WO2002022880A1

WO2002022880A1 - Compositions and methods for the identification of viral inhibitors of host cell protein synthesis shut-off

Info

Publication number: WO2002022880A1
Application number: PCT/US2001/028653
Authority: WO
Inventors: Bernard Roizman; Penelope Mavromara
Original assignee: University Of Chicago; Institute Pasteur Hellenque
Priority date: 2000-09-13
Filing date: 2001-09-13
Publication date: 2002-03-21

Abstract

Viruses have developed various ways to prevent shut off of host cell protein synthesis. The present invention discloses methods for using viral vectors to identify viral polypeptides that are responsible for inhibiting protein synthesis shut off. Specifically, a method is disclosed for using a herpes simplex virus that lacks a functional ICP 34.5 gene. One example shows the identification of NS5A, a protein found in Hepatitis C virus, as responsible for inhibiting shut off of host cell protein synthesis.

Description

DESCRIPTION

COMPOSITIONS AND METHODS FOR THE IDENTIFICATION OF VIRAL INHIBITORS OF HOST CELL PROTEIN SYNTHESIS SHUT-OFF

BACKGROUND OF THE INVENTION

This application claims benefit of the filing date of US Provisional Patent Application Serial No.60/232,582 filed on 09/13/2000 which is incorporated by reference herein.

I. Field of the Invention

The present invention relates to fields of molecular and cell biology generally, and more specifically, to the use of viral vectors as surrogate indicators of functions of genes that block antiviral host cell responses to infection.

II. Brief Description of the Prior Art

Control of host cell gene expression is an integral part of the life cycle of a virus.

Most viruses express RNA that is either double stranded or forms secondary structures that are partially double stranded. Such RNAs often induce an interferon-triggered host cell response that results in shut-off of protein synthesis, and thereby interferes with viral multiplication. The trigger for this response it thought to be a normally inactive protein, protein kinase R (PKR), that is activated by double-stranded RNA. The activated PKR phosphorylates the α subunit of the translation initiation factor eIF-2_α. Phosphorylated elF- 2_α totally blocks protein synthesis (Katze, 1995).

Not surprisingly, viruses that express RNAs that activate PKR, thereby inducing host cell protein synthesis shut-off, also have evolved strategies to block PKR shut-off protein synthesis. These strategies include degradation of PKR, blocking activation of PKR, preventing double stranded RNA from activating PKR, blocking activated PKR from phosphorylating eIF-2_α, and redirecting a cellular phosphatase to dephosphorylate eIF-2_α.

Though the process of viral inhibition of host cell protein synthesis shut-off is well established, it is not generally known which viral genes are involved in shutting off host response. In addition some viruses do not grow well in cell culture or do not lend themselves to manipulations that would allow the identification of the viral gene product responsible for inhibiting the host. Therefore, a method that (a) could identify which viral proteins are responsible for this effect and (b) would allow the direct measurements of this effect in cell culture would be very valuable. Knowledge of which viral proteins are responsible for shut- off of host cell anti-viral responses would enable screening of drugs against these targets. Such drugs not only will allow for improved therapies, including combination therapies, but they permit a greater specificity in treating of viral infection.

SUMMARY OF THE INVENTION

It is, therefore, an object of this invention to provide a method for identifying proteins that inhibit shut-off of cellular protein synthesis. In addition, it is an object of this invention to provide a method of screening for inhibitors of HCV infection.

In satisfying these goals, there is provided a method for identifying polypeptides that inhibit interferon/protein kinase R-mediated shut-off of infected cell protein synthesis comprising the steps of (i) providing a viral vector that does not encode an endogenous functional interferon/protein kinase R-mediated inhibitor of cellular protein synthesis shut- off; (ii) inserting into said viral vector a selected nucleic acid segment encoding a candidate polypeptide; (iii) infecting a suitable host cell; and (iv) measuring host cell protein synthesis; wherein increased infected host cell protein synthesis measured in (iv), relative to the host cell protein synthesis in a cell infected with a viral vector that does not encode an endogenous functional interferon/protein kinase R-mediated inhibitor of cellular protein synthesis shut- off, identifies said selected nucleic acid segment as encoding an inhibitor of interferon protein kinase-mediated cellular protein synthesis shut-off. In another embodiment of the invention, the additional step of measuring host cell protein synthesis in the absence of said selected nucleic acid segment is included along with steps (i)-(iv) above. In one specific embodiment of the invention, the cell is located in a non-human animal.

The viral vector may be selected from any number of viruses. Some examples include herpesvirus, vaccinia virus, influenza virus or poxvirus. Specific examples of herpesvirus that may serve as a viral vector include herpes simplex type I and type II viruses that lack functional 7^4.5 genes. The nucleic acid segment also may be selected from any number of nucleic acids. One of many possible sources of the nucleic acid segment is a viral cDNA library. Some examples of viruses that may be included in the viral cDNA library include hepatitis C, hepatitis B, hepatitis D, respiratory syncytial virus, measles, parainfluenza viruses, Epstein Barr virus, pseudorabies vims, varicella zoster virus, cytomegalo virus, and human herpesviruses No. 6 A, 6B, 7, and 8.

There are several techniques by which protein synthesis may be measured. Such methods include measurement by radioactive labeling of proteins and by expression of a marker protein expressed by said host cell.

In another embodiment of the invention, there is provided a viral vector that lacks a functional interferon/protein kinase R-mediated inhibitor of cellular protein synthesis shut- off, and comprises a heterologous nucleic acid segment that encodes a functional inhibitor of interferon/protein kinase-mediated host cell protein synthesis shut-of The viral vector may be any number of viruses. Specific examples include herpesvirus, vaccinia virus, influenza virus and poxvirus. Specific examples of herpesvirus that may serve as a viral vector include herpes simplex type I and type II viruses that lack functional γι34.5 genes. The nucleic acid segment also may be selected from any number of nucleic acids. One of many possible sources of the nucleic acid segment is a viral cDNA library. Some examples of viruses that may be included in the viral cDNA library include hepatitis C, hepatitis B, hepatitis D, respiratory syncytial virus, measles, parainfluenza viruses, Epstein Barr virus, pseudorabies virus, varicella zoster virus, cytomegalovirus, and human herpesviruses No. 6A, 6B, 7, and 8

As was also true for a previous embodiment, the viral vector may be selected from any number of viruses. Some examples include herpesvirus, vaccinia virus, influenza virus or poxvirus. Specific examples of herpesvirus that may serve as a viral vector include herpes simplex type I and type II viruses that lack functional γi34.5 genes. The nucleic acid segment also may be selected from any number of nucleic acids. One of many possible sources of the nucleic acid segment is a viral cDNA library. Some examples of viruses that may be included in the viral cDNA library include hepatitis C, hepatitis B, hepatitis D, respiratory syncytial virus, measles, parainfluenza viruses, Epstein Barr virus, pseudorabies virus, varicella zoster virus, cytomegalovirus, and human herpesviruses 6 A, 6B, 1, and 8.

Yet another embodiment of the invention provides an inhibitor of cellular interferon/protein kinase R-mediated protein synthesis shut-off identified according to the method comprising the steps of (i) providing a viral vector that does not encode an endogenous functional interferon protein kinase R-mediated inhibitor of cellular protein synthesis shut-off; (ii) inserting into said viral vector a selected nucleic acid segment encoding a candidate polypeptide; (iii) infecting a suitable host cell; and (iv) measuring host cell protein synthesis; wherein increased infected host cell protein synthesis measured in step (iv), relative to the host cell protein synthesis in a cell infected with a viral vector that does not encode an endogenous functional interferon/protein kinase R-mediated inhibitor of cellular protein synthesis shut-off, identifies said selected nucleic acid segment as encoding a inhibitor of interferon/protein kinase-mediated cellular protein synthesis shut-off

Another embodiment of the invention provides for a method of producing a polypeptide that inhibits interferon/protein kinase R-mediated shut-off This embodiment requires implementing the steps set forth in the previously described embodiment for a method of identifying an inhibitor of cellular interferon/protein kinase R-mediated protein synthesis shut-off, and then performing the additional step of producing the identified polypeptide. Another embodiment provides for the sequencing of the selected nucleic acid segment. The viral vector may be selected from any number of viruses. Some examples include herpesvirus, vaccinia virus, influenza virus or poxvirus.

Yet another embodiment of the invention provides for a method of screening for inhibitors of HCN infection comprising the steps of (i) providing a non-HCV viral vector that (a) lacks an endogenous functional interferon/protein kinase R-mediated inhibitor of cellular protein synthesis shut-off, and (b) comprises an nucleic acid segment encoding a functional ΝS5A polypeptide; (ii) providing a candidate inhibitor substance; and (iii) measuring host cell protein synthesis in a cell infected with said non-HCV viral vector in the presence of said candidate inhibitor substance, wherein reduction in the infected host cell protein synthesis of step (iii), relative to the infected host cell protein synthesis in the absence of said candidate inhibitor substance, identifies said candidate inhibitor substance as an inhibitor of NS5A inhibition of cellular protein synthesis shut-off. In another embodiment of the invention, the additional step of measuring host cell protein synthesis in the absence of said candidate inhibitor substance is included along with steps (i)-(iii) above. The candidate inhibitor substance may be provided to the cell after, before, or at the same time as infection with the non-HCN viral vector. One specific embodiment of the invention provides that the cell be located in a non-human animal. As is true with other embodiments of the invention, the viral vector may be selected from any number of viruses. Some examples include herpesvirus, vaccinia virus, influenza virus or poxvirus. Specific examples of herpesvirus that may serve as a viral vector include herpes simplex type I and type II viruses that lack functional 7^4.5 genes.

In yet another embodiment, in addition to the three steps previously disclosed for a method for screening for inhibitors of HCN infection, an additional step is added of measuring host cell protein synthesis, in the presence of said candidate inhibitor substance, in a cell infected with a non-HCV viral vector that comprises an endogenous functional interferon/protein kinase R-mediated inhibitor of cellular protein synthesis shut-off, wherein the absence of any effect non-HCV viral vector that comprises an endogenous functional interferon/protein kinase R-infected host cell protein synthesis identifies said candidate inhibitor substance as an ΝS5A-specific inhibitor. One specific embodiment provides that the host cell is located in a non-human animal.

The final embodiment provides for an inhibitor of HCV infection identified according to the method of (i) providing a non-HCV viral vector that (a) lacks an endogenous functional interferon/protein kinase R-mediated inhibitor of cellular protein synthesis shut-off and (b) comprises an nucleic acid segment encoding a functional NS5A polypeptide; (ii) providing a candidate inhibitor substance; and (iii) measuring host cell protein synthesis in a cell infected with said non-HCV viral vector in the presence of said candidate inhibitor substance; wherein reduction in the infected host cell protein synthesis of step (iii), relative to the infected host cell protein synthesis in the absence of said candidate inhibitor substance, identifies said candidate inhibitor substance as an inhibitor of NS5A inhibition of interferon/protein kinase- mediated cellular protein synthesis shut-off

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1. Schematic representation of the DNA sequence arrangement of the recombinant R3659 virus used in this study. The viral genome consists of two covalently linked components flanked by inverted sequences designated as ab and a'b'. The break in the line indicates a 500 nt deletion within the natural TK gene. The expanded regions represent the structure of the γ₁34.5 gene present in R3659. That is the tk gene has been used to replace both copies of 7^4.5. Last line represents the structure of the HSV/HCV sequences present in the shuttle vector pRB4878 used to construct the recombiant R3659-NS5a virus. The HCV NS5A ORF is under the control of the egr-1 promoter and HBV polyA regulatory sequences. This transcriptional cassette by homologous recombination replaces the tk gene of R3659 yielding the R3659-NS5A recombinant virus.

FIG. 2. Autoradiographic image of electrophoretically separated [³⁵S]methionine- labeled proteins prepared from lysates of SK-N-SH or Vero cells mock infected or virus infected. Replicate cultures of Vero (lanes 1-4 and 9-12) or SK-N-SH (lanes 5-8 and 13-16) cells were mock infected or infected with 5PFU of HSV-1(F), R3659 or R3659-NS5A. At 6 hrs (lanes 1-8) or 14 hrs (lanes 9-16) after infection the cultures were labeled with [³⁵S]methionine for two hours as described in Materials and Methods. After labeling, the infected cells were harvested, solubilized in an SDS-containing buffer and electrophoretically separated on a denaturing 12.5% polyacrylamide gel. The electrophoretically separated proteins were transferred to a nitrocellulose sheet and subjected to autoradiography.

FIG. 3. Photograph of electrophoretically separated proteins reacted with anti-eIF2-P antibody. Replicate cultures of Vero (lanes 1-4) or SK-N-SH (lanes 5-8) cells were mock infected or infected with 5PFU of HSV-l(F), R3659 or R3659-NS5A. At 8 hrs or 16 hrs after infection the cultures were harvested, solubilized in an SDS-containing buffer and electrophoretically separated on a denaturing 12.5% polyacrylamide gel. The electrophoretically separated proteins were transferred to a nitrocellulose sheet and reacted with a polyclonal antibody directed specifically against a phosphorylated eIF2a peptide. DETAILED DESCRIPTION OF THE INVENTION

I. Viral Vector for Identifying Viral Genes that Block Antiviral Host Responses to Infection

As stated previously, most cells contain an inactive enzyme known as protein kinase R, or PKR. Viral infection triggers the activation of PKR, in an interferon-dependent fashion, which in turn phosphorylates the α subunit of translation initiation factor eIF-2_α, which completely blocks protein synthesis. Unfortunately, viruses have adopted a variety of strategies to block PKR from shutting off protein synthesis, thereby facilitating infection. The present invention provides methods for the identification of viral polypeptides that are responsible for inhibiting interferon/protein kinase R-mediated shut-off of infected cell protein synthesis.

The present invention relies, in large part, on the provision of a viral vector that lacks a functional inhibitor of host protein synthesis shut-off. Using this vector in a fashion analogous to those employed in "promoter trap" experiments, one of skill in the art will insert a nucleic acid segment into the vector and determine the ability of that segment to compensate (in whole or in part) for the missing inhibitor function. Genes identified through this process can be used in subsequent experiments to screen for inhibitors of viral inhibition of host cell protein synthesis shut off.

While the present invention discusses the use of herpesvirus as a suitable vector, viral vectors also include, but are not limited to, vaccinia virus, influenza virus, poxvirus, and herpesvirus, or virtually any viral vector for which an endogenous functional inhibitor of protein synthesis shut-off has been identified, and can be deleted.

The invention, in its various aspects, is discussed in greater detail below.

II. Herpes Simplex Virus and ICP34.5

The present invention exemplifies a herpes simplex virus (HSV) vector. HSV, designated with subtypes 1 and 2, are enveloped viruses that are among the most common infectious agents encountered by humans, infecting millions of human subjects worldwide. These viruses cause a broad spectrum of disease which ranges from relatively insignificant to severe and life-threatening. The large, complex, double-stranded DNA genome encodes for dozens of different gene products, some of which are derived from spliced transcripts. In addition to virion and envelope structural components, the virus encodes numerous other proteins. HSV has evolved a gene, γ_j34.5 (also referred to herein as the ICP34.5 gene, i.e., as the gene encoding infected cell protein 34.5), whose product, the infected cell protein No. 34.5 (ICP34.5) that redirects phosphatase l_α to dephosphorylate eIF-2_α, thereby preventing the shut-off of host cell protein synthesis. Black et al. (1993); Caroll et al. (1993); Chiu et al. (1995); He et al. (1997).

The ICP34.5 gene maps in the sequences flanking the long unique sequence of HSV- 1 DNA and therefore is present in two copies per genome. Chou et al. (1986). The 263 amino acid protein encoded by the HSV-1(F) ICP34.5 gene consists of 3 domains, an amino terminal domain of 160 amino acid-domain, 10 repeats of three amino acids (Ala-Thr-Pro), and a 73 amino acid-carboxyl terminal domain. Chou et al. (1990). A stretch of 64 amino acids at the carboxyl terminus of the ICP34.5 gene is homologous to a corresponding stretch of amino acids of the carboxyl terminus of a murine protein known as MyD116 and a Chinese hamster protein known as GADD34. Chou et al. (1992); McGeoch et al. (1991).

The ICP34.5 gene lacks a canonical TATAA box, and the a sequence, its 5' transcribed non-coding domain, is very GC-rich, contains numerous repeats and lacks the features characteristic of HSV promoters. Mocarski et al. (1981); Mocarski et al. (1982); Chou et al. (1985). The a sequence contains the signals and the site of cleavage of the unit length DNA molecule from newly synthesized head to tail concatemers of viral DNA. Mocarski et al. (1982); Chou et al. (1985); Deiss et al. (1986); Varmuza et al. (1985); Vlazny et al. (1982). Specifically, the cleavage occurs within a direct repeat which flanks the a sequence. Mocarski et al. (1982). The transcription of the ICP34.5 gene is initiated in this repeat. Chou et al. (1986).

Infection of human cells with HSV in which both copies of the ICP34.5 gene are inactivated or deleted, but particularly of human neuroblastoma cell line SK-N-SH or primary human foreskin fibroblasts, results in nearly complete cessation of host cell protein synthesis replicative cycle. Chou et al. (1994). This total premature shutoff of protein synthesis is not seen in these cells when they are treated with inhibitors of viral DNA synthesis or in Vero cells. Chou et α/. (1992). The capacity to preclude total premature shutoff of protein synthesis maps in the carboxyl terminus domain of the ICP34.5 protein, which is homologous to the MyD116 protein. Chou et al. (1994). Indeed, the carboxyl terminus of MyD116 successfully substitutes for the corresponding domain of the ICP34.5 protein. The viruses lacking the ICP34.5 protein or unable to express the carboxyl terminus of the protein are totally avirulent in a murine encephalitis model of HSV-1 infections. Chou et al. (1990); Whitley et al. (1993); McKie et α/. (1994).

U.S. Patent No. 5,328,688, is herein incorporated by reference. The '688 patent discloses various herpes simplex virus vectors that encode functionally "null" ICP34.5 genes. For example, one virus contains an ICP34.5 gene having a stop codon in reading frame between a first and a last codon of a coding sequence of the ICP34.5 gene, and in particular, may have a stop codon at a fatEII site in the ICP34.5 gene of HSV-l(F), such as the site in herpes simplex virus designated R4009 (ATCC VR2278). Also disclosed is a herpes simplex virus that encodes an ICP34.5 gene having a deletion mutation, more particularly, an ICP34.5 gene lacking a portion of a coding sequence between ifatEII and Stωl sites in HSV-l(F), which portion may be 1000 base pairs in length, such as in a herpes simplex virus which is designated R3616 (ATCC VR2280).

One embodiment of the present invention uses the recombinant virus R3659 as the viral vector. Randall & Roizman (1997); Lagunoff & Roizman (1994). R3659 lacks the Sacl-Bglϊl sequence consisting of i?αmHI-Q encoding the thymidine kinase (tk) and Uι24 genes. A sequence consisting of the coding domain of the tk gene under control of the _α27 promoter replaced the i&tEII-StwI sequence of the BamHl S fragment encoding the ICP34.5 and ORF P genes. R3659 is only one example of a suitable HSV vector, and many other recombinant HSV lacking the ICP34.5 gene could also be used.

As discussed above, HSV lacking the ICP34.5 gene does not cause inhibition of host cell protein synthesis shut-off, and thus can be used as a vector to identify the genes of other viruses that perform this function. Another factor that makes HSV an attractive vector is the size and organization of the genome. Because HSV is relatively large, incorporation of multiple genes or expression cassettes is less problematic than in other smaller viral systems. In addition, the availability of different viral control sequences with varying performance (temporal, strength, etc.) makes it possible to control expression to a greater extent than in other systems. It also is an advantage that the virus has relatively few spliced messages, further easing genetic manipulations. Both HSV-1 and HSV-2 that lack the ICP34.5 gene may be used as viral vectors.

III. Insertion of Foreign DNA into a Viral Vector

Another aspect of the invention is the introduction, into a viral vector, of foreign DNA segments. These segments are derived from viruses (a) to be screened for inhibitors of host cell protein synthesis shut-off or (b) that are problematic in terms of culturing, where one seeks a better system in which to screen for modulators of viral inhibitors of host cell protein synthesis shut-off

In one embodiment of the invention, candidate nucleic acid segments are taken from a viral cDNA library. Specific examples of candidate nucleic acid segments that may be selected from viral cDNA libraries include, but are not limited to, segments selected from hepatitis C, hepatitis B, hepatitis D, respiratory syncytial virus, measles, parainfluenza viruses, Epstein Barr virus, pseudorabies virus, varicella zoster virus, cytomegalovirus, and human herpesviruses 6A, 6B, 7 and 8.. Methods for preparation of cDNA libraries are well known in the art. Alternatively, one may obtain genomic fragments of viral DNA. These fragments may be generated by restriction of viral genomes, random shearing, or other analogous means.

Manipulation of the herpesviral genome is well known in the art. U.S. Patents 4,769,331 and 5,288,641, incorporated by reference, disclose experimental details for creating mutations in HSV vectors, and for inserting DNA segments therein, including insertion of expressable DNA segments.

IV. Infection of Host Cells and Measurement of Protein Synthesis

Once a chimeric virus is created, one will infect a suitable host cell. The host cell may vary depending on the vector used. For HSV, host cells include Vero cells, SN-K-SH and rabbit skin. It also may prove beneficial to select a host cell that is suitable for growth of both the vector virus and the virus from which the heterologous DNA segment is selected. This should permit a more accurate reflection of normal infectious process and, in so doing, create a more faithful reproduction of the interaction between the putative inhibitor of host protein synthesis shut-off. The cell may be in vivo or in vitro. In vitro assays are well known in the art. Assays suitable for automation are desired. For example, 96-well trays may be employed in which several wells are reserved for controls while the remainder comprise test viruses. In vivo hosts may be either humans or animals. Suitable routes of administration are dependent on the type of viral vector utilized.

Protein synthesis in infected cell is measured. Measurements made be made at set intervals of time. Cellular protein synthesis may be measured by several methods. In one embodiment of the invention, protein synthesis is measured by the integral radioactive labeling of proteins. The amount of radioactivity can then be measured by various means, including gamma-counting, radio-immune precipitation, or any other suitable method.

In another embodiment, protein synthesis is measured by expression of a marker protein expressed by the host cell. For example, the host cell may optionally comprise an expression cassette encoding a marker protein, wherein expression of the marker protein is directed by a promoter that can be regulated by the interferon-PKR pathway. The marker may be luciferase, green fluorescent protein, β-galactosidase, or any other suitable protein.

Other formats also are provided. For instance, one may utilize a viral vector that lacks any protein synthesis shut-off inhibitor as negative controls. One may also utilize wild- type vector virus and wild-type virus corresponding to the inserted viral DNA segment as positive controls. The amounts of infecting virus may be altered in order to create the ideal system for inhibition of host cell protein synthesis shut-off.

V. Candidate Inhibitors

As used herein, the term "candidate substance" refers to any molecule that may potentially inhibit a viral protein's ability to inhibit host cell protein synthesis shut-off The candidate substance may be a protein or fragment thereof, a small molecule inhibitor, or even a nucleic acid molecule. It may prove to be the case that the most useful pharmacological compounds will be compounds that are structurally related to compounds which interact naturally with viral proteins that effect inhibition of protein synthesis shut-off Creating and examining the action of such molecules is known as "rational drug design," and include making predictions relating to the structure of target molecules. The goal of rational drug design is to produce structural analogs of biologically active polypeptides or target compounds. By creating such analogs, it is possible to fashion drugs which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules. In one approach, one would generate a three-dimensional structure for a molecule like NS5A or ICP34.5, and then design a molecule for its ability to interact with NS5A or ICP34.5. Alternatively, one could design a partially functional fragment of one of these proteins, thereby creating a competitive inhibitor. This could be accomplished by x-ray crystallography, computer modeling or by a combination of both approaches.

It also is possible to use antibodies to ascertain the structure of a target compound or inhibitor. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti- idiotypic antibodies to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of anti-idiotype would be expected to be an analog of the original antigen. The anti-idiotype could then be used to identify and isolate peptides from banks of chemically- or biologically-produced peptides. Selected peptides would then serve as the pharmacore. Anti-idiotypes may be generated using the methods described herein for producing antibodies, using an antibody as the antigen.

On the other hand, one may simply acquire, from various commercial sources, small molecule libraries that are believed to meet the basic criteria for useful drugs in an effort to "brute force" the identification of useful compounds. Screening of such libraries, including combinatorially generated libraries (e.g., peptide libraries), is a rapid and efficient way to screen large number of related (and unrelated) compounds for activity. Combinatorial approaches also lend themselves to rapid evolution of potential drugs by the creation of second, third and fourth generation compounds modeled of active, but otherwise undesirable compounds.

Candidate compounds may include fragments or parts of naturally-occurring compounds or may be found as active combinations of known compounds which are otherwise inactive. It is proposed that compounds isolated from natural sources, such as animals, bacteria, fungi, plant sources, including leaves and bark, and marine samples may be assayed as candidates for the presence of potentially useful pharmaceutical agents. It will be understood that the pharmaceutical agents to be screened could also be derived or synthesized from chemical compositions or man-made compounds. Thus, it is understood that the candidate substance identified by the present invention may be polypeptide, polynucleotide, small molecule inhibitors or any other compounds that may be designed through rational drug design starting from known inhibitors of hypertrophic response.

Other suitable inhibitors include antisense molecules, ribozymes, and antibodies

(including single chain antibodies), each of which would be specific for a target located within the calcineurin pathway. Such compounds are described in greater detail elsewhere in this document.

It will, of course, be understood that all the screening methods of the present invention are useful in themselves notwithstanding the fact that effective candidates may not be found. The invention provides methods for screening for such candidates, not solely methods of finding them.

VI. Hepatitis C Virus and Inhibitors of NS5A

The Hepatitis C virus (HCV) is a major human pathogen. It is a positive-stranded, enveloped virus which is evolutionarily related to the flaviviruses and pestiviruses. The HCV genome consists of a 5' untranslated region (5' UTR), a long open reading frame of between

9030 and 9099 nucleotides and a 3' UTR. The 3' end of the genome has been reported to contain a short stretch of poly(A) in type I strains, but this has not been confirmed in type II,

III or IV strains. It has been reported that sequences in the 5' UTR exhibit negative translational control over viral gene expression, but the major regulation of HCV gene expression comes from the proteolytic cleavages of the polyprotein. Various proteolytic cleavages of the polyprotein are essential for the efficient expression of replication-associated viral proteins after translation. The resultant replicase proteins interact with a region of between 27 and 45 nucleotides in the 3' UTR which functions as a structural cue for the initiation of viral genomic replication.

HCV is known to cause various liver diseases, and even has been associated with liver cancer. Although there have been considerable efforts to develop antiviral drugs to combat

HCV infection, these efforts have not been successful. The only approved therapies for HCV infection are based on the use of _α-interferon therapy, alone or in combination with ribavirin. However, the sustained response rate is low - only 30% to 40%. Development of novel therapies is critical and is the major objective of many pharmaceutical companies. One obvious target of such therapies is the viral gene products that block host response to infection. The primary problem is that HCV does not grow well in cell culture. Additionally, there are no experimental animal models - humans are the only known hosts. Alter, 1998. Thus, there has been only limited ability to examine HCV functions in the research context. It is known that HCV, like other viruses, shuts off host cell response to viral infection. Recent reports have implicated two HCV proteins, the glycoprotein E2 and a protein known as NS5A, in this function. Gale et al, 1997; 1998; Taylor, et al, 1999; Pawlotsky and Germanidis, 1999. However, these studies are based on in vitro assays and cannot be confirmed in the context of a viral infection.

The present invention has demonstrated, as dicussed in the Examples below, that the

NS5A gene encodes a protein responsible for HCV inhibition of host cell protein synthesis shut off. As part of this process, a chimeric virus was produced that carried the NS5A gene in the context of a herpesvirus vector. This chimeric virus provides an ideal vector for screening of inhibitors of the NS5A gene product. Thus, in another embodiment of the present invention, there are provided methods of screening for inhibitors of HCV infection. These screening methods will determine the ability of various candidate substances to inhibit NS5A from inhibiting host cell protein synthesis shut-off. Several assays are contemplated by the invention.

In a first assay, a host cell is infected with the chimeric virus. One then will measure host cell protein synthesis in a cell infected with NS5A in the presence and absence of a candidate inhibitor substance. The substance may be contacted with the cell prior to, at the same time, or after the infection with the chimeric virus. In some cases, the candidate inhibitor substance may simply be contacted with the cell. In other situations, depending on the nature and putative mechanism of action, the candidate inhibitor substance may be modified to provide improved results. For example, where expression of an antisense molecule or a protein is required, a nucleic acid will be placed into an expression construct. Further, any nucleic acid may advantageously be formulated in liposomes or as virally- encapsulated expression vehicles.

Once the candidate inhibitor substance has been provided to a cell that is infected with the chimeric virus, the evaluation of protein synthesis is undertaken. Depending on the type of assay, it is possible to automate this process and test hundreds of candidates at the same time. For example, 96-well trays may be employed in which several wells are reserved for controls while the remainder comprise test substances, usually with each substance being tested at several different amounts.

A candidate inhibitor is in fact an inhibitor of protein synthesis if cellular protein synthesis in the infected cell in presence of the candidate inhibitor is less than protein synthesis measured in a cell infected with the viral vector containing the nucleic acid segment encoding NS5A, but in which the candidate inhibitor is not present.

The present invention also provides for a method for determining the specificity of a candidate inhibitor in inhibiting host cell protein synthesis shut-off. This is accomplished by comparing the ability of the substance to promote host cell protein synthesis shut off in a cell infected with NS5A as compared with its ability to promote shut-off in a cell infected with a virus that encodes an inhibitor of protein synthesis shut-off that is not NS5A. If the candidate inhibitor promotes cellular protein synthesis shut-off in a cell infected with NS5 A, but does not effect the same level of protein synthesis in a cell infected with an inhibitor protein synthesis shut-off, then the candidate inhibitor is an NS5A specific inhibitor (or relatively so).

While discussed in the context of HCV and the NS5A gene, the present invention supports the use of virtually any chimeric virus that contains a heterologous inhibitor of host cell protein synthesis shut-off.

VII. Examples

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1: MATERIALS AND METHODS

Cells and Viruses. Vero, SN-K-SH and rabbit skin cells (RSC) were grown in Dulbecco's modification of Eagle minimal essential medium (DMEM) containing 10% (SN- K-SH) or 5% of fetal bovine serum (Vero, RSC). HSN-1 is a prototype HSV-1 strain. Recombinant R3659 contains a 500 nt deletion within the natural tk gene and a chimeric a27- TK gene in place of both copies of ICP34.5 and has been described before. Langunoff & Roizman (1995). For the construction of the recombinant R3659-ΝS5A virus, intact R3659 viral DNA and pHPI738 plasmid DNA were cotransfected in RSC. The recombinant virus was isolated by plating the transfection progeny in 143tk- cells maintained in media containing bromodeoxyuridine to select against the thymidine kinase. Viral DNA was isolated from Vero cells infected with plaque purified viral stocks and analyzed by PCR and Southern blot analysis.

Plasmids. Plasmid pHPI738 was made by inserting a blunt ended Hindlϊl fragment containing the NS5A ORF from HCV genotype 1 a into a blunt ended Kpnl site of shuttle vector pBR4878. The orientation is such so that the NS5A is under the control of the egr-1 promoter.

Antibodies. Rabbit polyclonal antibodies directed against a synthetic 13-residue phosphorylated rat eIF2a peptide were obtained from Research Genetics (HuntsviUe, Alabama). Polyclonal anti-NS5A antibodies were raised in rabbits using a purified GST- NS5A chimeric protein synthesized in E.coli.

Immunoblots. Infected or uninfected cell lysates were electrophoretically separated in a 12% denaturing polyacrylamide gel, electrically transferred onto a nitrocellulose sheet, and reacted with anti-NS5A polyclonal antibody. The protein bands reacting with the antibody were visualized by using an enhanced chemiluminescence (ECL) system (Pierce,

Rockford, Illinois).

In Vivo Protein Labeling. Infected SK-N-SH or Vero cells in 25 cm² flasks were incubated in 1 ml of medium 199V lacking methionine but supplemented with 50 μCi of [35S]methionine for two hours before harvest. The cells were then rinsed twice with PBS-A (phosphate-buffered saline), scraped in 1 ml of ice-cold PBS-A, pelleted, solubilized in disruption buffer, boiled, electrophoretically separated on a denaturing 12.5% polyacrylamide gel, electrically transferred to a nitrocellulose sheet and subjected to autoradiography on Kodak3 XAR5 film or immunoblotting. EXAMPLE 2: RESULTS

Construction of HSV-1 Recombinant Viruses Lacking the ICP34.5 Gene and Expressing the NS5A Protein from HCV-la

The DNA sequence arrangement of recombinant virus R3659 is described elsewhere. Randall & Roizman (1997) and is shown schematically in FIG. 1. R3659 lacks a 500- nucleotide segment in the sequence of the TK gene and contains a chimeric _α27-TK gene in place of both copies of the ICP34.5 gene. The recombinant R3659-NS5A virus was constructed by homologous recombination between intact R3659 DNA and a DNA plasmid containing the NS5A gene of the HCV in place of the _α27-TK gene within the ICP34.5 gene. Consequently, in R3659-NS5A recombinant virus the 34.5 gene has been replaced by the HCV-la NS5A gene.

Expression of NS5A Rescues the Shut-Off of Protein Synthesis in Cells Infected with HSV Recombinants Lacking Both Copies of the ICP34.5 Gene and Expressing NS5A

The purpose of these experiments was to examine whether HCV NS5A protein expressed in the context of ICP34.5^" HSV recombinant is able to overcome the translation block imposed in HSV infected cells in the absence of ICP34.5.

Replicate 10-cm cultures of Vero or SK-N-SH were either mock infected or infected with 5 PFU of HSV-l(F), R3659 or R3659-NS5A recombinant viruses. The cultures were labeled with 35S-methionine at 6 hours and 14 hours after infection for 2 hours, solubilized, electrically separated in a 12% denaturing polyacrylamide gel and processed for autoradiography as described previously.

The results are shown in FIG. 2. At 16 hours after infection there was a dramatic inhibition of protein synthesis in SN-K-SH cells infected with the R3659 virus whereas partial prevention of protein expression was observed at 8 hours after infection. Chou et al. (1995). In contrast, protein synthesis was restored in SN-K-SH cells infected with the R3659-NS5A recombinant virus both at early and late times post infection. In fact, the level of protein synthesis in cells infected with the NS5A expressing viruses could not be differentiated from that of cells infected with HSV-l(F). Consistent with previous data, protein synthesis in Vero cells infected with all HSV viruses were sustained both at 8 hours and 16 hours after infection. It is concluded that the NS5A protein from the HCV genotype restored protein synthesis in cells infected with HSV recombinants lacking both copies of the ICP34.5 gene.

Expression of the NS5A Protein Inhibits the Phosphorylation of the eIF2_α in cells infected with HSV Recombinants Lacking the 34.5 Gene and Expressing NS5A

The purpose of these experiments was to examine whether the restoration of the shut- off of protein synthesis in cells infected with the ICP34.5^" recombinant HSV virus expressing NS5A was determined at the level of the eIF-2_α phosphorylation. In this experiment, replicate cultures of Vero, SN-K-SH and HepG2 cells were mock infected or exposed to 5PFU of HSV-l(F), R3659, or R3659-NS5A. The cultures were harvested 8 hours or 16 hours after infection, solibilized and electrophoretically separated in a 12% denaturing polyacrylamide gel, electrically transferred to a nitrocellulose membrane and then reacted with a polyclonal antibody specific in recognizing only the phosphorylated form of the elF- ^■Ax-

As shown in FIG. 3, the anti-NS5A polyclonal antibody reacted strongly with a protein with an apparent Mr of 36,000 corresponding to the phosphorylated form of the elF- 2_α in mock infected SK-N-SH cells and in SK-N-SH cells infected with R3659 virus. In contrast, the lysates of SK-N-SH cells infected with the R3659-NS5A or HSV- 1(F) failed to react with the antibody to phosphorylated eIF-2_α both at 8 hours and 16 hours post infection. Interestingly, the level of phosphorylated eIF-2_cj in Vero cells infected with NS5 A protein blocks the phosphorylation of the eIF-2_α. Thus, NS5A effectively replaced the γ₁34.5 gene in enabling the virus to maintain protein synthesis and by extension, uninterrupted viral replication.

The conclusions of the experiment are as follows. First, HSV can act as a vector for the expression of cDNA encoding the NS5A protein. Second, NS5A substitutes, functionally, for both copies of the 7^4.5 genes in that the 7_J34.5^" virus expressing NS5A blocks the shut- off of protein synthesis characteristic of 7^4.5^" vector. Third, the consequences of infection of component cells by the 7^4.5^" mutants is the phosphorylation of eIF-2_α. In cells infected with the 7_!34.5^" vector carrying the NS5A protein, eIF-2_α is not phosphorylated even though the mechanisms by which HCV and HSV attain the same end result shut-off of host response to infection are very different.

All of the COMPOSITIONS and/or METHODS disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the COMPOSITIONS and/or METHODS and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference:

Alter, H.J., Natural clinical and clinical aspects of hepatitis C virus infection. In: Schinazi & Sommadossi eds. Therapies for viral hepatitis London International Medical Press, p. 45-79, 1998. Black et al, Degradation of the interferon-induced 68,000 Mr protein kinase by poliovirus requires RNA. J. Virol. 67:791-800, 1993.

Carroll et al, Recombinant vaccinia virus K3L gene product prevents activation of double- stranded RNA-dependent, initiation factor 2α-specific protein kinase. J. Biol Chem. 268:12837-2842, 1993. Chou et al, Cell 41:80, 1985. Chou et al., J. Virol. 57:629, 1986.

Chou et al, J. Virol. 64:1014-1020, 1990.

Chou et al, Mapping of herpes simplex virus-1 neurovirulence to 7^4.5, a gene nonessential for growth in cell culture. Science 250: 1262-1266, 1990. Chou et al, Proc. Nat'l Acad. Sci. USA 89:3266-3270, 1992. Chou et al , Proc. Nat 1 Acad. Sci. USA 91 :5247-5251 , 1994. Chou et α/., J Virol. 66:8304-8311, 1994.

Chou et al, Association of a M_r 90,000 phosphoprotein with protein kinase PKR in cells exhibiting enhanced phosphorylation of translation initiation factor eIF-2 and premature shutoff of protein synthesis after infection with 7ι34.5^~ mutants of herpes simplex virus 1. Proc. Nat 'I Acad. Sci. USA 92 : 10516- 10520, 1995.

Deiss et al, J. Virol, 59:605, 1986.

Enomoto et al, Mutations in the NS5A gene and response to interferon in patients with chronic hepatitic C virus lb infection. N. Engl J. Med. 334:77-81. 1996. Gale et al, Evidence that hepatitis C virus resistance to interferon is mediated through expression of the PKR protein kinase by the nonstructural 5 A protein. Virology

230:217-227, 1997. Gale et al, Control of PKR protein kinase by hepatitis C virus nonstructural 5 A protein: molecular mechanism of kinase regulation. Mol Cell. Biol. 18:5208-5218, 1998 Gale & Katze, Molecular mechanisms of interferon resistance mediated by viral-directed inhibition of PKR, the interferon-induced protein kinase.v Pharmacol. Ther. 78:29-46,

1998. He et al, The γι34.5 protein of herpes simplex virus 1 complexes with protein phosphatase lα to dephosphorylate the α subunit of the eIF-2 translation initiation factor and preclude the shutoff of protein synthesis by double stranded RNA activated protein kinase

(PKR). Proc. Nat 'I Acad. Sci. USA 94:843-848, 1997. Katze, M., Regulation of interferon-induced PKR: can viruses cope? Trends in Microbiol. 3:75-

78, 1995. Lagunoff & Roizman, The regulation of synthesis and properties of the protein product of the open reading frame P of the herpes simplex virus 1 genome. J. Virol 69:3615-3623,

1995. McGeoch et al, Nature 353:609, 1991. McKie et a/., J Gen. Virol 75:733-741, 1994. Mocarski et al, Proc. Nat'l Acad. Sci. USA 78, 7047, 1981. Mocarski et al, Cell 31:89, 1982. Pawlotsky & Germanidis, The non-structural 5A protein of hepatitis C virus. J Viral

Hepatology 6:343-356, 1999. Randall & Roizman, Transcription of the derepressed ORF P of herpes simplex virus 1 precludes the expression of the antisense 7^4.5 gene and may account for the attenuation of the mutant virus. J. Virol. 71:7,750-7,757, 1997. Taylor et al, Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein.

Science 285:107-110, 1999. Varmuza et al, Cell 41 :793, 1985. Vlazny et al, Proc. Nat'l. Acad. Sci. USA 79:1423, 1982. Whitley et al, J. Clin. Invest. 91:2837-2843, 1993.

Claims

1. A method for identifying polypeptides that inhibit interferon/protein kinase R- mediated shut-off of infected cell protein synthesis comprising:

(i) providing a viral vector that does not encode an endogenous functional interferon/protein kinase R-mediated inhibitor of cellular protein synthesis shut-off;

(ii) inserting into said viral vector a selected nucleic acid segment encoding a candidate polypeptide;

(iii) infecting a suitable host cell; and

(iv) measuring host cell protein synthesis,

wherein increased infected host cell protein synthesis measured in step (iv), relative to the host cell protein synthesis in a cell infected with a viral vector that does not encode an endogenous functional interferon/protein kinase R-mediated inhibitor of cellular protein synthesis shut-off, identifies said selected nucleic acid segment as encoding a inhibitor of interferon/protein kinase-mediated cellular protein synthesis shut-off.

2. The method of claim 1, wherein said viral vector is a herpesvirus, vaccinia virus, influenza virus or poxvirus.

3. The method of claim 2, wherein said viral vector is herpesvirus.

4. The method of claim 3, wherein said herpesvirus is heφes simplex virus type I that lacks a functional ₁34.5 gene.

5. The method of claim 3, wherein said herpesvirus is herpes simplex virus type II that lacks a functional 7₁34.5 gene.

6. The method of claim 1, wherein said nucleic acid segment is from a viral cDNA library.

7. The method of claim 6, wherein said viral cDNA library is derived from hepatitis C, hepatitis B, hepatitis D, respiratory syncytial virus, measles, parainfluenza viruses, Epstein Barr virus, pseudorabies virus, varicella zoster virus, cytomegalovirus, and human heφesviruses 6 A, 6B, 7 and 8.

8. The method of claim 1, wherein protein synthesis is measured by radioactive labeling ofproteins.

9. The method of claim 1, wherein protein synthesis is measured by expression of a marker protein expressed by said host cell.

10. The method of claim 1, further comprising the step of measuring host cell protein synthesis in the absence of said selected nucleic acid segment.

11. The method of claim 1 , wherein said cell is located in a non-human animal.

12. A recombinant viral vector that:

(i) lacks a functional interferon/protein kinase R-mediated inhibitor of cellular protein synthesis shut-off; and

(ii) comprises a heterologous nucleic acid segment that encodes a functional inhibitor of interferon/protein kinase-mediated host cell protein synthesis shut- off.

13. The recombinant viral vector of claim 12, wherein said vector is a heφesvirus, vaccinia virus, influenza virus or poxvirus.

14. The recombinant vector of claim 13, wherein said viral vector is heφesvirus.

15. The recombinant vector of claim 14, wherein said heφesvirus is heφes simplex virus type I that lacks a functional 7134.5 gene.

16. The recombinant vector of claim 14, wherein said heφesvirus is heφes simplex virus type II that lacks a functional 7134.5 gene.

17. The recombinant vector of claim 12, wherein said nucleic acid segment is from a viral cDNA library.

18. The recombinant vector of claim 17, wherein said viral cDNA library is derived from hepatitis C, hepatitis B, hepatitis D, respiratory syncytial virus, Epstein Barr virus, pseudorabies virus, varicella zoster virus, cytomegalovirus, and human heφesvirus 6.

19. An inhibitor of cellular interferon/protein kinase R-mediated protein synthesis shut- off identified according to the method comprising:

(iii) infecting a suitable host cell; and

(iv) measuring host cell protein synthesis,

wherein increased infected host cell protein synthesis measured in step (iv), relative to the host cell protein synthesis in a cell infected with a viral vector that does not encode an endogenous functional interferon/protein kinase R-mediated inhibitor of cellular protein synthesis shut-off, identifies said selected nucleic acid segment as encoding a inhibitor of interferon/protein kinase-mediated cellular protein synthesis shut-off

20. A method for producing a polypeptide that inhibits interferon/protein kinase R- mediated shut-off of cellular protein synthesis comprising:

(iii) infecting a suitable host cell; and (iv) measuring infected host cell protein synthesis, wherein increased infected host cell protein synthesis, relative to the infected host cell protein synthesis in a cell infected with a viral vector that does not encode an endogenous functional interferon/protein kinase R-mediated inhibitor of cellular protein synthesis shut-off, identifies said selected nucleic acid segment as encoding a inhibitor of interferon/protein kinase-mediated cellular protein synthesis shut-off; and

(v) producing the identified polypeptide.

21. The method of claim 20, further comprising sequencing said selected nucleic acid segment.

22. The recombinant viral vector of claim 20, wherein said vector is a heφesvirus, vaccinia virus, influenza virus or poxvirus.

23. A method of screening for inhibitors of HCV infection comprising:

(i) providing a non-HCV viral vector that

(a) lacks an endogenous functional interferon/protein kinase R-mediated inhibitor of cellular protein synthesis shut-off; and

(b) comprises an nucleic acid segment encoding a functional NS5A polypeptide;

(ii) providing a candidate inhibitor substance; and

(iii) measuring host cell protein synthesis in a cell infected with said non-HCV viral vector in the presence of said candidate inhibitor substance,

wherein reduction in the infected host cell protein synthesis of step (iii), relative to the infected host cell protein synthesis in the absence of said candidate inhibitor substance, identifies said candidate inhibitor substance as an inhibitor of NS5A inhibition of cellular protein synthesis shut-off.

24. The method of claim 23, further comprising the step of measuring host cell protein synthesis in the absence of said candidate inhibitor substance.

25. The method of claim 23, wherein said non-HCV viral vector is a heφesvirus, vaccinia virus, influenza virus or poxvirus.

26. The method of claim 25, wherein said non-HCV viral vector is heφesvirus.

27. The method of claim 26, wherein said heφesvirus is heφes simplex virus type I that lacks a functional 7₁34.5 gene.

28. The method of claim 26, wherein said heφesvirus is heφes simplex virus type II that lacks a functional 7₁34.5 gene.

29. The method of claim 23, wherein said candidate inhibitor substance is provided to said cell after infection with said non-HCV viral vector.

30. The method of claim 23, wherein said candidate inhibitor substance is provided to said cell before infection with said non-HCV viral vector.

31. The method of claim 23, wherein said candidate inhibitor substance is provided to said cell at the same time as infection with said non-HCV viral vector.

32. The method of claim 23, wherein said host cell is located in a non-human animal.

33. The method of claim 23, further comprising measuring host cell protein synthesis, in the presence of said candidate inhibitor substance, in a cell infected with a non-HCV viral vector that comprises an endogenous functional interferon/protein kinase R- mediated inhibitor of cellular protein synthesis shut-off, wherein the absence of any effect non-HCV viral vector that comprises an endogenous functional interferon/protein kinase R-infected host cell protein synthesis identifies said candidate inhibitor substance as an NS5A-specific inhibitor.

34. The method of claim 33, wherein said host cell is located in a non-human animal.

35. An inhibitor of HCV infection identified according to the method comprising:

(i) providing a non-HCV viral vector that:

(a) lacks an endogenous functional interferon/protein kinase R-mediated inhibitor of cellular protein synthesis shut-off; and (b) comprises an nucleic acid segment encoding a functional NS5A polypeptide;

(ii) providing a candidate inhibitor substance; and

wherein reduction in the infected host cell protein synthesis of step (iii), relative to the infected host cell protein synthesis in the absence of said candidate inhibitor substance, identifies said candidate inhibitor substance as an inhibitor of NS5A inhibition of interferon/protein kinase-mediated cellular protein synthesis shut-off