geneticin from Gibco / BRL. Anti-NGFR antibody was obtained from ATCC (HB-8737) as hybridoma 2
0.4 (US Pat. Nos. 4,786,593 and 4,855,241). Two anti-CD4 antibodies (L77 and L120) were labeled by Becton Di
Obtained from Ckenson.

C. Transduction and GSE Selection A library prepared from HL-60 cells according to the method in Section A of this Example was
The packaging cell line PA317 (ATCC CRL 9078) was transfected and converted to retrovirus for infection of OM10.1 cells. pLN
The CX vector library was co-cultured and selected using G418. pLNG
The FRM vector library was spinoculated (90 at 1200 xg
Centrifuge for minutes in the presence of filtered retroviral supernatant; Dunn et al., 19
99, Gene Therapy 6: 133-137), N
Selected by FACS sorting of GFR ⁺ populations. After selection, OM10.1 cells with the entire RFE library were incubated with 10 U / mL TNF-α at 37 ° C. and after 24 hours stained with antibody and sorted for CD4 expression.
Genomic DNA from CD4 ⁺ cells was purified using vector-derived primers,
It was used for amplification of the insert by PCR. The amplified mixture was digested with EcoRI and BamHI and cloned back into the retroviral vector. The selection was repeated for a further round.

In addition, a standardized RFE library made from HeLa cells was loaded with CEM-s.
s cells were transfected and the neo resistant population was given a TCID ₅₀ of 3000/1.
^Six cells were infected with HIV-1 _IIIB . This RFE library is
Presented by 50 × 10 ⁶ independent recombinant clones. Four and seven days after infection, purified anti-CD4 monoclonal antibody L77 (Becton Dicki
(obtained from Nson) at 5 μg / mL was added to prevent the formation of syncytia. The formation of syncytia is believed to be prevented by blocking the interaction between gp120 expressed on the surface of infected cells and CD4 on the surface of uninfected cells. Antibody L77 does not prevent HIV infection of cells. 10 to 12 days after infection, the CD4 ⁺ , p24 ⁻ cell population showing non-infected cells was selected. Genomic DNA from CD4 ⁺ , p24 ⁻ cells was purified and used for PCR amplification of inserts using vector-derived primers. The amplified mixture was digested with EcoRI and BamHI and cloned back into the retroviral vector. The selection was repeated for a further round.

D. Immunofluorescence and Flow Cytometry For immunofluorescent staining of CD4 ⁺ cells for selection, 10 ⁷ cells were treated twice with assay buffer (500 ml PBS, 1 ml 0.5 mM EDT pH8).
A, washed with 0.5 ml of 10% sodium azide and 10 ml of fetal bovine serum) and resuspended in 500 μL of PBS, to which 50 μL of anti-CD4 antibody (Q4) was added.
120PE, obtained from Sigma, St. Louis, MO). After incubating at 4 ° C. for 30 minutes, 5 mL of assay buffer was added and the cells were added at 1200 rp.
Centrifuge for 4 minutes at m. Cells are washed twice with assay buffer, then F
It was selected by ACS. The above procedure was performed under sterile conditions.

To determine p24 expression in HIV infected cells, cells were first washed twice with assay buffer. Approximately 10 ⁶ cells were suspended in 100 μL of assay buffer and 2 mL of Ortho PermeaFix.
Mix with the solution (obtained from Ortho Diagnostics) and incubate for 40 minutes at room temperature. After centrifugation at 1200 rpm for 4 minutes at 4 ° C., the cells were washed with 2 ml of wash buffer (500 mL PBS, 25 mL fetal bovine serum, 1.5 mL).
% Bovine serum albumin and 0.0055% EDTA) for 10 minutes at room temperature. After centrifugation, the cells are resuspended in 50 μL wash buffer for 4 minutes for 20 minutes.
° C. 1: 500 were mixed with IgG _2a dilution and then incubated for 30 minutes at 4 ° C. with anti-p24 antibody of 5~10μL (KC57-FITC, available from Coulter). The cells were then washed twice with wash buffer and analyzed by flow cytometry.

For selection of NGFR ⁺ cells, 10 ⁷ cells were washed twice with assay buffer, resuspended in 200 μL assay buffer and 5% normal mouse serum and 2 mL anti-NGFR −. PE antibody was added. After incubating at 4 ℃ for 30 minutes, 5mL
Assay buffer was added and cells were centrifuged at 1200 rpm for 4 minutes. Cells were washed twice with assay buffer and then sorted by FACS.

E. GSE Recovery and Sequence Analysis Genomic DNA was prepared by cell pelleting 0.1% Triton X in 1 × PCR buffer.
Isolated from a population of selected OM10.1 or CEM-ss cells with putative GSE by resuspending in -100, 20 μg / mL proteinase K, incubating at 55 ° C. for 1 hour, and boiling for 10 minutes. . Genomic DNA,
The vector-derived primers were used for PCR amplification, cloned into retroviral vectors, and cloned into E. coli using techniques known in the art. E.coli. Individual plasmids were transformed into E. coli using the Qiagen Plasmid Kit. coli
Purified from clones. The insert was sequenced by the dideoxy procedure (obtained from Pharmacia Biotech's AutoRead Sequencing Kit) to obtain a Pharmacia LKB A. L. F. Flush with a DNA sequencer. The array is
Analyzed using the DNASTAR program.

F. Identification of GSEs Two selection strategies were used to isolate human-derived GSEs with HIV suppressor activity from RFE libraries. One strategy was chosen for GSE to suppress the productive infection of cells by HIV. The second strategy was selected for GSE that suppressed the induction of latent retrovirus in OM10.1 cells. OM10.1 cells
When treated with NF-α and stained with an antibody specific for the cell surface molecule CD4, rapid C
A decrease in D4 expression was observed (Figure 1). In contrast, the majority of non-induced OM10.1 cells maintained CD4 expression. Activation of latent virus in OM10.1 cells by TNF-α results in production of the viral protein gp120,
It is believed that it binds to cytosolic CD4, thereby preventing its translocation to the cell surface. Loss of CD4 ⁺ OM10.1 cells is also associated with increased production of viral protein p24 in cells following TNF-α induction.

Identifying the GSE obtained from the cDNA displaying the expressed human cell gene,
For decoding, using HIV provirus activation in OM10.1 cells,
It was isolated from an RFE library made from HL-60 cells. After transfection of the entire library into the packaging cell line, retroviruses with the library were used to co-culture or spinoculate OM10.
． One cell was infected and NGFR selection was performed to ensure retention of the viral vector. Treatment of infected cells with TNF-α resulted in a small number of residual CD4 ⁺ cells being treated with anti-CD4.
Detected by antibody and sorted by FACS. GSE contained in these cells
Was recovered by PCR amplification and its nucleotide sequence was determined.

A total of 20 GSEs were isolated using OM10.1 and CEM-ss cells by the two selection strategies described above. Six of these GSEs are NADH
CDNA of a gene encoding different subunits of the dehydrogenase enzyme complex
It was shown to have considerable sequence identity with NA. For example, CF-315 (
SEQ ID NO: 1) is an antisense molecule that inhibits HIV replication, GSE, which in its sense orientation has sequence identity with the gene encoding ND6, a subunit of the mitochondrial enzyme NADH dehydrogenase. Chomyn et al., 1988, Science 234: 614). CF-31
5 was further shown to protect uninfected human T cells from proliferative HIV-1 infection (FIG. 2). In this experiment, a retroviral vector, pLNGFRM, containing a CF-315 nucleic acid molecule was introduced into CEM-ss cells and then NGFR.
A selection was made. A vector containing plasmid DNA (designated LNGFRM) was used as a negative control. NGFR ⁺ cells were 99% CD4 ⁺ , then they were infected with HIV-1 _SF2 with a TCID ₅₀ of 1000. Infected cells, 1 of infection
Removed after 1, 14, 18, 21, 25, 28, 32, 35 and 39 days, H
As an index of IV infection, it was stained with a fluorescent anti-p24 antibody. FIG. 2 shows that CF-315 shows that human T by HIV-1 _SF2 was compared to the negative control of vector plasmid DNA.
It is shown to inhibit infection of cells.

CF-319 (SEQ ID NO: 2) represses HIV replication in the sense orientation and NADH
GSE, which has considerable sequence identity with another portion of the gene encoding the ND6 subunit of dehydrogenase. CF-101 (SEQ ID NO: 3) also exhibits its HIV suppressive activity in the sense orientation and has considerable sequence identity with the gene encoding the ND2 subunit of NADH dehydrogenase (Chomyn et al.,
1985, Nature 314: 592). CF-117 (SEQ ID NO: 4) is
It suppresses HIV infection as an antisense molecule and, in its sense orientation, has considerable sequence identity with the gene encoding the ND6 subunit of NADH dehydrogenase. CF-025 (SEQ ID NO: 5) suppresses HIV infection as an antisense molecule and, in its sense orientation, has considerable sequence identity with the gene encoding the ND2 subunit of NADH dehydrogenase. CF-128 (SEQ ID NO: 6)
Suppresses HIV infection in the sense orientation and has considerable sequence identity with the gene encoding the ND4 subunit of NADH dehydrogenase.

Both selection strategies lead to GSEs with considerable sequence identity with different subunits of NADH dehydrogenase, an enzyme complex that plays an important role during HIV infection, and thus this complex or its Methods of down-regulating expression of either subunit can be used to block HIV replication in infected cells.

Selection of GSEs from a HeLa cell library using productive infection of CEM-ss cells isolated 12 additional nucleic acid molecules with significant sequence identity to other cellular genes. It was CF-004 (SEQ ID NO: 7) suppresses HIV infection as a sense molecule and has considerable sequence identity with the gene encoding human 2-oxoglutarate dehydrogenase (Koike, 1995, Gene 1).
58: 261-266; Koike et al., 1992, Proc. Natl. Aca
d. Sci. USA 89: 1963-1967). CF-113 (SEQ ID NO: 8
) Suppresses HIV infection as a sense molecule, and human M2 type pyruvate kinase /
It has considerable sequence identity with the gene encoding the cytosolic thyroid hormone binding protein (Kato et al., 1989, Proc. Natl. Acad. Sci.
． USA 86: 7861-7865). CF-204 (SEQ ID NO: 9) suppresses HIV infection as an antisense molecule and has considerable sequence identity with the gene encoding human calnexin in the sense orientation (David et al., 1993, J. Am.
Biol. Chem. 268: 9585-9592). CF-001 (SEQ ID NO: 10) suppresses HIV infection as an antisense molecule and has considerable sequence identity in its sense orientation with the gene encoding human ADP-ribosylation factor 3 (
Tsai et al., 1991, J. Am. Biol. Chem. 266: 23053-230
59). CF-273 (SEQ ID NO: 11) suppresses HIV infection as a sense molecule and has considerable sequence identity with the gene encoding human eukaryotic translation initiation factor 3 (Merrick and Hershey, 1996, Translationa).
I Control, Cold Spring Harbor, NY, 31-69). C
F-311 (SEQ ID NO: 12) suppresses HIV infection as an antisense molecule,
In its sense orientation, it has considerable sequence identity with the gene encoding human protein tyrosine phosphatase (Kim et al., 1996, Oncogene 13).
: 2275-2279). CF-313 (SEQ ID NO: 13) suppresses HIV infection as a sense molecule and has considerable sequence identity with the gene encoding human protein tyrosine phosphatase (Keyse and Emslie, 1992,
Nature 359: 644-647). CF-210 (SEQ ID NO: 14) is
It suppresses HIV infection as a sense molecule and has considerable sequence identity with genes encoding herpesvirus-associated ubiquitin-specific proteases (Everet
et al., 1997, EMBO J. et al. 16: 566-577). CF-266 (SEQ ID NO: 15) suppresses HIV infection as an antisense molecule and shares significant sequence identity with the gene encoding human eIF4B in its sense orientation (Milb.
urn et al., 1990, EMBO J. et al. 9: 2783-2790). CF-302
(SEQ ID NO: 16) suppresses HIV infection as an antisense molecule and, in its sense orientation, has considerable sequence identity with the gene encoding human CD44 (St.
amenkovic et al., 1991, EMBO J. et al. 10: 243-248). C
F-317 (SEQ ID NO: 17) suppresses HIV infection as an antisense molecule,
In its sense orientation, it has considerable sequence identity with the gene encoding human phosphatidylinositol 3-kinase (Volinia et al., 1995, EMBO.
J. 14: 3339-3348). CF-286 (SEQ ID NO: 18) suppresses HIV infection as a sense molecule and has considerable sequence identity with the gene encoding human elongation factor-1α (Uetsuki et al., 1989, J. Biol. Che.
m. 264: 5791-5798).

Furthermore, the two isolated GSEs did not share sequence homology with any known genes by sequence comparison. CF-061 (SEQ ID NO: 19) was selected on OM10.1 cells. CF-280 (SEQ ID NO: 20) was selected on CEM-ss cells. These two nucleic acid molecules represent parts of an unknown human cell gene that play a role in supporting HIV infection.

FIG. 3 shows the above-mentioned 4 in preventing the proliferative infection of CEM-ss cells by HIV.
2 illustrates the capabilities of two exemplary GSEs. GSE was isolated as a fragment of a distinct cellular gene, but they all inhibited HIV infection as indicated by a decrease in p24 levels in infected cells when compared to controls. Furthermore, FIG. 4 shows the HIV inhibitory activity of CF-001, which functions as an antisense molecule.

Using the productive infection of CEM-ss cells, selection of GSE from a HeLa cell library isolated 21 additional nucleic acid molecules with significant sequence identity to other cellular genes. It was CF-537 (SEQ ID NO: 25) interferes with the HIV life cycle in the sense orientation, human bone morphogenetic protein-1 (BMP1-6; Wo
Zney et al., 1988, Science 242: 1528-1534) is a GSE with considerable sequence identity to the gene encoding it. The complement of SEQ ID NO: 25 is represented by SEQ ID NO: 26. CF-320 (SEQ ID NO: 27) interferes with HIV infection in the sense orientation and causes double-strand break DNA repair gene protein (McKay).
Et al., 1996, Genomics 36: 305-315), which is a GSE with considerable sequence identity to the gene. The complement of SEQ ID NO: 27 is represented by SEQ ID NO: 28. CF-321 (SEQ ID NO: 29) interferes with HIV replication in the sense orientation and induces rat guanine releasing protein (Burton et al., 1993, Nat.
ure 361: 464-467) is a GSE with considerable sequence identity to the gene encoding it. The complement of SEQ ID NO: 29 is represented by SEQ ID NO: 30. C
F-322 (SEQ ID NO: 31) interferes with HIV replication in the sense orientation and is an anti-growth factor protein (BTG-1; Rouault et al., 1992, EMBO J, 11:
1663-1670), which has significant sequence identity with the gene encoding GS
It is E. The complement of SEQ ID NO: 31 is represented by SEQ ID NO: 32. CF-332 (
SEQ ID NO: 33) interferes with HIV replication as an antisense molecule and is associated with lymphocyte-specific protein 1 (Jongstra-Bilen et al., 1990, J. Immun.
ol. 144: 1104-1110) is a GSE with considerable sequence identity to the gene encoding the same. The complement of SEQ ID NO: 33 is represented by SEQ ID NO: 34.
CF-335 (SEQ ID NO: 35) interferes with HIV replication as an antisense molecule and is a protein phosphatase 2A protein (Mayer et al., 1991, Bio.
Chemistry 30: 3589-3597), which is a GSE with considerable sequence identity to the gene encoding it. The complement of SEQ ID NO: 35 is SEQ ID NO: 36.
Indicated by. CF-42 (SEQ ID NO: 37) is a GSE that interferes with HIV replication in the sense orientation and has considerable sequence identity with the gene encoding the squalene synthetase protein (Summers et al., Ibid.). The complement of SEQ ID NO: 37 is represented by SEQ ID NO: 38. CF-50 (SEQ ID NO: 39) also has HI in the sense orientation.
It exhibits V-repressive activity and has considerable sequence identity with the gene encoding the squalene synthetase protein (Summers et al., Ibid.). The complement of SEQ ID NO: 39 is represented by SEQ ID NO: 40. CF-527 (SEQ ID NO: 41) interferes with HIV replication and eukaryotic release factor 1 protein (ERF-1; Andjelkovic et al.,
1996, EMBO J .; 15; 7156-7167), which is a peptide derived from those having considerable sequence identity with the gene encoding GES. The complement of SEQ ID NO: 41 is shown by SEQ ID NO: 42. CF-528 (SEQ ID NO: 43) is
GTP-binding proteins (Zigman et al., 1
993, Endocrinology 133: 2508-2514), which is a GSE with considerable sequence identity to the gene encoding it. The complement of SEQ ID NO: 43 is represented by SEQ ID NO: 44. CF-529 (SEQ ID NO: 45) interferes with HIV replication as an antisense molecule, and the importin β subunit protein (
Gorlic et al., 1995, Curr Biol. 5: 383-392) is a GSE with considerable sequence identity to the gene encoding it. The complement of SEQ ID NO: 45 is represented by SEQ ID NO: 46. CF-531 (SEQ ID NO: 47) interferes with HIV replication as an antisense molecule and induces cell adhesion molecule L1 protein (LCAM).
1; Hlavin et al., 1991, Genomics 11: 416-23), which is a GSE with considerable sequence identity to the gene. The complement of SEQ ID NO: 47 is represented by SEQ ID NO: 48. CF-545 (SEQ ID NO: 49) is a GSE that interferes with HIV replication as an antisense molecule and has considerable sequence identity with the gene encoding the U-snRNP-associated cyclophilin protein (Horowitz et al., Ibid.). The complement of SEQ ID NO: 49 is represented by SEQ ID NO: 50.
The complement of SEQ ID NO: 49 is represented by SEQ ID NO: 50. CF-547 (SEQ ID NO: 5
1) is a GSE that interferes with HIV replication as an antisense molecule and has considerable sequence identity with the gene encoding the resepin endoprotease protein (GenBank Accession No. U03644). The complement of SEQ ID NO: 51 is represented by SEQ ID NO: 52. CF-619 (SEQ ID NO: 53) interferes with HIV replication in the sense orientation and is Arg / Abl interacting protein (ArgBP2A; GenBan).
kSE having significant sequence identity with the gene encoding deposit number AF049884). The complement of SEQ ID NO: 53 is represented by SEQ ID NO: 54. CF-
620 (SEQ ID NO: 55) is a GSE that interferes with HIV replication as an antisense molecule and has considerable sequence identity with the gene encoding the keratin-releasing protein (IFN-γ regulation; Flohr et al., Ibid.). The complement of SEQ ID NO: 55 is shown by SEQ ID NO: 56. CF-624 (SEQ ID NO: 57) is HIV in the sense orientation.
GSE, which interferes with replication and has considerable sequence identity with the gene encoding the p18 protein (Zhu et al., Ibid.). The complement of SEQ ID NO: 57 is SEQ ID NO: 5.
Shown by 8. CF-630 (SEQ ID NO: 59) is HI as an antisense molecule.
Interferes with V replication and p40 protein (Mayer et al., 1988, Biochim.
GSE with considerable sequence identity to the gene encoding Biophys Acta 1395: 301-308). The complement of SEQ ID NO: 59 is represented by SEQ ID NO: 60. CF-579 (SEQ ID NO: 61) interferes with HIV replication in the sense orientation and is associated with glucosidase αII protein (GenBank Accession No. AJ0).
GSE, which has considerable sequence identity with the gene encoding 00332). The complement of SEQ ID NO: 61 is shown by SEQ ID NO: 62. CF-287 (SEQ ID NO: 77) interferes with HIV replication in the sense orientation and is a Na ⁺ -D-glucose cotransport regulatory protein (Lambotte et al., 1996, DNA Cell Biol.
15: 769-77), which has considerable sequence identity with the gene encoding GS
It is E. The complement of SEQ ID NO: 77 is represented by SEQ ID NO: 78. CF-622 (
SEQ ID NO: 87) interferes with HIV replication as an antisense molecule and is a Rox protein (Meroni et al., 1997, EMBO J. 16: 2892-2906).
It is a GSE with considerable sequence identity to the gene that encodes. Sequence number 8
The complement of 7 is represented by SEQ ID NO: 88.

Figures 7, 8 and 9 show the ability of the seven exemplary GSEs described immediately above in preventing proliferative infection of CEM-ss cells by HIV. GSE was isolated as a fragment of a distinct cellular gene, but they all blocked HIV infection as indicated by a decrease in p24 levels in infected cells when compared to controls.

Example 2 This example demonstrates inhibition of HIV infection by NADH dehydrogenase inhibitors.

The NADH dehydrogenase inhibitors Amital (obtained from Sigma, St. Louis, MO) and Mofaroten (Uchida et al., 1994, Int. J.C.
ancer 58: 891-897) was diluted in sterile culture medium and used according to the indicated concentrations.

OM10.1 cells were cultured in RPMI1640 glucose-free medium prior to and during incubation with NADH dehydrogenase inhibitor and TNF-α induction. Inhibitors were added to the cells and then induced with TNF-α 1-2 hours later. Expression of CD4 by cells was assessed by anti-CD4 antibody staining and flow cytometry after incubation at 37 ° C for 24 hours.

Human peripheral blood leukocytes (PBL) were isolated using Ficoll-Hypaque density gradient separation. Cells were washed twice and RPMI + 10% FBS + 2% human AB serum + penicillin / streptomycin / glutamine + 100 units / mL IL-.
The cells were resuspended in 2 at a concentration of 0.5 × 10 ⁶ cells / mL. Then, set PBL to 0
． Activated with 5 μg / mL phytohemagglutinin and placed in a humidified incubator at 37 ° C./5% CO ₂ . After activation for 2 days, 10 ⁶ cells, a TCID ₅₀ 1000, with HIV-1 _SF33, various concentrations Mofaroten (0 [mu] M
1 μM, 0.5 μM, 0.1 μM and 0.05 μM).
Separate sets of uninfected samples were also kept as controls under the same concentration of mofarotene. Samples were analyzed by flow cytometry 4 and 6 days post infection. Cells were started for CD3 expression (for T cells). Expression of CD4 and virus p24 and CD4 was then examined by bivariate dot plot.

Two compounds with known NADH dehydrogenase inhibitory activity were selected, as several GSEs were selected and shown to have considerable sequence identity with cellular genes encoding different subunits of NADH dehydrogenase. It was tested for its ability to suppress HIV infection. FIG. 5 shows that amital dose-dependently increased OM1 as a result of its ability to retain CD4 expression by TNF-α-induced OM10.1 cells.
It shows that the induction of latent HIV provirus in 0.1 cells was inhibited. In the same assay, mofarotene, which down-regulates mitochondrial gene expression, also inhibited HIV-1 induction at even lower concentrations (Fig. 6). At 100 nM mofarotene, 80% of the cells retained CD4 expression and more than 80% of the cells remained viable. Moreover, when p24 expression was measured as an indicator by HIV infection, both amital and mofarotene suppressed intracellular p24 levels in treated cells compared to untreated controls.

[0196] NADH dehydrogenase inhibitor, to test the ability to prevent the productive infection of T cells by HIV, the Human PBL were isolated and infected with HIV _SF33 in the presence of Mofaroten various concentrations. CD3 ⁺ T cells, CD4 and viral p2
4 was analyzed and the results are shown in Table 1.

[0197]

[Table 1] Four days after infection, HIV-infected P with or without mofarotene
There were few detectable differences between the BLs. Mofaroten alone is a CD
It did not significantly change the proportion of 4 ⁺ T cells, nor did it change the expression level of CD3 on the cell surface.

[0198] On day 6, with respect to the ratio of ratios and p24 ⁺ CD4 ⁺ T cells CD4 ⁺ T cells, no significant differences were detected. In HIV-infected samples without mofarotene, CD
33% of 4 ⁺ T cells were p24 ⁺ and only 15% of T cells were CD4 ⁺ . In the HIV uninfected control sample, approximately 32% of T cells were CD4 ⁺ , which was
It suggests dramatic CD4 ⁺ T cell depletion due to IV infection. In HIV-infected samples with 1 μM mofarotene, only 10% of CD4 ⁺ T cells were p24 ⁺ .
In addition, approximately 25% of T cells are CD4 ⁺ , which causes mofarotene to
It shows that the inhibition of CD4 ⁺ T cell depletion due to infection was inhibited. The level of protection by mofarotene as measured by the ratio of p24 ⁺ CD4 ⁺ T cells and CD4 ⁺ cells in the CD3 ⁺ T cell population disappeared with decreasing concentrations of mofarotene. Mofaroten alone increased the proportion of CD4 ⁺ T cells in uninfected samples, but had minimal effect (up to 4%). At day 6, mofarotene did not change the expression level of CD3 on the cell surface. Thus, NADH dehydrogenase inhibitors prevented HIV productive infection of primary cultures of human T cells.

Example 3 This example describes the construction of a PBMC library and the isolation of GSE from it.

A. PBMC library Cell-derived random fragment library (RFL) was used as a peripheral blood mononuclear cell (PBMC
) -Derived cDNA. Four buffy coats were obtained from four healthy blood donors, PBMCs were purified by Ficoll gradient centrifugation, then PHA (1
μg / mL). Cells were removed 5, 10 and 24 hours after PHA was added and total RNA was isolated by Trizol extraction. All time points for all donors were then pooled. Polyadenylated mRNA was purified from total RNA by passing through an oligo-dT column. cDNA from polyadenylated RNA to cDNA
Synthesis was performed using the Gibco Superscript Choice system for NA synthesis (Gibco BRL, Rockville, MD). cDNA for Diatche
nko et al. (1996, Proc. Natl. Acad. Sci. USA 93,
6025-6030, 1996), PCR-Select cDNA subtraction kit (CA) based on the suppressive subtractive hybridization method.
Clontech, Palo Alto, CA). The primers used for standardization were: 5'-TAGGGCTCGAGCCGCCACCATG-3 '(Xho5'S
No. 2: SEQ ID NO: 97) 5'-ATCCCTGCAGGTCACTCACTCA-3 '(Sse3'A
No. S1: SEQ ID NO: 98) The standardized random fragment was digested with XhoI and Sse8387I, purified on a Quick Spin column (obtained from Qiagen, Valencia, CA), and the SseI / XhoI of the dicistronic retroviral vector EMCVNgfrMPBIN.
Ligated to the site. This vector is based on Dunn et al. (1999, Gene T).
herapology 6: 130-137) based on LXSNgfr. The modifications are based on Moloney murine leukemia virus LTR as described in Baum et al. 1995 (J. Vir.
ol. 69: 7541-7547) and the SV.
The 40 promoter was replaced with an encephalomyocarditis virus (EMCV) internal ribosome entry site isolated from the plasmid pCITE (obtained from Amersham, Arlington Heights, IL). The ligation mixture was then transformed into competent cells. All plasmids were purified from approximately 50 million clones.

B. Isolation of GSE PBMC libraries were screened using the method described above in Section 1F. All 14 GSEs were isolated using CEM-ss cells. CF-674
(SEQ ID NO: 69) is a GSE that interferes with HIV replication in the sense orientation and has significant sequence identity with the gene encoding human translationally regulated oncoprotein 1 (GenBank Accession No. HUMCH13C4A). The complement of SEQ ID NO: 69 is represented by SEQ ID NO: 70. CF-675 (SEQ ID NO: 71) is another GSE with considerable sequence identity to the gene encoding human translationally regulated oncoprotein 1 (GenBank Accession No. HUMCH13C4A). The complement of SEQ ID NO: 71 is
This is shown by SEQ ID NO: 72. CF-679 (SEQ ID NO: 83) is another GSE with significant sequence identity to the gene encoding human translationally regulated oncoprotein 1 (GenBank Accession No. HUMCH13C4A). The complement of SEQ ID NO: 83 is represented by SEQ ID NO: 84. CF-693 (SEQ ID NO: 75) interferes with HIV replication in the sense orientation and is a NEF interacting protein (GenBank Accession No. HS).
It is GSE with considerable sequence identity to the gene encoding U83843). The complement of SEQ ID NO: 75 is represented by SEQ ID NO: 76. CF-660 (SEQ ID NO: 79) interferes with HIV replication as an antisense molecule and is associated with human hsp90 chaperone protein (Rebbe et al., 1989, J Biol Chem 264).
: 15006-15011), which is a GSE with considerable sequence identity to the gene encoding the same. The complement of SEQ ID NO: 79 is represented by SEQ ID NO: 80. CF-6
53 (SEQ ID NO: 67) interferes with HIV replication as an antisense molecule and
-1α protein (Obata et al., 1988, J Virol. 62: 4381.
˜4386) is a GSE with considerable sequence identity to the gene encoding it. The complement of SEQ ID NO: 67 is represented by SEQ ID NO: 68. CF-676 (SEQ ID NO: 63) interferes with HIV replication as an antisense molecule and has considerable sequence identity with the gene encoding the α-enolase protein (Yu et al., 1997, Genome Res. 7: 353-358). , GSE. The complement of SEQ ID NO: 63 is represented by SEQ ID NO: 64. CF-675 (SEQ ID NO: 65) is another GSE with considerable sequence identity to the gene encoding the alpha enolase protein. The complement of SEQ ID NO: 65 is represented by SEQ ID NO: 66. CF-673 (SEQ ID NO: 73) is a GSE that interferes with HIV replication as an antisense molecule and has considerable sequence identity with the gene encoding the BBC-1 satellite DNA protein (GenBank Accession No. AJ223209). The complement of SEQ ID NO: 73 is represented by SEQ ID NO: 74. CF-672 (SEQ ID NO: 81) is another GSE that interferes with HIV replication as an antisense molecule and has considerable sequence identity with the gene encoding the BBC-1 satellite DNA protein. SEQ ID NO: 81
The complement of is shown by SEQ ID NO: 82. CF-681 (SEQ ID NO: 85) interferes with HIV replication as an antisense molecule, and is associated with FK506 binding protein A1 (Ma
ki et al., 1990, Proc. Natl. Acad. Sci. USA 87: 5
440-5443), with significant sequence identity to the gene encoding GSE
Is. The complement of SEQ ID NO: 85 is represented by SEQ ID NO: 86. CF-683 (SEQ ID NO: 89) is a GSE that interferes with HIV replication as an antisense molecule and has considerable sequence identity with the gene encoding the β signal sequence receptor protein (Chinen et al., Ibid.). The complement of SEQ ID NO: 89 is represented by SEQ ID NO: 90. CF-684 (SEQ ID NO: 91) is a GSE that interferes with HIV replication in the sense orientation and has considerable sequence identity with the gene encoding the human tumor imaginal disc protein (Schilling et al., Ibid.). The complement of SEQ ID NO: 91 is represented by SEQ ID NO: 92. CF-685 (SEQ ID NO: 93) shows a cluster of GSEs that interferes with HIV replication as an antisense molecule and has considerable sequence identity with the gene encoding the cell surface heparin binding protein (Liu et al., Ibid.).
The complement of SEQ ID NO: 93 is represented by SEQ ID NO: 94. Another member of the cluster is CF-686 (SEQ ID NO: 95), which also has considerable sequence identity with the gene encoding the cell surface heparin binding protein (Liu et al., Ibid.). The complement of SEQ ID NO: 95 is represented by SEQ ID NO: 96.

Example 4 This example describes the identification of GSE, which inhibits translocation of HIV protein Rev.

The methods generally described by Stauber et al. (1998, Virology 251: 38-48) are used to identify GSEs that inhibit the translocation of RevHIV proteins in cells. Plasmids CF-24 and CF-367 (vectors without insert) and CF-203, CF-261, CF-527, CF-5
29, CF-537, CF-545, CF-619, CF-653, CF-65
9, CF-660, CF-662 and CF-674 are transfected into cells expressing Rev protein fused to green fluorescent protein (Rev-GFP). Transport of the Rev-GFP fusion protein between the nucleus and cytoplasm of cells transfected with a plasmid containing a GSE nucleic acid sequence was determined using confocal microscopy to
Determine if it is inhibited by the expression of SE. Observations using cells transfected with GSE are compared to cells transfected with vector alone.

Although various embodiments of the invention have been described in detail, it will be apparent to those skilled in the art that variations and adaptations of those embodiments will occur. However, it is expressly understood that such modifications and adaptations are within the scope of the invention as set forth in the following claims.

[Sequence list]

[Brief description of drawings]

FIG. 1 shows the percentage of CD4 ⁺ OM10.1 cells that disappear after TNF-α induction; TNF-induced cells, − ■ −; non-induced cells, − ◆ −.

FIG. 2 shows CF-3 after infection with HIV-1 _SF2 at TCID ₅₀ of 1000.
The ratio of intracellular p24 ⁺ CEM-ss cells containing 15 sequences (SEQ ID NO: 1) is shown.
CE containing C-315 construct or control vector DNA (designated LNGFRM)
M-ss cells (10 ⁶ ) were harvested on designated days post infection, stained with FITC-conjugated anti-p24 monoclonal antibody and analyzed by flow cytometry. Mock infected cells,-◇-; LNGFRM vector infected cells,-□-; and C-
315 infected cells, -Δ-.

FIG. 3 shows various G after infection with HIV-1 _SF2 at TCID ₅₀ of 1000.
SE: CF-004 (SEQ ID NO: 7), CF-025 (SEQ ID NO: 5), CF-11
Intracellular p24 ⁺ C, including 3 (SEQ ID NO: 8) and CF-204 (SEQ ID NO: 9)
The ratio of EM-ss cells is shown. Controls include mock-infected, vector (LNGFRM) -infected and HIV-infected CEM-ss cells.

FIG. 4 shows CF-0 after infection with HIV-1 _SF2 at TCID ₅₀ of 1000.
The ratio of intracellular p24 ⁺ CEM-ss cells containing 01 (SEQ ID NO: 10) is shown. Controls include mock-infected and vector (LNGFRM) infected cells.

FIG. 5 shows that CD4 ⁺ OM10.
The ratio of 1 cell is shown. TNF induction,-▲-; not induced by TNF,-■-.

FIG. 6 shows CD4 ⁺ OM10 after treatment with mofarotene after TNF-α induction.
． The ratio of 1 cell is shown. TNF induction,-■-; not induced by TNF,-◆-.

FIG. 7 shows various G after infection with HIV-1 _SF2 at TCID ₅₀ of 1000.
SE: CF-527 (SEQ ID NO: 41), CF-529 (SEQ ID NO: 45) and C
The ratio of intracellular p24 ⁺ CEM-ss cells containing F-531 (SEQ ID NO: 47) is shown.

FIG. 8 shows GSE after infection with HIV-1 _SF2 at TCID ₅₀ of 1000.
Fig. 6 shows the ratio of intracellular p24 ⁺ CEM-ss cells containing CF-579 (SEQ ID NO: 61). Controls include mock-infected, vector (LNGFRM) -infected and RevM10-transfected CEM-ss cells.

FIG. 9: Different G after infection with HIV-1 _SF2 at TCID ₅₀ of 1000.
SE: CF-619 (SEQ ID NO: 53), CF-620 (SEQ ID NO: 55) and C
Fig. 8 shows the ratio of intracellular p24 ⁺ CEM-ss cells containing F-624 (SEQ ID NO: 57). Controls include mock-infected, vector (LNGFRM) -infected and RevM10-transfected CEM-ss cells.

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (Reference) A61P 31/18 C12N 1/15 4C084 C07K 14/47 1/19 4C086 C12N 1/15 1/21 4H045 1 / 19 9/16 1/21 9/24 5/10 C12Q 1/02 9/16 1/68 Z 9/24 G01N 33/15 Z C12Q 1/02 33/50 Z 1/68 C12N 15/00 ZNAA G01N 33 / 15 5/00 A 33/50 A61K 37/02 (81) Designated countries 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, MZ, SD, SL SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL , IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72 ) Inventor Dan, Stephen Jay. 94041 Mountain View Paul Avenue 80F Term (California, USA) 2G045 AA40 4B024 AA01 AA14 BA07 BA63 CA04 HA01 HA14 HA17 4B050 DD11 LL01 4B063 QA18 QQ08 QQ21 QQ43 QR62 QR77 QS25 QS34 4B065 CA44A44 CA02 CA44 A02 CA44 A02 CA53 CA62 DA27 DC01 NA14 ZC552 4C086 AA02 AA03 EA16 MA01 MA04 NA14 ZC55 4H045 AA10 AA30 CA40 DA50 EA29 EA53

Claims (29)

[Claims] 1. Bone morphogenetic protein-1, double-strand break DNA repair gene protein, rat guanine nucleotide-releasing protein, anti-proliferative factor (BTG-
1), lymphocyte-specific protein 1, protein phosphatase 2A, squalene synthetase, eukaryotic release factor 1, GTP binding protein, importin β subunit, cell adhesion molecule L1, U-snRNP associated cyclophilin, recepin, Arg / Abl Interacting protein (ArgBP2A), keratin-related protein, p18 protein, p40 protein, glucosidase II, α enolase, macrophage inflammatory protein 1α, translation control tumor protein 1 (TCTP)
1), BBC1, Nef interacting protein, Na ⁺ -D-glucose cotransporter gene protein, hsp90 chaperone protein, FK506 binding protein A1, Rox, β signal sequence receptor, tumor primordium protein, cell surface heparin binding An isolated nucleic acid molecule encoding a gene fragment or its complement, encoding a protein selected from the group consisting of proteins and homologues thereof, said nucleic acid molecule being operably linked to regulatory sequences, the host The isolated nucleic acid molecule, wherein expression of said nucleic acid molecule in a cell blocks infection by HIV. 2. The nucleic acid molecule according to claim 1, wherein the inhibition of HIV infection is measured by continuous CD4 expression in cells after HIV infection. 3. The nucleic acid molecule according to claim 1, wherein the inhibition of infection by HIV is measured by the reduction of viral p24 expression in cells after HIV infection. 4. The nucleic acid molecule comprises SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 2.
9, sequence number 31, sequence number 33, sequence number 35, sequence number 37, sequence number 39
, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49,
Sequence number 51, sequence number 53, sequence number 55, sequence number 57, sequence number 59, sequence number 61, sequence number 63, sequence number 65, sequence number 67, sequence number 69, sequence number 71, sequence number 73, sequence number 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85; SEQ ID NO: 87; SEQ ID NO: 89; SEQ ID NO: 91: SEQ ID NO: 93, SEQ ID NO: 95, complements of any of these sequences And a nucleic acid sequence selected from the group consisting of and homologues thereof. 5. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is a human nucleic acid molecule. 6. The nucleic acid molecule is CF-315, CF-319, CF-10.
1, CF-117, CF-025, CF-128, CF-004, CF-113
, CF-204, CF-001, CF-273, CF-311, CF-313,
CF-210, CF-266, CF-302, CF-317, CF-286, C
F-061, CF-280, CF-537, CF-320, CF-321, CF
-322, CF-332, CF-335, CF-42, CG-50, CF-52
7, CF-528, CF-529, CF-531, CF-545, CF-547
, CF-619, CF-620, CF-624, CF-630, CF-579,
CF-676, CF-675, CF-653, CF674, CF-675, CF
-673, CF-693, CF-287, CF-658, CF-672, CF-
679, CF-681, CF-622, CF-683, CF-684, CF-6
The nucleic acid molecule according to claim 1, which is selected from the group consisting of 85, and CF-686. 7. An expression vector comprising the nucleic acid molecule according to claim 1. 8. A blocking composition comprising one or more nucleic acid molecules of claim 1. 9. A host cell comprising the nucleic acid molecule of claim 7. 10. Bone morphogenetic protein-1, double-strand break DNA repair gene protein, rat guanine nucleotide-releasing protein, anti-growth factor (BTG).
-1), lymphocyte-specific protein 1, protein phosphatase 2A, squalene synthetase, eukaryotic release factor 1, GTP binding protein, importin β
Subunit, cell adhesion molecule L1, U-snRNP-associated cyclophilin, recepin, Arg / Abl interacting protein (ArgBP2A), keratin-related protein, p18 protein, p40 protein, glucosidase II, α enolase, macrophage inflammatory protein 1α, translation control tumor Protein 1 (TCT
P1), BBC1, Nef interacting protein, Na ⁺ -D-glucose cotransporter gene protein, hsp90 chaperone protein, FK506 binding protein A1, Rox, β signal sequence receptor, tumor imaginal discriminant protein, cell surface heparin binding. An isolated protein comprising a peptide or a fragment of less than full length of a protein selected from the group consisting of proteins and homologues thereof, wherein said protein blocks infection by HIV. 11. The protein comprises SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, sequence. No. 45, SEQ ID No. 47, SEQ ID No. 4
9, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59
, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69,
Sequence number 71, sequence number 73, sequence number 75, sequence number 77, sequence number 79, sequence number 81, sequence number 83, sequence number 85; sequence number 87; sequence number 89; sequence number 91: sequence number 93, sequence number. The protein according to claim 10, which is selected from the group consisting of proteins encoded by the complementary sequences of nucleic acid sequences selected from the group consisting of 95, and homologues thereof. 12. The protein of claim 10, wherein the protein comprises a peptide. 13. The blocking composition comprises an isolated cell-derived protein or fragment thereof that blocks HIV infection, or a mimetope thereof, or an isolated cell-derived nucleic acid molecule that is operably linked to a regulatory sequence to block HIV infection. , And HI
Said HIV blocking composition comprising a protective compound selected from the group consisting of inhibitors of target gene products identified by their ability to block V infection. 14. The cell-derived protein is bone morphogenetic protein-1,
Double-strand break DNA repair gene protein, rat guanine nucleotide-releasing protein, anti-proliferative factor (BTG-1), lymphocyte-specific protein 1, protein phosphatase 2A, squalene synthetase, eukaryotic releasing factor 1, GTP binding protein, import Tin β subunit, cell adhesion molecule L1, U-snRNP
Associated cyclophilin, recepin, Arg / Abl interacting protein (ArgB
P2A), keratin-related protein, p18 protein, p40 protein, glucosidase II, α enolase, macrophage inflammatory protein 1α, translationally regulated oncoprotein 1 (TCTP1), BBC1, Nef interacting protein, N
a ⁺ -D-glucose cotransport regulator gene protein, hsp90 chaperone protein, FK506 binding protein A1, Rox, β signal sequence receptor,
14. The composition of claim 13, comprising a fragment that is less than full length of a protein selected from the group consisting of adult tumor primordia proteins, cell surface heparin binding proteins and homologues thereof. 15. The cell-derived protein is CD44, calnexin, M
14. A composition according to claim 13, comprising a fragment shorter than the full length of a protein selected from type 2 pyruvate kinase, glucosidase II, macrophage inflammatory protein 1α, BBC1, FK506 binding protein A1, cell surface heparin binding protein and homologues thereof. object. 16. The cell-derived protein comprises SEQ ID NO: 25 and SEQ ID NO: 27.
, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37,
Sequence number 39, sequence number 41, sequence number 43, sequence number 45, sequence number 47, sequence number 49, sequence number 51, sequence number 53, sequence number 55, sequence number 57, sequence number 59, sequence number 61, sequence number 63, sequence number 65, sequence number 67, sequence number 69, sequence number 71, sequence number 73, sequence number 75, sequence number 77, sequence number 79, sequence number 81, sequence number 83, sequence number 85; sequence number 87; Sequence number 8
9; SEQ ID NO: 91: SEQ ID NO: 93, SEQ ID NO: 95, and a composition according to claim 13, encoded by a complementary sequence of a nucleic acid selected from the group consisting of homologues thereof. 17. The composition according to claim 13, wherein the cell-derived protein is a peptide. 18. The cell-derived nucleic acid molecule comprises bone morphogenetic protein-1,2
Double-strand break DNA repair gene protein, rat guanine nucleotide-releasing tamper
Quality, anti-proliferative factor (BTG-1), lymphocyte-specific protein 1, protein protein
Sphatase 2A, squalene synthetase, eukaryotic release factor 1, GTP-binding protein
Protein, importin β subunit, cell adhesion molecule L1, U-snRNP
Combined cyclophilin, lecepin, Arg / Abl interacting protein (ArgBP
2A), keratin-related protein, p18 protein, p40 protein, glucine
Cosidase II, α enolase, macrophage inflammatory protein 1α, translational regulation
Oncoprotein 1 (TCTP1), BBC1, Nef interacting protein, Na ⁺ -D-glucose cotransport regulator gene protein, hsp90 chaperonta
Protein, FK506 binding protein A1, Rox, β signal sequence receptor, tumor
From ulcer primordium protein, cell surface heparin-binding protein and its homologues
Corresponds to fragments shorter than the full length of the gene encoding the protein selected from the group
14. The composition of claim 13, wherein 19. The cell-derived nucleic acid molecule comprises SEQ ID NO: 25, SEQ ID NO: 27,
Sequence number 29, sequence number 31, sequence number 33, sequence number 35, sequence number 37, sequence number 39, sequence number 41, sequence number 43, sequence number 45, sequence number 47, sequence number 49, sequence number 51, sequence number 53, sequence number 55, sequence number 57, sequence number 59, sequence number 61, sequence number 63, sequence number 65, sequence number 67, sequence number 69, sequence number 71, sequence number 73, sequence number 75, sequence number 77, Sequence number 7
9, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85; SEQ ID NO: 87; SEQ ID NO: 89
SEQ ID NO: 91: SEQ ID NO: 93, SEQ ID NO: 95, a composition according to claim 13 comprising a nucleic acid sequence selected from the group consisting of the complements of any of these and homologues thereof. 20. The cell-derived nucleic acid molecule comprises SEQ ID NO: 9, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 61, SEQ ID NO: 67, SEQ ID NO: 73, SEQ ID NO: 85, SEQ ID NO: 93, SEQ ID NO: 95, and their complements. 14. The composition of claim 13, comprising a nucleic acid sequence selected from the group consisting of somatic bodies and homologues thereof. 21. The inhibitor is bone morphogenetic protein-1, double-strand breaks D.
NA repair gene protein, rat guanine nucleotide-releasing protein, anti-proliferative factor (BTG-1), lymphocyte-specific protein 1, protein phosphatase 2A, squalene synthetase, eukaryotic releasing factor 1, GTP binding protein, importin β subunit , Cell adhesion molecule L1, U-snRNP associated cyclophilin, resepin, Arg / Abl interacting protein (ArgBP2A), keratin-related protein, p18 protein, p40 protein, glucosidase II, α enolase, macrophage inflammatory protein 1α, translation control tumor protein 1 (TCTP1), BBC1, Nef interacting protein, Na ⁺ -D-glucose cotransporter gene protein, hsp90 chaperone protein,
14. The composition of claim 13, which inhibits the activity of a protein selected from the group consisting of FK506 binding protein A1, Rox, β signal sequence receptor, adult tumor primordium protein and cell surface heparin binding protein. 22. The composition of claim 13, wherein the composition further comprises a component selected from the group consisting of excipients and carriers. 23. The inhibitor is bone morphogenetic protein-1, double-strand breaks D.
NA repair gene protein, rat guanine nucleotide-releasing protein, anti-proliferative factor (BTG-1), lymphocyte-specific protein 1, protein phosphatase 2A, squalene synthetase, eukaryotic releasing factor 1, GTP binding protein, importin β subunit , Cell adhesion molecule L1, U-snRNP associated cyclophilin, resepin, Arg / Abl interacting protein (ArgBP2A), keratin-related protein, p18 protein, p40 protein, glucosidase II, α enolase, macrophage inflammatory protein 1α, translation control tumor protein 1 (TCTP1), BBC1, Nef interacting protein, Na ⁺ -D-glucose cotransporter gene protein, hsp90 chaperone protein,
14. The composition of claim 13, which inhibits the activity of a protein selected from the group consisting of FK506 binding protein A1, Rox, β signal sequence receptor, adult tumor primordium protein and cell surface heparin binding protein. 24. A method of protecting host cells from HIV infection, comprising introducing an effective amount of the composition of claim 13. 25. The composition is introduced into a host cell in vitro.
The method according to 3. 26. The composition is introduced into a host cell in vivo.
The method described in. 27. A method of treating an HIV infection, comprising administering to the individual a therapeutically effective amount of the composition of claim 13 to inhibit the HIV infection. 28. A method of treating an HIV infection, comprising administering to the individual a therapeutically effective amount of the composition of claim 13 to prevent the HIV infection. 29. A method of selecting an inhibitor, comprising: (a) exposing a mammalian cell to a test compound; (b) measuring the expression of a cellular gene or the activity of its encoded product in said cell. And (c) selecting a compound that downregulates the expression of said gene or interferes with the activity of its encoded product, wherein said cellular gene is a bone morphogenic protein- 1, double-strand break DNA repair gene protein, rat guanine nucleotide-releasing protein, antiproliferative factor (
BTG-1), lymphocyte-specific protein 1, protein phosphatase 2A,
Squalene synthetase, eukaryotic release factor 1, GTP binding protein, importin β subunit, cell adhesion molecule L1, U-snRNP associated cyclophilin,
Resepine, Arg / Abl interacting protein (ArgBP2A), keratin-related protein, p18 protein, p40 protein, glucosidase II, α
Enolase, macrophage inflammatory protein 1α, translational regulatory oncoprotein 1 (
TCTP1), BBC1, Nef interacting protein, Na ⁺ -D-glucose cotransport regulator gene protein, hsp90 chaperone protein, FK50
6. The method as described above, which encodes a protein selected from the group consisting of 6-binding protein A1, Rox, β signal sequence receptor, adult tumor primordium protein and cell surface heparin-binding protein.