WO2011003071A1

WO2011003071A1 - Methods of modulating hepatitis c virus infection

Info

Publication number: WO2011003071A1
Application number: PCT/US2010/040918
Authority: WO
Inventors: T. Jake Liang; Qisheng Li; Abraham L. Brass; Stephen Elledge
Original assignee: US Department of Health and Human Services
Current assignee: US Department of Health and Human Services
Priority date: 2009-07-02
Filing date: 2010-07-02
Publication date: 2011-01-06
Anticipated expiration: 2012-01-02

Abstract

The present invention relates to the discovery that numerous factors produced by the host cell modulate (i.e., increase or decrease) hepatitis C virus (HCV) infection. Such host factors can be proviral host factors or antiviral host factors. The present invention provides a method to use these host factors to identify compounds that modulate viral infection. Also provided are inhibitory compounds as well as enhancing compounds that modulate viral infection. Such inhibitory or enhancing compounds act on host factors of the present invention, or on biochemical pathways comprising such host factors. Also provided are methods to inhibit or enhance viral infection, using such inhibitory compounds or enhancing compounds, respectively, as well as methods to protect a patient from viral infection by administering such inhibitory compounds to the patient.

Description

METHODS OF MODULATING HEPATITIS C VIRUS INFECTION Field of the Invention The present invention relates to methods of identifying inhibitors or enhancers of hepatitis C virus infection, and the use of such inhibitors in protecting patients from hepatitis C virus infection.

Background of the Invention

Hepatitis C virus (HCV) is an important cause of liver disease worldwide. The virus causes a chronic infection resulting in progressive liver damage. Hepatitis C virus (HCV) enters hepatocytes via interactions between the viral envelope proteins, El and E2, and four known host receptors, CD81, Claudin-1, SBR-I and occludin (Ploss, Nature 457, 2009, pp 882-886). Subsequent to entry, host ribosomes bind to the internal ribosomal entry site (IRES) of the HCV genome, and translate viral polyproteins on the rough endoplasmic reticulum (ER) (Moradpour (2007) Nat Rev Microbiol 5, 453-463). Host and viral proteases process the polyprotein into both structural (core, and envelope proteins, El and E2) and nonstructural proteins (p7, NS2-3, NS3, NS4A, NS4B, NS5A, and NS5B, (Lindenbach (2005) Nature 436, 933-938; WoIk (2008) J Virol 82, 10519-10531.). Oligomerization of NS4B distorts the host ER into membranous webs, which house HCV replication complexes (RCs), within which the RNA-dependent RNA polymerase, NS5B, transcribes viral genomic RNAs (Moradpour (2007) Nat Rev Microbiol 5, 453-463.). Progeny viral genomes generated in the RCs are translocated to lipid droplet-containing organelles and assemble into virions, which then traffic to the cell surface for release (Miyanari (2007) Nat Cell Biol 9, 1089-1097.).

However, while progress has been made in the areas of entry and replication, the later stages of the viral lifecycle, e.g., assembly, budding, maturation and secretion, remain less explored.

The in vitro study of HCV replication has benefited from the use of the replicon system, consisting of sub-genomic or whole genome viral RNAs stably expressed in permissive tissue culture cells. Using the replicon system, several recent functional genomic screens, using either partial or genome-wide siRNA libraries, have identified a number of host factors involved in HCV replication (Tai (2009) Cell Host Microbe 5, 298-307; Supekova (2008) J Biol Chem 283, 29-36; Ng (2007) Hepatology 45, 1413-1421.). However, the replicon system is unable to address the role of host factors in the entire HCV lifecycle. The infection-competent HCV cell culture system (HCVcc) recapitulates the complete HCV lifecycle, thereby permitting a greater range of host- viral interactions to be studied (Wakita (2005) Nat Med 11, 791-796; Zhong (2005) Proc Natl Acad Sd U S A 102, 9294-9299; Lindenbach (2005) Science 309, 623-626; Tellinghuisen (2007) J Virol 81, 8853-8867.)); see also PCT International Publication WO 2007/013882 A2, published Feb 1, 2007. Two efforts have employed the HCVcc system using limited candidate gene siRNA screens of either 65 or 140 curated targets. (Randall (2007) Proc Natl Acad Sd USA 104, 12884-12889; Berger (2009) PNAS.).

There remains a need to identify factors involved during the HCV life-cycle, and to obtain compounds to protect individuals from HCV infection.

Summary of the Invention

The present invention identifies, for the first time, numerous host factors that are involved in modulating hepatitis C infection. Because these host factors are involved in modulating viral infection, they represent new targets that can be used to identify compounds capable of inhibiting or enhancing viral infection. Thus the present invention relates to methods of using host factors of the present invention to identify compounds that modulate viruses of the present invention. The invention further relates to compounds that interact with host factors of the present invention, or biological pathways comprising host factors of the present invention, in such a way as to inhibit or enhance viral infection. The present invention also relates to methods to inhibit or enhance viral infection using inhibitory or enhancement compounds, and methods to protect a patient from viral infection using such compounds.

One embodiment of the present invention is a method to identify a compound that modulates viral infection, the method comprising assaying a candidate compound for the ability to interact with a host factor of the present invention in such a manner as to modulate viral infection. In a preferred embodiment, the virus is hepatitis C virus.

One embodiment of the present invention is a method to identify a compound that modulates viral infection, the method comprising; (a) contacting a candidate compound with at least one isolated host factor under conditions suitable for formation of a complex between said compound and said host factor; and,

(b) detecting the presence of a complex, if present; wherein the presence of said complex indicates that the test compound modulates viral nfection.

Another embodiment of the present invention is a method to identify a compound that modulates viral infection, the method comprising;

(a) contacting a candidate compound with at least one isolated host factor under conditions suitable for allowing an interaction between the compound and the at least one host factor; and

(b) determining if said interaction results in modification of said host factor; wherein modification of the host factor indicates that the test compound modulates viral nfection. Still another embodiment of the present invention is a method to identify a compound hat modulates viral infection, which comprises:

(a) combining a candidate compound with a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1 under conditions suitable for such candidate compound to interact with such host factor, (b) assaying for the presence of such interaction between such candidate compound and such host factor and;

(c) determining if such an interaction decreases or increases viral infectivity, wherein a decrease or increase in viral infectivity indicates that the compound modulates viral infection. In one embodiment, an inhibitory compound of the present invention modulates a pathway comprising a host factor of the present invention by interacting with said host factor, thereby inhibiting viral infection. In another embodiment, an enhancing compound of the present invention modulates a pathway comprising a host factor of the present invention by interacting with said host factor, hereby enhancing viral infection.

Another embodiment of the present invention is an inhibitory compound for inhibiting nfection by a virus of the present invention, wherein the inhibitory compound interacts with a host factor of the present invention, thereby inhibiting the viral infection. In one mbodiment, an inhibitory compound of the present invention modulates a pathway comprising a host factor of the present invention by interacting with said host factor, thereby nhibiting viral infection, hi another embodiment, an enhancing compound of the present nvention modulates a pathway comprising a host factor of the present invention by nteracting with said host factor, thereby enhancing viral infection.

One embodiment of the present invention is a method to inhibit viral infection, the method comprising contacting a cell infected with the virus with an inhibitory compound that nteracts with a host factor of the present invention in such a manner as to inhibit viral nfection. Another embodiment of the present invention is a method to enhance viral nfection, the method comprising contacting an infected cell with an enhancing compound hat interacts with a host factor in such a manner as to increase the amount of virus produced. n a preferred embodiment, the cell is infected with hepatitis C virus.

Another embodiment is a method to protect a patient from virus infection, comprising administering to a patient an inhibitory compound that modulates a pathway comprising a host factor of the present invention in such a manner as to inhibit viral infection.

The embodiments include a method to inhibit viral infection, wherein such a method comprises contacting a cell with an inhibitory compound that interacts with a host factor encoded by a nucleic acid molecule listed in Table 5 or with a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1, thereby inhibiting viral infection. In certain embodiments, such a method comprises contacting a patient with such an inhibitory compound. Such a patient can be infected with such a virus. In certain embodiments such a virus is a Flavi virus, such as a hepatitis C virus.

The embodiments include a method to inhibit viral infection, wherein such a method comprises contacting a cell with an inhibitory compound that modulates a pathway comprising a host factor encoded by a nucleic acid molecule listed in Table 5 or a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1, therebynhibiting viral infection. In certain embodiments, such a method comprises contacting a patient with such an inhibitory compound. Such a patient can be infected with such a virus. n certain embodiments such a virus is a Flavivirus, such as a hepatitis C virus. The embodiments include a method to protect a patient from hepatitis C virus nfection, wherein such a method comprises administering an inhibitory compound that nteracts with a host factor encoded by a nucleic acid molecule listed in Table 5 or with a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1. In certain embodiments, such a method can provide prophylactic protection. In certain embodiments, such a method can provide therapeutic protection.

The embodiments include a method to protect a patient from hepatitis C virus nfection, wherein such a method comprises administering an inhibitory compound that modulates a pathway comprising a host factor encoded by a nucleic acid molecule listed in Table 5 or a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1. In certain embodiments, such a method can provide prophylactic protection. In certain embodiments, such a method can provide therapeutic protection.

The embodiments include a method to enhance viral infection, wherein such a method comprises contacting a cell with an enhancing compound that interacts with a host factor encoded by a nucleic acid molecule listed in Table 5 or with a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1, thereby enhancing viral infection. In certain embodiments such a virus is a Flavivirus, such as a hepatitis C virus.

The embodiments include a method to enhance viral infection, wherein such a method comprises contacting a cell with an enhancing compound that modulates a pathway comprising a host factor encoded by a nucleic acid molecule listed in Table 5 or a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1 , thereby enhancing viral infection. In certain embodiments such a virus is a Flavivirus, such as a hepatitis C virus.

The embodiments include an inhibitory compound that interacts with a host factor encoded by a nucleic acid molecule listed in Table 5 or with a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1, thereby inhibiting viral infection. The embodiments include an inhibitory compound that modulates a pathway comprising a host factor encoded by a nucleic acid molecule listed in Table 5 or a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1, therebynhibiting viral infection. In certain embodiments such a virus is a Flavivirus, such as a hepatitis C virus. The embodiments include an enhancing compound that interacts with a host factor encoded by a nucleic acid molecule listed in Table 5 or with a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1, thereby enhancing viral nfection. The embodiments include an enhancing compound that modulates a pathway comprising a host factor encoded by a nucleic acid molecule listed in Table 5 or a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1 , thereby enhancing viral infection. In certain embodiments such a virus is a Flavivirus, such as a hepatitis C virus.

The embodiments include a method to identify a compound that inhibits viral nfection, wherein such a method comprises assaying a candidate compound for the ability to nteract with a host factor in such a manner as to inhibit such an infection, wherein such a host factor is encoded by a nucleic acid molecule listed in Table 5 or is encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1. In certain embodiments such a virus is a Flavivirus, such as a hepatitis C virus. The embodiments include an inhibitory compound identified by such a method. The embodiments include a method to identify a compound that inhibits viral infection, wherein such a method comprises: (a) combining a candidate compound with a host factor encoded by a nucleic acid molecule listed in Table 5 or with a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1 under conditions suitable for such a candidate compound to interact with such a host factor; (b) assaying for the presence of such an interaction; and (c) determining if such an interaction results in a decrease in the amount of virus obtained upon infection of a cell with such a virus; wherein a decrease in the amount of virus indicates that such a compound inhibits viral infection. In certain embodiments such a virus is a Flavivirus, such as a hepatitis C virus. The

embodiments include an inhibitory compound identified by such a method. The embodiments include a method to identify a compound that inhibits viral infection, wherein such a method comprises assaying a candidate compound for the ability to modulate a pathway comprising a host factor encoded by a nucleic acid molecule listed in Table 5 or a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1, wherein the ability to modulate such a pathway identifies a compound that inhibits viral infection. In certain embodiments such a virus is a Flavivirus, such as a hepatitis C virus. The embodiments include an inhibitory compound identified by such a method. The embodiments include a method to identify a compound that enhances viral infection, wherein such a method comprises assaying a candidate compound for the ability to interact with a host factor in such a manner as to enhance such an infection, wherein such a host factor is encoded by a nucleic acid molecule listed in Table 5 or is encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1. In certain embodiments such a virus is a Flavivirus, such as a hepatitis C virus.

The embodiments include a method to identify a compound that enhances viral infection, wherein such a method comprises: (a) combining a candidate compound with a host factor encoded by a nucleic acid molecule listed in Table 5 or with a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1 under conditions suitable for such a candidate compound to interact with such a host factor; (b) assaying for the presence of such an interaction; and (c) determining if such an interaction results in an increase in the amount of virus obtained upon infection of a cell with such a virus; wherein an increase in the amount of virus indicates that such a compound enhances viral infection. In certain embodiments such a virus is a Flavivirus, such as a hepatitis C virus. The

embodiments include an inhibitory compound identified by such a method.

The embodiments include a method to identify a compound that enhances viral infection, wherein such a method comprises assaying a candidate compound for the ability to modulate a pathway comprising a host factor encoded by a nucleic acid molecule listed in Table 5 or a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1, wherein the ability to modulate such a pathway identifies a compound that enhances viral infection. In certain embodiments such a virus is a Flavivirus, such as a hepatitis C virus. The embodiments include an inhibitory compound identified by such a method.

The embodiments include a host factor encoded by a nucleic acid molecule listed in Table 2. The embodiments include a host factor encoded by a nucleic acid molecule listed in Table 11, Table 12, Table 13 or Table 14. The embodiments include a host factor encoded by a nucleic acid molecule comprising SEQ ID NO:79. The embodiments include a host factor comprising amino acid sequence SEQ ID NO:80. The embodiments include one of more inhibitory compounds listed in Table 3. The embodiments include one or more of the following inhibitory compounds: colchicine, cytochalasin, EIPA, rottlerin, UO 126 or wortmannin. The embodiments include one or more of the following inhibitory compounds: brefeldin A or golgicide A. The embodiments include an inhibitory compound comprising a small interfering RNA (siRNA). Such siRNA can be one or more of the siRNAs listed in Table 4.

Brief Description of the Figures Fig. 1 Effect of siRNA screen on HCV infected cells Fig. 2 Network of connections between HCV proteins

Fig. 3 Map of interactions between HCV host factors from siRNA screen and host factors that interact with HCV core components

Fig. 4 Common network modules amongst various Flaviviridae

Fig. Sl Effect of siRNA treatment on stages of HCV infection and cellular molecular function

Fig. 5 Effect of siRNA silencing of IKKα gene expression on HCV infection

Fig. 6 (A) Effect of siRNA silencing of expression of various NF-kβ pathway-related factors on HCV RNA levels. (B) Relative knock-down efficiency of various host factors by the siRNAs. Fig. 7 (A) Effect of expression of IKKα dominant negative mutant (HA-IKKaKA) on HCV RNA levels. (B) Western blot illustrating levels of HA-IKKaKA.

Fig. 8 Effect of siRNA silencing of IKKα expression on (A) intracellular HCV RNA levels and (B) extracellular HCV RNA levels. (C) Relative silencing efficiency of IKKα expression as measured by quantitative PCR. Fig. 9 (A) Effect of siRNA silencing of IKKα expression on (A) viral entry of an HCV pseudovirus and (B) RNA replication. Fig 10 Effect of (A) rotterlin, (B) colchicines, (C) EIPA, (D) wortmannin, (E) cytochalasin, r (F) U0126 on HCV entry.

Fig. 11 Effect of (A) rottlerin or (B) EIPA on HCV RNA levels. Fig. 12 Effect of (A) golgicide or (B) brefeldin on HCV RNA levels.

Detailed Description of the Invention

Before the present invention is further described, it is to be understood that this nvention is not limited to particular embodiments described, as such may, of course, vary. It s also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present nvention will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, the singular forms a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is urther noted that the claims may be drafted to exclude any optional element. As such, this tatement is intended to serve as antecedent basis for use of such exclusive terminology as solely," "only" and the like in connection with the recitation of claim elements, or use of a negative" limitation.

It should be understood that as used herein, the term "a" entity or "an" entity refers to one or more of that entity. For example, a host factor refers to one or more host factors. As such, the terms "a", "an", "one or more" and "at least one" can be used interchangeably. Similarly the terms "comprising", "including" and "having" can be used interchangeably.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by eference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is appreciated that certain features of the invention, which are, for clarity, described n the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described n the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present invention and are disclosed herein just as if each and every combination was ndividually and explicitly disclosed. In addition, all sub-combinations are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub- combination was individually and explicitly disclosed herein.

The present invention generally relates to the discovery that numerous host factors are involved in modulating (e.g., inhibiting or enhancing) hepatitis C virus (HCV) infection. The present invention relates to methods of using host factors of the present invention to identify compounds that modulate viral infection. Also encompassed are compounds that interact with host factors of the present invention, and the use of such compounds to modulate infection by viruses of the present invention. The instant invention is novel since, prior to the inventors' surprising discovery, it was not appreciated that host factors disclosed herein were involved in modulating viral infection. Thus, host factors of the present invention represent new tools for identifying compounds that can modulate viral infection. The present invention also teaches novel methods for protecting (e.g., prophylactically or therapeutically) a patient from viral infection, and in particular hepatitis C virus infection. As defined herein, a host factor of the present invention is a molecule produced by the host cell that modulates HCV infection. Host factors are biological molecules, examples of which include proteins, peptides, lipids, nucleic acid molecules, carbohydrates, lipoproteins, glycoproteins, metabolic products, and the like. As used herein, a host factor modulates HCV infection if, when the host factor-related activity is altered (e.g., reduced or eliminated, or increased) the amount of virus is decreased or increased. Accordingly, host factors can be proviral or antiviral, and the amount of virus produced during an infection is influenced by interactions between viral components, proviral host factors and antiviral host factors. A proviral host factor is a molecule that positively influences virus production. Consequently when the amount or activity of a proviral host factor is reduced or eliminated, the amount of virus produced is decreased. For example, 3-hydroxy-3-methylgluttaryl CoA reductase is known to be required for replication of HCV and thus is a proviral host factor (Ye, PNAS, Dec. 23, 2003, ppl5865-15870). An antiviral host factor is a molecule that negatively influences virus production. Consequently when the amount or activity of an antiviral host factor is reduced or eliminated, the amount of virus produced is increased. One example of an antiviral host factor is interleukin 7 receptor β. Host factors of the present invention can be identified by those skilled in the art using the screening methods disclosed herein, e.g., siRNA screening.

Certain host factors of the present invention or the nucleic acid molecules encoding such host factors are listed in one or more of the Tables included in this disclosure. These nucleic acid molecules are characterized in various ways appreciated by those skilled in the art, e.g., Entrez Gene Symbol, Entrez Gene ID, Genbank Accession Number, Gene Name, SEQ ID NO: (or SEQ NO). In certain embodiments, host factors of the present invention are encoded by the nucleic acid molecules listed in Table 1 , with their corresponding nucleic acid sequence SEQ ID NO:s. Preferred host factors are those encoded by nucleic acid molecules listed in Table 2. hi some embodiments, host factors are described by their amino acid sequences as listed in Table 1. Certain host factors are encoded by nucleic acid molecules listed in Table 5. Certain host factors are encoded by a nucleic acid molecule listed in Table 5 or are encoded by a nucleic acid molecule having a SEQ ID NO listed in Table 1. Certain host factors involved in viral entry are encoded by nucleic acid molecules listed in Table 11. Certain host factors involved in viral IRES-mediated translation are encoded by nucleic acid molecules listed in Table 12. Certain host factors involved in viral replication are encoded by nucleic acid molecules listed in Table 13. Certain host factors involved in viral trafficking, assembly and/or release are encoded by nucleic acid molecules listed in Table 14. In some embodiments, host factors are encoded by nucleic acid molecules listed in Table 11, Table 12, Table 13 or Table 14. hi certain embodiments, a host factor is encoded by a nucleic acid molecule comprising SEQ ID NO:79. In certain embodiments, a host factor comprises amino acid sequence SEQ ID NO:80.

As used herein, the phrases viral infection, viral infectivity, infection by a virus of the present invention, viral propagation, and the like, refers to the ability of a virus to carry out all steps in the viral life cycle, resulting in the production of infectious particles. Such a life cycle comprises a variety of steps including, for example, attachment, uncoating,ranscription, translation, protein processing, replication of nucleic acid molecules, assembly of viral particles, intracellular transport of viral particles, budding, release and the like. Other teps may also be included depending on the virus. One embodiment of the present invention is a method to identify a compound that modulates viral infection, the method comprising assaying a candidate compound for the ability to interact with a host factor of the present invention in such a manner as to modulate viral infection. In a preferred embodiment, the virus is hepatitis C virus. In a preferred embodiment, the method identifies a compound that inhibits viral infection by assaying a candidate compound for the ability to interact with a host factor of the present invention in such a manner as to inhibit viral infection. In another preferred embodiment, the method dentifies a compound that enhances viral infection by assaying a candidate compound for the ability to interact with a host factor of the present invention in such a manner as to enhance viral infection. As used herein, the phrases modulate viral infection, modulate infection by a virus of he present invention, modulate viral infectivity, modulate viral propagation, and the like, refers to increasing or decreasing the amount of virus present in an infected cell or patient relative to the amount of virus present in a cell or patient that has not been treated using the disclosed methods or compounds. Also encompassed is the ability to prevent viral infection. It should be appreciated that the terms amount and concentration can be used

nterchangeably. An amount of virus can also be referred to as a titer. It is also understood by those of skill in the art that the amount of virus can refer to the total number of viral particles, or it can refer to the number of viral particles that are infectious, i.e. capable of carrying out the viral life cycle, including the ability to effect another cycle of infectious particle formation. For example, in a given population of virus particles, some or all of the particles may be unable to carry out a specific step in its life cycle (e.g., attachment or entry) due to a deficiency in a molecule needed to perform that step. While the number of particles in the population may be large, the number of infectious particles could be small to none. Thus the amount of virus determined by counting virus particles may differ from that determined by measuring functional virus in, for example, a plaque assay. Accordingly methods of the present invention can be used to identify compounds that affect the total number of viral particles produced, as well as the number of infectious viral particles produced. Appropriate methods of determining the amount of virus are understood by those skilled in the art and nclude, but are not limited to, directly counting virus particles, titering virus in cell culture e.g., plaque assay), measuring the amount of viral protein(s), measuring the amount of viral nucleic acids, or measuring the amount of a reporter protein, e.g., luciferase.

Modulation of viral infection can result in a partial reduction in the amount of virus, or it can result in complete elimination of virus from a cell or patient or in prevention of viral nfection. In one embodiment of the present invention, the amount of virus is reduced by at east 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at east 70%, at least 80%, at least 90%, or at least 95%. In another embodiment, the amount of virus is reduced by a factor of at least 10, at least 100, at least 1000, or at least 10,000. In one embodiment the viral infection is completely inhibited (i.e., there are no infectious particles). n another embodiment, the amount of virus is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In a particular embodiment of the present, the amount of virus is ncreased by a factor of at least 10, at least 100, at least 1000, or at least 10,000. As used herein, a candidate compound is any compound which may have the ability o modulate viral infection by interacting with a host factor of the present invention, but that has not yet been tested for such activity. Candidate compounds encompassed by the present nvention include, but are not limited to, proteins, peptides, antibodies, lipids, nucleic acid molecules, small interfering RNA' s (siRNA's), carbohydrates, sugars, lipoproteins, small molecule compounds and combinations thereof. Such compounds can be isolated from nature (e.g., isolated from organisms) or they can be produced in a laboratory (e.g., recombinantly or synthetically). Also encompassed are compounds that are combinations of natural and synthetic molecules. Methods to isolate or produce recombinant or synthetic candidate compounds are known to those skilled in the art. Candidate compounds that are shown to interact with a host factor, or a pathway comprising a host factor, thereby inhibiting viral infection are referred to as inhibitory compounds. Thus, as used herein, an inhibitory compound of the present invention is any molecule that inhibits (e.g., reduces, eliminates or prevents) a virus of the present invention by interacting with a pro viral host factor of the present invention to reduce the host factor's amount or activity, or by interacting with an antiviral host factor of the present invention to increase that host factor's amount or activity. Inhibitory compounds of the present invention also encompass any molecule that inhibits a virus of the present invention by interacting with a pathway comprising a host factor of the present invention in such a manner as to inhibit viral infection.

Candidate compounds that are shown to interact with a host factor, or a pathway comprising a host factor, to enhance viral infection are referred to as enhancing compounds. Thus, as used herein, an enhancing compound of the present invention is any molecule that enhances infection of a virus of the present invention by interacting with an antiviral host factor of the present invention to reduce that host factor's amount or activity, or by interacting with a pro viral host factor of the present invention to increase that host factor's amount or activity. Enhancing compounds of the present invention also encompass any molecule that enhances infection of a virus of the present invention by interacting with a pathway comprising a host factor of the present invention in such a manner as to enhance viral infection.

As defined herein, a virus of the present invention is any virus, the infectivity of which is modulated by a host factor of the present invention (i.e., a host factor that modulates HCV infection), or a pathway comprising a host factor of the present invention. Using the methodology taught herein, one of skill in the art can identify viruses of the present invention. Such viruses include those in a family selected from the group consisting of adenoviridae, herpesviridae, pappillomaviridae, polyomaviridae, poxviridae, parvoviridae, hepadnaviridae, retroviridae, arenaviridae, bornaviridae, bunyaviridae, filoviridae, orthomyxoviridae, paramyxoviridae, rhabdoviridae, arteriviridae, astroviridae, caliciviridae, coronaviridae, flaviviridae, hepatitis-E-like viruses, picornaviridae, and togaviridae that require a host factor of the present invention. A preferred virus is one from the Flaviviridae family of viruses, which includes the genera Flavivirus, Pestisvirus, and Hepacivirus.

Suitable Flavi viruses for practicing the instant invention include Gadget's Gully virus, Kadam virus, Kyasanur Forrest disease virus, Langat virus, Omsk hemorrhagic fever virus, Tick-borne encephalitis virus, Louping ill virus, Aroa virus, Dengue viruses 1-4, Kedougou virus, Cacipacore virus, Koutango virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Usutu virus, West Nile virus, Yaounde virus, Kokobera virus group, Kokobera virus, Bagaza virus, Ilheus virus, Israel turkey meningoencephalomyelitis virus, Ntaya virus, Tembusu virus, Zika virus, Banzi virus, Bouboui virus,Edge Hill virus, Jugra virus, Saboya virus, Sepik virus, Uganda S virus, Wesselsbron virus, Yellow fever virus, Entebbe bat virus, Yokose virus, Apoi virus, Cowbone Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja virus, San Perlita virus, Bukalasa bat virus, Carey Island virus, Dakar bat virus, Montana myotis leukoencephalitis virus, Phnom Penh bat virus, Rio Bravo virus, hepatitis C virus, e.g., hepatitis C virus genotypes 1-6, and GB virus A and B. A preferred virus is a member of the Hepacvirus genus of viruses, with hepatitis C virus being

particularly preferred. As used herein, the term interact indicates that the candidate compound and the host factor come into sufficient physical proximity so that the compound affects the host factor in such a manner as to inhibit or enhance viral infection. Preferred compounds are those that selectively interact with a host factor of the present invention. As used herein, the terms selectively, selective, specifically, and the like, indicate the compound has a greater affinity for the host factor than it does for molecules unrelated to the host factor. One type of interaction is a binding interaction. In such an interaction the compound associates with the host factor to form a complex. An example of complex formation is the association of an antigen with an antibody or a drug with its target. According to the present invention, association of a compound with a host factor can be reversible or non-reversible. One useful measure of the strength of binding between a compound and a host factor is the dissociation constant (Kd). Formation of a complex may or may not result in modification of the host factor. Methods of measuring and analyzing interactions, including binding interactions, between a compound and a host factor are known by those of skill in the art. hi some instances, the interaction between a compound and a host factor can result in modification of the host factor. In these instances, the host factor and the compound may or may not form a complex. For example, a compound may have proteolytic activity, and may cleave the host factor thereby reducing the amount of host factor present, or affecting any activity possessed by the host factor. Examples of host factor modifications include, but are not limited to, cleavage, phosphorylation, myristylation, ligation, and the like. Suitable techniques for assaying candidate compounds for their ability to interact with host factors of the present invention are known to those skilled in the art. Such assays can be in vitro or in vivo assays. Examples of useful assays include, but are not limited to, an enzyme-linked immunoassay, a competitive enzyme-linked immunoassay, a

radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a lateral flow assay, a flow-through assay, an agglutination assay, a particulate-based assay (e.g., using particulates such as, but not limited to, magnetic particles or plastic polymers, such as latex or polystyrene beads), an immunoprecipitation assay, an immunoblot assay (e.g., a western blot), a phosphorescence assay, a flow-through assay, a chromatography assay, a polyacrylamide gel electrophoresis (PAGe)-based assay, a surface plasmon resonance assay, a spectrophotometry assay, a particulate-based assay, and an electronic sensory assay. Such assays are well known to those skilled in the art. In one embodiment, an assay can be performed in cells in culture or it can be performed in a whole animal. Assays can be designed to give qualitative, quantitative or semi-quantitative results, depending on how they are used and the type of result that is desired.

One embodiment of the present invention is a method to identify a compound that modulates viral infection, the method comprising;

(a) contacting a candidate compound with at least one isolated host factor under conditions suitable for formation of a complex between said compound and said host factor; and,

(b) detecting the presence of a complex, if present; wherein the presence of said complex indicates that the test compound modulates viral infection. Suitable methods for detecting the formation of a complex between a candidate compound and a host factor have been disclosed herein. For example, utilizing an assay technology disclosed herein, a compound can be immobilized on a substrate and the immobilized compound then brought into contact with a host factor under conditions suitable for formation of a complex. Following a wash step to eliminate non-specific binding, the presence of a complex can be detected using a detectable marker. Examples of detectable markers include, but are not limited to, a radioactive marker, a colorimetric marker, a fluorescent marker, and a chemiluminescent marker. In some instances, the detectable marker is directly linked (e.g., covalently) to the host factor. Alternatively, the complex can be detected by using a detection molecule that binds to the host factor, and that is labeled with a detectable marker. A detection molecule is any molecule that binds to the host factor, and can be used detect its presence. Examples of such molecules include, but are not limited to, antibodies, ligand, and nucleic acid molecules. Alternatively, the host factor can be immobilized on a substrate and a candidate compound introduced under conditions suitable for formation of a complex. Similar methodology to that described above can then be used to detect binding of the candidate compound to the immobilized host factor. In one

embodiment, the Kd of a complex formed by a host factor of the present invention and a compound that selectively interacts with the host factor is at least 10^-4, 10^-5, 10^'6, 10^'7, 10^'8, 10^'9, 10^-10, 10^-11 or 10^-12. In another embodiment of the present invention, binding of a host factor to a compound that selectively interacts with the host factor is irreversible. In one embodiment, a compound forms a covalent bond with the host factor. In one embodiment of the present invention, a host factor is encoded by a nucleic acid molecule having a SEQ ID NO. listed in Table 1. In another embodiment, a host factor is encoded by a nucleic acid molecule listed in Table 2. Ln certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table 5. hi certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table 5 or encoded by a nucleic acid molecule having a SEQ ID NO listed in Table 1. In certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table 11, Table 12, Table 13 or Table 14. In certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table 11. In certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table 12. In certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table 13. hi certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table 14. In certain embodiments, a host factor is encoded by a nucleic acid molecule having SEQ ID NO:79. In certain embodiments, a host factor comprises amino acid sequence SEQ ID NO:80.

Another embodiment of the present invention is a method to identify a compound that modulates viral infection, the method comprising; (a) contacting a candidate compound with at least one isolated host factor under conditions suitable for allowing an interaction between the compound and the at least one host factor; and

(b) determining if said interaction results in modification of said host factor; wherein modification of the host factor indicates that the test compound modulates viral infection.

As noted elsewhere, modifications to the host factor include changes to physical properties such as, for example, size and charge. In one embodiment, the step of determining includes determining if the host factor has been altered by, for example, enzymatic cleavage, ligation, esterification, polymerization, phosphorylation, myristylation, acetylation, deacetylation, alkylation, glutamylation, hydroxylation, sulfation, or combinations thereof. Other types of modifications known by those skilled in the art are also encompassed. In one embodiment, a host factor is encoded by a nucleic acid molecule having a SEQ ID NO. listedn Table 1. In another embodiment, the host factor is encoded by a nucleic acid moleculeisted in Table 2. In certain embodiments, a host factor is encoded by a nucleic acid molecule isted in Table 5. In certain embodiments, a host factor is encoded by a nucleic acid molecule isted in Table 5 or encoded by a nucleic acid molecule having a SEQ ID NO listed in Table

I . In certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table

I I, Table 12, Table 13 or Table 14. In certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table 11. In certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table 12. In certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table 13. In certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table 14. In certain embodiments, a host factor is encoded by a nucleic acid molecule having SEQ ID NO:79. In certain embodiments, a host factor comprises amino acid sequence SEQ ID NO:80.

In some instances a host factor can possess activity, such as enzymatic activity. For example, one host factor identified using the methods disclosed herein is encoded by the AGP AT3 gene, which encodes a protein having O-acetyltransferase activity. In instances where the host factor has enzymatic activity, interaction with a compound can increase, decrease, or completely eliminate, the enzymatic activity of the host factor. Thus in one embodiment, compounds that modulate viral infection are identified by contacting a host factor with a compound, and measuring the level of host factor-related activity. In such a method, the level of host factor-related activity is compared to the level of host factor-related activity measured in the absence of the compound. Suitable assays for measuring host factor- related activity can be determined by those skilled in the art, and depend on the type of activity being measured. In another embodiment, candidate compounds that form a complex with a host factor, or that modify a host factor can be further assayed for their ability to modulate viral infection. Such assays can be conducted in cells in culture or they can be conducted using whole animals. For example, the amount of virus produced by cells in culture in the presence and absence of a compound that interacts with a host factor can be compared. Similarly, a candidate compound that interacts with a host factor can be administered to an animal infected with a virus, and the amount of virus produced determined. One embodiment of the present invention is a method to identify a compound that modulates viral infection, which comprises (a) combining a candidate compound with a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1 under conditions suitable for such candidate compound to interact with such host factor, (b) assaying for the presence of such interaction between such candidate compound and such host factor and (c) determining if such an interaction decreases or increases viral infectivity, wherein a decrease or increase in viral infectivity indicates that the compound modulates viral infection. One embodiment is a method to identify a compound that modulates viral infection, which comprises (a) combining a candidate compound with a host factor encoded by a nucleic acid molecule listed in Table 5 or with a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1 under conditions suitable for such candidate compound to interact with such host factor, (b) assaying for the presence of such interaction between such candidate compound and such host factor and (c) determining if such an interaction decreases or increases viral infectivity, wherein a decrease or increase in viral infectivity indicates that the compound modulates viral infection. One embodiment is a method to identify a compound that modulates viral infection, which comprises (a) combining a candidate compound with a host factor encoded by a nucleic acid molecule listed in Table 5 under conditions suitable for such candidate compound to interact with such host factor, (b) assaying for the presence of such interaction between such candidate compound and such host factor and (c) determining if such an interaction decreases or increases viral infectivity, wherein a decrease or increase in viral infectivity indicates that the compound modulates viral infection. In one embodiment, viral infectivity is decreased, indicating that the compound inhibits viral infection. In another embodiment, viral infectivity is increased, indicating that the compound enhances viral infection. Viral infectivity can be measured by determining the amount of virus, as described herein. The interaction can be a binding interaction in which a complex is formed between the candidate compound and host factor or it can be an interaction in which the host factor is modified.

In some instances, a host factor of the present invention is known to interact with existing compounds (e.g., drugs) used to treat non-HCV related disease. Some of these compounds have already been shown to modulate HCV infection. For example, the cholesterol-lowering drug simvastatin, which is known to reduce the activity of the host factor diacylglycerol-2-acetyltransferase (Waterman, Diabetes (2002);51:1708-13), has been shown to inhibit HCV propagation. However, the potential of many existing compounds to modulate HCV infection was not appreciated since, prior to the present invention, it was not known that the targets of these compounds (i.e., host factors that interact with these compounds) were involved in modulating HCV infection. For example, FLT4 targeted by the drugs sunitinib and sorafenib, as well as TOPl, targeted by the drugs topotecan, camptothecan and related drugs, were not previously known to modulate HCV infection. In view of the inventor's discovery, these drugs are promising candidates for compounds capable of modulating infection by HCV and related viruses. Thus, one embodiment of the present invention is a method to inhibit viral infection by contacting a cell infected with the virus or susceptible to the virus with a compound selected from Table 3. Table 3 provides examples of host factors of the present invention, and pathways comprising such host factors, that appear to have some association with existing drugs used to treat non-HCV related disease. To the inventors' knowledge, host factors and pathways listed in Table 3 were previously unknown to be involved in modulating HCV infection. Thus drugs listed in Table 3 have previously unappreciated potential to modulate viral infection by interacting with host factors or pathways comprising one or more of such host factors.

It will be appreciated by one of skill in the art that one or more host factors can interact with each other as part of (or as an entire) pathway, and thus assays of the present invention can comprise more than one host factor. Therefore, the present invention also discloses pathways that modulate viral infectivity. Thus one embodiment of the present invention is a method to identify a compound that modulates viral infection, the method comprising assaying a candidate compound for the ability to modulate a pathway comprising a host factor of the present invention in such a manner as to modulate viral infection. In a preferred embodiment, the virus is a hepatitis C virus. In a preferred embodiment, the method identifies a compound that inhibits viral infection by assaying a candidate compound for the ability to modulate a pathway comprising a host factor of the present invention in such a manner as to inhibit viral infection. In another preferred embodiment, the method identifies a compound that enhances viral infection by assaying a candidate compound for the ability to modulate a pathway comprising a host factor of the present invention in such a manner as to enhance viral infection. As used herein, the term pathway refers to a sequence of biochemical molecules and/or related activities (e.g., reactions) that result in the production of an end product. The end product can be a biological molecule, such as a protein, or it can be an activity, such as an enzymatic activity (e.g., a phosphorylation event). As an example, the glycolytic pathway consists of several enzymatic proteins, the end product of which is pyruvate. As a further example, the rumor growth factor beta (TGF-beta) signaling pathway comprises several proteins that interact in a sequential manner, resulting in regulation of gene expression.

Examples of other biological pathways are known by those of skill in the art. A pathway of the present invention comprises at least one host factor of the present invention. It is appreciated by those of skill in the art that the end result of one pathway can be an activity that effects, or modulates, another pathway. The interaction of biological pathways is referred to in the art as a network. Examples of pathways and networks of the present invention are shown in Figures 2, 3 and 4. Because of the inter-related nature of biological pathways, it will be appreciated by those of skill in the art that modulation of one pathway frequently results in modulation of one or more other pathways. Thus methods and compounds of the present invention can modulate viral infection by modulating a pathway that directly affects viral infection, or a pathway that indirectly affects viral infection. As used herein, a pathway that directly modulates viral infection is one in which the host factors that comprise the pathway directly interact with components of the virus. In contrast, a pathway that indirectly modulates viral infection is one in which the end result of the pathway does not directly interact with components of the virus, but affects another pathway that directly modulates viral infection.

As used herein, to modulate a pathway refers to changing, varying, or altering the activity of that pathway by altering the amount of a host factor associated with the pathway, or the level of activity (e.g., enzymatic activity) resulting from a host factor or a pathway. As used herein, modulation can refer to an increase (e.g., up-regulation) or a decrease (e.g., down-regulation) in the amount of a host factor or activity. Regardless, the end result of modulating a host factor or pathway of the present invention is modulation of viral infection. In one embodiment, such viral infection is preferably inhibited. Table S3 lists pathways of the present invention.

The present invention also includes inhibitory compounds and enhancing compounds identified by the methods disclosed herein. Thus one embodiment of the present invention is an inhibitory compound for inhibiting infection by a virus of the present invention, wherein the inhibitory compound interacts with a host factor of the present invention, thereby inhibiting the viral infection. For example, an inhibitory compound can inhibit infection either by decreasing the amount or activity of a proviral host factor or by increasing the mount or activity of an antiviral host factor. A further embodiment of the present invention s an inhibitory compound for inhibiting infection by a virus of the present invention, whereinhe inhibitory compound modulates a pathway comprising a host factor of the present nvention, thereby inhibiting viral infection. For example, an inhibitory compound cannhibit infection either by decreasing the activity of a pathway required for viral infection, or by increasing the activity of a pathway that inhibits viral infection. Another embodiment of he present invention is an enhancing compound for enhancing infection by a virus of the present invention, wherein the enhancing compound interacts with a host factor of the present nvention, thereby enhancing the viral infection. For example, an enhancing compound can enhance infection either by increasing the amount or activity of a proviral host factor or by decreasing the amount or activity of an antiviral host factor. A further embodiment of the present invention is an enhancing compound for enhancing infection by a virus of the present nvention, wherein the enhancing compound modulates a pathway comprising a host factor of he present invention, thereby enhancing viral infection. For example, an enhancing compound can enhance infection either by increasing the activity of a pathway required for viral infection, or by decreasing the activity of a pathway that inhibits viral infection. As has been discussed herein, useful compounds can be isolated from a natural source or they can be synthetic. Examples of useful inhibitory and enhancing compounds include, but are not imited to, proteins, peptides, antibodies, lipids, lipoproteins, nucleic acid molecules (e.g., microRNA and small interfering RNAs), an organic molecule, a synthetic molecule, small molecule compounds, and combinations thereof.

One example of an inhibitory compound is an antibody that binds a host factor encoded by a nucleic acid molecule having a SEQ ID NO. listed in Table 1 in such a manner as to inhibit viral infection. One example of an enhancing compound is an antibody that binds a host factor encoded by a nucleic acid molecule having a SEQ ID NO. listed in Table 1 in such a manner as to enhance viral infection. One example of an inhibitory compound is an antibody that binds a host factor encoded by a nucleic acid molecule listed in Table 5 in such a manner as to inhibit viral infection. One example of an enhancing compound is an antibody that binds a host factor encoded by a nucleic acid molecule listed in Table 5 in such a manner as to enhance viral infection.

One example of a compound of the present invention is a small interfering RNA (siRNA). These molecules are short (approximately 20-30 nucleotides) double stranded RNA molecules that, in association with the RNA-induced silencing complex (RISC), pair with complementary messenger RNA resulting in the cleavage of the messenger RNA molecule, thereby reducing expression of the target gene. siRNAs useful for practicing the instant invention are listed in Table 4. siRNAs can be proviral or antiviral, depending on the host factor they target. In one embodiment, a siRNA interacts with the same host factor as that targeted by a siRNA listed in Table 4 in such a manner as to inhibit viral infection. The siRNA's can interact with the host factor at the same site, at overlapping sites, or at completely different sites.

In one embodiment, an inhibitory compound of the present invention modulates a pathway comprising a host factor of the present invention by interacting with said host factor, thereby inhibiting viral infection. In another embodiment, an enhancing compound of the present invention modulates a pathway comprising a host factor of the present invention by interacting with said host factor, thereby enhancing viral infection. Preferred host factors with which inhibitory compounds or enhancing compounds can interact are those encoded by a nucleic acid molecule listed in Table 1. In certain embodiments, host factors with which inhibitory compounds or enhancing compounds can interact are those encoded by a nucleic acid molecule listed in Table 5. In certain embodiments, host factors with which inhibitory compounds or enhancing compounds can interact are those encoded by a nucleic acid molecule listed in Table 1 or in Table 5. Types of interactions have been discussed previously. In one embodiment, the compound binds to the host factor with a Kd of at least 10^-4, 10^-5, 10^-6, 10^'7, 10^-8, 10^'9, 10^'10, 10^-11 or 10^-12. In another embodiment, the binding is irreversible. In another embodiment the compound modifies the host factor thereby modulating its activity. In one embodiment of the present invention, a compound that interacts with a host factor of the present invention reduces the amount of virus produced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%. In another embodiment, the amount of virus is reduced by a factor of at least 10, at least 100, at least 1000, or at least 10,000. In one embodiment the viral infection is completely inhibited.

Also contemplated is the use of methods and compounds of the present invention to modulate infection by viruses of the present invention. Modulation of viral infection can be effected in a patient infected with HCV, or it can be effected in cells in culture (e.g., tissue culture). Thus one embodiment of the present invention is a method to inhibit viral infection, the method comprising contacting a cell infected with the virus with an inhibitory compound that interacts with a host factor of the present invention in such a manner as to inhibit viral nfection. In a preferred embodiment, the cell is infected with hepatitis C virus. In some circumstances it may be desirable to increase the amount of virus produced during annfection. For example, during the production of a vaccine, it would be useful to be able toncrease the yield of virus obtained from a culture or animal. Thus one embodiment of the present invention is a method to enhance viral infection, the method comprising contacting annfected cell with an enhancing compound that interacts with a host factor in such a manner as to increase the amount of virus produced. In a preferred embodiment, the cell is infected with hepatitis C virus.

As used herein, the term contacting refers to bringing the compound and the cell into proximity so that the compound is capable of interacting with a host factor of the present nvention. Such contacting can be achieved by introducing the compound to the cell when he cell is in a tissue culture environment, or it can be achieved when the cell is present in a whole body. Consequently contacting the compound with the infected cell can be achieved hrough introducing the compound into a patient, for example, through an oral medication, an njection or other route of administration. The compound can interact with and remain on outside of the cell, or it can enter the cell and interact with a host factor within the cell.

Regardless, the end result of such contact is modulation of a pathway comprising a host factor of the present invention.

One embodiment of the present invention is a method to inhibit viral infection, the method comprising contacting a cell with an inhibitory compound that modulates a pathway comprising a host factor of the present invention in such a manner as to inhibit viral infection. Another embodiment of the present invention is a method to enhance viral infection, the method comprising contacting a cell with an enhancing compound that modulates a pathway comprising a host factor of the present invention in such a manner as to enhance viral infection. Suitable host factors are those encoded by a nucleic acid molecule having a SEQ ID NO. listed in Table 1. Preferred host factors are those encoded by nucleic acid molecules listed in Table 2. In certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table 5. Li certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table 5 or encoded by a nucleic acid molecule having a SEQ ID NO listed in Table 1. hi certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table .11, Table 12, Table 13 or Table 14. In certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table 11. In certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table 12. In certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table 13. In certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table 14. In certain embodiments, a host factor s encoded by a nucleic acid molecule having SEQ ID NO:79. In certain embodiments, a host factor comprises amino acid sequence SEQ ID NO: 80. As discussed previously, an inhibitory compound of the present invention is useful for nhibiting infection by a virus of the present invention. Consequently such a compound can be used to protect a patient from viral infection, such as hepatitis C infection. As used herein, he term protect refers to prophylactic as well as therapeutic use. Thus, one embodiment of he present invention is a method to prevent viral infection in a patient capable of being nfected by a virus of the present invention by administering a pharmaceutical composition comprising an inhibitory compound that interacts with a host factor of the present invention n such a manner as to inhibit viral infection.

Another embodiment is a method to reduce the amount of virus in a patient infected with a virus, said method comprising administering a pharmaceutical composition comprising an inhibitory compound that interacts with a host factor of the present invention. In one embodiment, the patient is infected with Flavivirus. In a preferred embodiment, the patient is nfected with hepatitis C virus.

Another embodiment is a method to protect a patient from virus infection, comprising administering to a patient an inhibitory compound that modulates a pathway comprising a host factor of the present invention in such a manner as to inhibit viral infection. Suitable host factors are those encoded by a nucleic acid molecule having a SEQ ID NO. listed in Table 1. A preferred host factor is one encoded by a nucleic acid molecule listed in Table 2. hi certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table 5. In certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table 5 or encoded by a nucleic acid molecule having a SEQ ID NO listed in Table 1. In certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table 11, Table 12, Table 13 or Table 14. In certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table 11. In certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table 12. In certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table 13. In certain embodiments, a host factor is encoded by a nucleic acid molecule listed in Table 14. In certain embodiments, a host factor is encoded by a nucleic acid molecule having SEQ ID NO:79. In certain embodiments, a host factor omprises amino acid sequence SEQ ID NO:80. In a preferred embodiment, the patient isnfected with hepatitis C virus.

Examples of inhibitory compounds include, but are not limited to, inhibitory ompounds that inhibit viral entry, viral translation, viral replication or viral trafficking, assembly or release. In certain embodiments, an inhibitory compound inhibits viral entry. In ertain embodiments, an inhibitory compound inhibits viral translation. In certain embodiments, an inhibitory compound inhibits viral replication. In certain embodiments, an nhibitory compound inhibits viral trafficking, assembly or release. Examples of preferred nhibitory compounds are listed in Table 3. Table 3. Drugs and Small Molecule Inhibitors that Affect Pathways and Host Factors of the Present Invention

A. Drugs

* - data obtained on July 22, 2009, from the PharmacoGenomics Knowledge Base maintained by Stanford University at http://www.pharmgkb.org/

T) = the product of the gene is the direct drug target

B. Small Molecule Inhibitors

- all data obtained July 22, 2009

In one embodiment, a preferred inhibitory compound is a siRNA, such as those listed in Table 4.

Table 4. Small Interfering RNA's that Interact with Host Factors of the Present Invention

In certain embodiments, an inhibitory compound can be colchicine, cytochalasin, EIPA, rottlerin, UO 126, wortmannin or a mixture of two or more of such compounds, hi certain embodiments, an inhibitory compound can be brefeldin A, golgicide A or a mixture thereof. LQ certain embodiments, an inhibitory compound can be colchicine. In certain embodiments, an inhibitory compound can be, cytochalasin. In certain embodiments, an inhibitory compound can be EIPA. In certain embodiments, an inhibitory compound can be rottlerin. In certain embodiments, an inhibitory compound can be UO 126. In certain embodiments, an inhibitory compound can be wortmannin. hi certain embodiments, an inhibitory compound can be brefeldin A. In certain embodiments, an inhibitory compound can be golgicide A.

Another embodiment of the present invention is a pharmaceutical composition. As used herein, a pharmaceutical composition comprises an inhibitory compound that interacts with a host factor of the present invention and a pharmaceutically acceptable vehicle, such as a diluent, carrier, excipient, adjuvant or a combination thereof. Selection of such a vehicle is known to those skilled in the art. As used herein the term "patient" refers to an animal infected with a virus of the present invention. The animal can be a human or a non-human animal. A preferred animal o treat is a mammal.

A compound of the present invention, or a pharmaceutical composition thereof can be administered to a patient by a variety of routes, including, but limited to, by injection (e.g., ntravenous, intramuscular, subcutaneous, intrathecal, intraperitoneal), by inhalation, by oral e.g., in a pill, tablet, capsule, powder, syrup, solution, suspension, thin film, dispersion or emulsion.), transdermal, transmucosal, pulmonary, buccal, intranasal, sublingual,

ntracerebral, intravaginal rectal or topical administration or by any other convenient method known to those of skill in the art.

The amount of a compound of the present invention and/or a pharmaceutical composition thereof that will be effective can be determined by standard clinical techniques known in the art. Such an amount is dependent on, among other factors, the patient being reated, including, but not limited to the weight, age, and condition of the patient, the ntended effect of the compound, the manner of administration and the judgment of the prescribing physician.

A compound of the present invention, or a pharmaceutical composition thereof, can be administered alone or in combination with one or more other pharmaceutical agents, ncluding other compounds of the present invention. A compound can be administered or applied per se or as pharmaceutical compositions. The specific pharmaceutical composition depends on the desired mode of administration, as is well known to the skilled artisan.

In a preferred embodiment, a patient is protected using a composition comprising an inhibitory compound that inhibits viral infection by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%. In another embodiment, the amount of virus is reduced by a factor of at least 10, 100, 1000, or 10,000. In one embodiment the viral infection is completely inhibited.

The present invention also includes kits to identify a compound that modulates viral infection. One embodiment is a kit to identify an inhibitory compound, the kit comprising a means for determining if a candidate compound is capable of inhibiting viral infection by interacting with a host factor of the present invention. Another embodiment is a kit to identify an enhancing compound, the kit comprising a means for determining if a candidate compound is capable of enhancing viral infection by interacting with a host factor of the present invention. Kits can also comprise means for identifying both inhibitory compounds and enhancing compounds. Suitable and preferred means are disclosed herein. As such, a kit can also comprise host factors of the present invention. The kit can also contain associated reagents and components, such as, but not limited to, buffers, labels, containers, inserts, tubings, vials, syringes and the like.

The following Examples are provided for the purpose of illustration and are not intended to limit the scope of the invention.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the embodiments, and are ot intended to limit the scope of what the inventors regard as their invention nor are they ntended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, emperature, etc.) but some experimental errors and deviations should be accounted for.

Standard abbreviations may be used. For example, amino acids can be denoted by either the tandard 3 -letter or 1 -letter code.

Example 1.

This Example illustrates the identification of host factors required for completion of he HCV life-cycle. iRNA screen: To identify host factors required for HCV infection, a high-throughput RNAi- based screen was undertaken on an arrayed library targeting 19,470 genes (Dharmacon iARRAY siRNA Library (Human Genome, G-005000-05, Thermo Fisher Scientific, Lafayette, CO, the remaining 1,651 pools of the original 21,121 gene library were not creened because the vast majority have been retired due to revised refseq annotation). Part one of the screen: siRNAs were transiently transfected into the Huh 7.5.1 cells at a 50 nM final concentration, using a reverse transfection protocol employing 0.44% Oligofectamine (Invitrogen, Carlsbad, CA) in a 384-well format (384 well, black plastic, clear bottomed assay plate, Corning 3712). The Oligofectamine was diluted in Opti-MEM (Invitrogen) and allowed to incubate ten minutes. The lipid solution was then aliquoted into the wells (9 ul/well) using a Wellmate liquid handing robot (Thermo Fisher Matrix). The plates were spun down at 1,000 RPM and the arrayed siRNAs were added robotically, 1.5 μL of a 1 μM stock solution per well. After a twenty minute incubation, 800 Huh 7.5.1 cells were added per well, in 20 μL of Dulbecco's modified minimal essential media (DMEM, Invitrogen), supplemented with 15% fetal bovine serum (FBS, Invitrogen). The plates were next spun at 1000 RPM and then placed in a tissue culture incubator at 37⁰C and 5% CO2. The subsequent day, 5 μL of fresh complete media (10% FBS) was added to the outer two wells at the plate margins to decrease edge effects. After 72 h of siRNA-mediated gene knockdown the medium was removed and the cells were infected with the JFH-I genotype 2a virus, at an MOI of approximately 0.2-0.4 in 40 μL DMEM with 10% FBS (which translated to 12-20%nfected cells at 48 h post infection as determined by HCV core staining and Hoechst 33342 DNA staining of nuclei). After an additional 48 h incubation (when silencing is still operative), 30 ul of media was removed and replica plated onto a new 384 well plate, which had received 2500 Huh 7.5.1 cells per well 24 h prior to viral supernatant addition (beginning of part two of screen). The "part one" cells were then fixed with 4% Formalin, permeabilized with 0.3% Triton-XIOO (Sigma) in Dulbecco's Phosphate Buffered Saline (D-PBS, nvitrogen) containing 3% FBS and 3% bovine serum albumin Fraction V (BSA, Sigma Aldrich), then stained for HCV core, using purified anti-HCV core monoclonal antibody produced from the anti-HCV core 6G7 hybridoma cells generously provided by Drs. Harry Greenberg and Xiaosong He, Stanford University) diluted in D-PBS with 1% BSA. The cells were then incubated with an Alexa 488 goat anti-mouse secondary at 1:1,000 in D-PBS with 1% BSA (Al 1001, Invitrogen), and stained for DNA with Hoechst 33342 at 1 :5,000-10,000 Invitrogen). Each step was followed by two washes with 30 μL D-PBS. The cells were then maged on an automated Image Express Micro (IXM) microscope (Molecular Devices) at 4X magnification, using two wavelengths, 488 nm to detect HCV infected cells expressing core, and 350 nm for nuclear DNA bound by Hoechst 33342. Images were then analyzed using the Metamorph Cell Scoring software program (Molecular Devices Inc.) to determine the total cells per well, and the percentage of core positive cells in each well (percent infected). A negative control (NT, siCONTROL Non-Targeting siRNA #2, Dharmacon D-OO 1210-02), and positive control siRNA SMARTpools against CD81 (Dharmacon SMARTpool M- 017257-02) and ApoE (Dharmacon SMARTpool M-006470-00) were present on each plate. In addition wells containing either buffer alone, or an siRNA pool directed against Polo like kinase one (PLKl, Dharmacon) were present on all plates transfected. The screen was performed in triplicate.

TaqMan Real-Time PCR Analysis: To determine the amount of HCV RNA, total RNA was extracted with QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA) from 140 μL of culture medium, or with RNeasy Mini Kit (Qiagen) from whole cell lysate. Copy numbers of HCV RNA were determined by quantitative PCR with the probe and primers listed below, using the TaqMan EZ RT-PCR CORE REAGENTS (Applied Biosystems) on an ABI 7500 Real Time PCR System (Applied Biosystem). PCR parameters consisted of 1 cycle of 50°C X 2 min, then 60 °C X 30 min, and 95°C X 3:30 min, followed by 50 cycles of PCR at 95 °C X 0 s, and 62 °C X 1 min. The relative amount of HCV RNA was normalized to the internal ontrol Human 18S rRNA (Applied Biosystems). HCV TaqMan probe used was 6-FAM CTGCGGAACCGGTGAGTACACTAMRA (IDT, Coralville, IA). HCV primer sequences were 5' CGGGAGAGCCATAGTGG, and 3' AGTACCACAAGGCCTTTCG. To quantify he expression levels of CD 81 and ApoE, total RNA was prepared from whole cell lysate with RNeasy Mini Kit according to manufacturer's instructions. Complementary DNA cDNA) was synthesized from total RNA with First Strand cDNA Synthesis Kit (Roche, ndianapolis, IN). The mRNA expression levels of CD81 and ApoE were quantified by quantitative PCR using a TaqMan Gene Express Master Mix (Applied Biosystems) on an ABI 7500 Real Time PCR System. PCR parameters were 1 cycle of 50°C X 2 min, then 95°C X 10 min, followed by 50 cycles of PCR at 95 °C X 15 s, and 60 °C X 1 min. Each reaction was performed in duplicate, and the relative amount of target gene mRNA was normalized to he internal control Human 18S rRNA. The primers and probes used were TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA). Accession Numbers: CD81 HsOOl 74717-ml, and ApoE Hs00171168-ml. iRNA screen: Part one: siRNAs were transiently transfected into the Huh 7.5.1 cells at a 50 nM final concentration, using 0.44% Oligofectamine (Invitrogen, Carlsbad, CA) in a 384- well format (384 well, black plastic, clear bottomed assay plate, Corning 3712). The next day, 5 μL of fresh complete media (10% FBS) was added to the outer two wells at the plate margins to decrease edge effects. After 72 h of siRNA-mediated gene knockdown, the medium was removed and the cells were infected with the JFH-I genotype 2a virus, at an MOI of approximately 0.2-0.4 in 40 μL DMEM with 10% FBS. After 48h, 30 μl of media was removed and replica plated onto a new 384-well plate with 2500 Huh 7.5.1 cells per well plated 24 h earlier for part 2. The "part one" cells were then fixed with 4% Formalin and stained with anti-HCV core 6G7 monoclonal antibody provided by Drs. Harry Greenberg and Xiaosong He, (Stanford University) using an Alexa 488 goat anti-mouse secondary antibody at 1:1,000 (AI lOOl, Invitrogen) and cells were imaged on an automated Image Express Micro (IXM) microscope (Molecular Devices) Images were then analyzed using the Metamorph Cell Scoring software program (Molecular Devices Inc.) Part two: As noted, to search for host factors whose depletion leads to defects in producing HCV infectious articles, 30 μL of conditioned media containing JFH-I HCV virus from each well in the first ound screen was removed prior to fixation and transferred to a new well containing ninfected Huh 7.5.1 cells. 48 h later these cells were stained for HCV core expression and maged identically to samples in part one described above.

siRNA pools were classified as hits (decreased infection) if the average of the riplicate plates showed that the percentage of core positive cells was less than 50% of the plate mean, and cell number was not less than 50% of the mean of the plate. Pools which ncreased infection by greater than 150% of the plate mean were also selected as hits increased infection).

Preferred host factors identified by the disclosed screens are listed in Table 2. Table 2. Preferred Host Factors

The validation round screen in which the four individual oligos comprising each pool were placed into separate wells, and screened again using identical methods as above. siRNA pools were considered validated if two or more of the individual oligos scored (50% or less infected cells (decreased infection) or 150% or greater infected cells (increased infection)) as compared to the negative control wells on the plate, in either both part one and two or part two alone, and the cell number was not less than 40% of the average of the negative control wells on the plate. The percent of infected cells relative to controls, as well as the normalized cell numbers for each of the individual genes that confirmed with two or more siRNAs is provided in Table Sl. Visual spot inspections of control images were done throughout the screen to confirm the accuracy of the automated imaging and cell scoring systems.

Table SlB

Cell lines and culture conditions: Huh 7.5.1 cells were grown in DMEM supplemented with 10% FBS.

Viral propagation and titration: JFH-I was propagated and viral infectivity was titrated as reviously described (Wakita (2005) Nat Med 11, 791-796; Kato (2007) J Virol 81, 4405-4411.).

Bioinformatic Meta-analysis:

Enrichment analyses: siRNA screen hits were classified into molecular function and biological process categories according to the Panther classification system, and statistical enrichment of hese categories were assessed for the set of hit genes relative to their representation in the global et of genes examined in the siRNA screen. P-values were computed using the hypergeometric distribution, implemented in the R statistical computing language.

Network analyses: Networks were generated with data extracted from interactome screens and urated literature entries in HPRD (Keshava (2009) Nucleic Acids Res 37, D767-772.) and proteome-wide mapping efforts from the I-MAP project examining HCV-host protein nteractions (de Chassey (2008) MoI Syst Biol 4, 230.). Network constructions were implemented n the Perl programming language and incorporate graph theoretical representations by abstracting gene products as nodes and interactions as edges.

A two-part screen was used to identify host proteins required for virus infection. This creen utilizes the HCVcc system, which consists of the infectious JFH-I genotype 2a virus and he Huh 7.5.1 human hepatocellular cell line (Brass (2008) Science 319, 921-926.).

In the first part of the screen, Huh 7.5.1 cells were transfected with small siRNAs for 72 hours, and then challenged with JFH-I virus. After 48 h the cells were stained and imaged for expression of the HCV core protein as a marker^* for productive viral infection (Fig IA). To find host factors involved in later stages of viral infection, we undertook part two of the screen, by exposing fresh Huh 7.5.1 cells to culture supernatant from part one for 48 h, then detecting core protein expression. This approach detects proteins needed for the complete viral lifecycle, from viral-host receptor binding to the successful completion of a second round of viral infection. In addition, genes functioning in either anti-viral responses or safeguarding the cells against the stress of infection, may be detected in this screen. The screen was optimized using siRNAs against the host proteins, CD81 (part one) and ApoE (part two). ApoE, a host protein involved in lipoprotein biosysnthesis, has been shown to e required for infectious particle formation (Hao (2008) Nature 454, 890-893; Huang (2007) Proc Natl Acad Sci U S A 104, 5848-5853; Chang (2007) J Virol 81, 13783-13793.). siRNAs gainst CD81 resulted in inhibition of part one infection (5-6 fold, Fig IA, SlA, B). siRNAs argeting ApoE, however, did not affect infection in part one, but did inhibit HCV infection in art two by 2-3 fold, in keeping with its previously assigned role in infectious particle formation Fig IA, SlA, B). The levels of infection were verified using quantitative RT-PCR and found to e directly proportional to the levels determined microscopically (Fig Sl C, D). The image-based latform was then used for a genetic screen with a commercially available siRNA whole-genome brary (Dharmacon siGenome, 19,470 pools of four siRNAs per gene). Pools were classified as its (decreased infection) if the percentage of infected cells was less than 50% of the plate mean n either part one and two, or in part two alone, and did not decrease cell numbers to less than 0% of the plate mean in part one. Using these criteria we selected 407 pools for further alidation (2% of the total genes screened, Fig IB & Table S2). siRNA pools whose transfection ncreased core protein expression by greater than 150% of the plate mean in either part one or wo, were also selected (increased infection) for validation (114 pools, 0.6%, Fig 1C & Table 2). hi the validation round, we tested the four non-overlapping siRNAs from each pool eparately, and found that 263 out of the 521 total pools selected (50%) were confirmed with at east two siRNAs, decreasing the likelihood of off-target effects (Table Sl, Materials and Methods, the list of all genes scoring in the primary round is shown in Table S2 (Echeverri (2006) Nat Methods 3, 777-779.). Forty-five genes decreased viral infection with multiple siRNAs predominantly in part two, implicating them primarily in late-stage viral infection (Table Sl).

siRNA screen bioinformatics and meta-analysis.

Among the host factors whose knockdown decreased HCV infection in the primary screen, we recovered both positive controls, CD81 and ApoE, and over thirty proteins previously implicated by functional genetic studies to be needed for the replication of HCV or West Nile virus (WNV), a related Flavivirus (Table Sl and S2, (Yang (2008) J Virol 82, 5269-5278. Flisiak (2008) Hepatology 47, 817-826.). The screen recovered 6 of the 26 HCV-associated host proteins determined by Randall et. al. to decrease JFH-I levels by greater than 3-fold after intensive siRNA targeting (Table S2, (Randall (2007) Proc Natl Acad Sci U S A 104, 12884- 2889.)). Among these shared candidates are the kinases MAPKl and Rafl/MAPK3₅ the RNA- inding protein, ELAVLl and the RNA-helicase, DDX3X. Proteomic studies, including the HCV infection mapping project (I-MAP) and experiments by Randall et al., have reported a irect physical interaction between Rafl/MAPK3, Stat3 and DDX3X and one or more of the HCV-encoded proteins (Randall (2007) Proc Natl Acad Sci U S A 104, 12884-12889; de Chassey (2008) MoI Syst Biol 4, 230.). This screen detected 15 of 96 factors recently reported by ai et. al. in a whole-genome siRNA screen using an HCV sub-genomic replicon platform (Table 2, (Tai (2009) Cell Host Microbe 5, 298-307.)); the common genes include the kinases PI4KA nd NUAK/SNARK, and the enzymes HASl, CTSF, and EDS. NUAK/SNARK, an AMPK- elated kinase functioning in the cellular stress response, was one of nine host factors found by Ng et. al. to be necessary for HCV replicon function in a siRNA screen targeting -4,000 genes Ng (2007) Hepatology 45, 1413-1421; Kuga (2008) Biochem Biophys Res Commun 317, 1062- 066.). A critical role for PI4KA was also apparent in an HCVcc-based screen using a preelected library of genes implicated in endocytic trafficking (Berger (2009) PNAS.). In that tudy, 7 of the 140 genes tested were needed by HCV, including the actin-regulating kinase, ROCK2, which we also recovered in our screen (Berger (2009) PNAS; Riento K (2003) Nat Rev MoI Cell Biol 4, 446-456.). Consistent with Tai et al., we observed that multiple sub-units ARCNl, COPA, and COPBl) of the Golgi-associated retrograde transport complex, coatomer 1 COPl), were needed for JFH-I infection, albeit with cell numbers sufficiently decreased to reclude these genes from further validation (Table S2 (Tai (2009) Cell Host Microbe 5, 298- 07.)). The essentialness of two of the COPl subunits (COPA and COPB2) for cell viability, as well as for HCV replication, was similarly observed in the trafficking-focused screen (Berger (2009) PNAS.).

Interestingly, the study detected 14 genes in common with those previously found in an RNAi screen for human proteins required for WNV infection, among them the enzymes

BCKDHA, RPS6KL1, and USPl 1 (Table S2 (Krishnan (2008) Nature 455, 242-245.)). The WNV siRNA screen was designed to detect host factors involved in viral entry through genome replication, and thus would not find genes detected in part two of our study. However, the small degree of overlap (4.9%), between the host genes needed by these related viruses could arise in part from viral-specific host factor requirements, as well as differences in experimental methodologies. For example, none of the HCV siRNA screening efforts have identified the ER- ssociated-degradation pathway (ERAD) as important for viral replication, even though multiple omponents of this pathway were required for WNV replication (Krishnan (2008) Nature 455, 42-245.), indicating differential dependencies.

Analysis for functionally enriched gene categories demonstrates a significant number of HCV host factors reside in the nucleus (82, p=0.002). This includes ribonucleoprotein complex omponents (e.g. RBM22, SRRM2, UBA52), and transcription factors (e.g. BRFl, E2F2, RCC5, FOXE3, MLXIPL, SMAD5, SMAD6, TRRAP, WWTRl). Classification of the host enes into higher level molecular function and biological process categories showed significant nrichment for kinases (14 in total, p=0.01), factors involved in protein metabolism and modification (p=0.03), oncogenesis (p=0.006), nucleic acid binding (p=0.01) and nucleic acid metabolism (p=0.03, Figure SlE and F, and Table S3).

We encountered several pathways and macromolecular complexes consistent with known phases of the viral lifecycle. As noted, HCV entry depends on the presence of four specific host cell receptors, and our screen recovered two of the four, CD81 and claudin 1 (Evans (2007) Nature 446, 801-805.). Our results confirm recent experiments which identified cyclophilin A (CypA) as the specific host cell peptidyl-prolyl cis-trans isomerase required for HCV infection (Yang (2008) J Virol 82, 5269-5278.). Therefore, CypA is the likely target of the anti-HCV non-immunosuppressive cyclosporin analogue, Debio-025, thus demonstrating that functional genomics can identify high-yield leads for host-directed anti-viral therapies (HDAVs) (Flisiak (2008) Hepatology 47, 817-826.). Depletion of three of five components of the signal peptidase complex (SPCSl, 2 and 3), which is believed to process the HCV envelope proteins El and E2 in the rER (Lindenbach (2005) Nature 436, 933-938.), inhibited infection specifically in part two, validating both the role of this complex in HCV pathogenesis (P=O-HeIO^-4) and the design of this portion of the screen. Studies have shown the importance of the specific glycosylation of the El and E2 viral envelope proteins for HCV entry (Goffard (2003) Biochimie 85, 295-301.), suggesting an explanation for the role of OST48/DDOST, a component of the rER oligosaccharyltransferase complex, which we detected as a late- acting HCV host factor. Previous efforts to find HIV-dependency factors (HDFs) similarly identified OST48 as being required for late-stage viral replication, consistent with its playing an important role in glycosylation of the viral envelope spike (Brass (2008) Science 319, 921-926.).

Previously unappreciated HCV dependencies were also detected. Components of the nucleolus were well represented in the nuclear-associated factors enriched for by the screen (TOPl, TCOFl, PNOl, NOP2, NOL6, NOP5/NOP58, NOC4L, HEATRl and IMP4). TCOFl physically associates with nucleolar proteins, and is the gene responsible for Treacher Collins Syndrome, a severe congenital craniofacial developmental disease (Sakai D (2009) Int J Biochem Cell Biol 41, 1229-1232.). Knockout mice deficient in TCOFl display ribosome biogenesis deficiencies suggesting that cells depleted in TCOFl may be unable to meet the increased demand for protein production required by HCV (Sakai (2009) Int J Biochem Cell Biol 41, 1229-1232.). In addition, Cherry et al. demonstrated that the IRES-mediated transcription of the picornavirus, Drosophila C, was dependent on the levels of specific ribosomal sub-units (Cherry (2005) Genes Dev 19, 445-452.). Decreased levels of nucleolar factors may adversely impact HCV IRES function by altering ribosomal subunit levels or modifications. Four of nine components of the Ccr4-Not complex (CNOTl, CNOT2, CNOT3, and CNOT6L) were required for HCV infection. The Ccr4-Not complex is a global regulator of gene expression that functions in transcription and polyadenylation (Collart (2004) Prog Nucleic Acid Res MoI Biol 77, 289-322.). Mutation of each of the components of the orthologous yeast Ccr4-Not complex has shown that the each subunit regulates the expression of a largely unique set of genes (Azzouz (2009) RNA 15, 377- 383; Cui (2008) MoI Genet Genomics 279, 323-337.). Therefore, the components identified in this screen may control distinct sets of HCV host factors. Interestingly, three mitochondrial ribosomal subunits (MRPLl 5, 38 and 48) and several mitochondrial- associated proteins (BCKDHA, CYBA, HCCS₃ PECR, PEMT, PPTC7, PTCDl, SURFl (Pagliarini (2008) Cell 134, 112-123.)) were found to be required for viral infection.

Integration of HCV-host factor Interactions

To provide a more comprehensive view of HCV-host interactions, we integrated the functional genomic results of our study with the proteome-wide mapping and literature mining data of the HCV infection mapping project (I-MAP), undertaken by Chassey et. al. (Fig 2, (de Chassey B (2008) MoI Syst Biol 4, 230.)). The I-MAP utilized parallel yeast two-hybrid protein interaction screens employing HCV proteins as prey and two human cDNA libraries as bait. The resulting protein-protein interaction (PPI) network was then augmented and extended using expertly curated literature mining and a large human PPI database (de Chassey (2008) MoI Syst Biol 4, 230.). In addition to providing functional evidence for the importance of nine host factors found by the I-MAP (CHUK, CTGF, DDX3X, PI4KA, RAFl₃ STAT3, FBLN5, RNF31, SMURF2), this network extension analysis revealed numerous first-order (direct) interactions between 63 HCV host factors found in this screen and those factors found in the I-MAP (enrichment p=0.0001, Fig 2). This places the candidates detected using functional genomics into the interaction neighborhood of the HCV proteome (core, E₃ F₃ p7 and NS components, Fig

3).

HCV₃ WNV and dengue virus belong to the Flaviviridae family, whose members include the human pathogens Japanese encephalitis virus and yellow fever virus (Murray (2008) Nat Rev Microbiol 6, 699-708; Lindenbach (2003) Adv Virus Res 59, 23-61.). We explored the possibility of uncovering common features presented by host-Flaviviridae interactions. Among the overlapping hits and first-order interactions between host factors required for HCV, WNV and dengue virus siRNA screens (Krishnan (2008) Nature 455, 242-245.), a striking enrichment of key pathway components and shared common network modules involved in TGF-β signaling (p=0.0002), ErbB signaling (p=0.002), MAPK signaling (p=0.004), focal adhesion (p=0.006) and ubiquitin-mediated proteolysis (p=0.04) was observed (Fig 4A). These could represent potentially important common functional modules utilized by Flaviviridae. An intriguing observation that may delineate the portion of the viral lifecycle that is dependent on TGF-β, is the recurrence of common network modules associated with TGF-β signaling (p<0.0005) when we examined overlapping siRNA hits and interactions from this screen and those reported using the HCV replicon system (Fig 4B₅ C (Tai (2009) Cell Host Microbe 5, 298-307.)).

An estimated 25%-30% of HIV-infected individuals are chronically co-infected with HCV (Andersson (2006) Clin Liver Dis 10, 303-320, viii.). Defining host factors and pathways that both HIV and HCV depend on could suggest a common treatment strategy. Comparing the host factors recovered from the HCVcc and HFV siRNA screens reveals ten genes, DDX3X, DNAJBl, ETFl, HEATRl, MAP4, NMTl, OST48, SPCS3, SUV420H1 and Rap9p40/RABEPK that are needed for replication of both viruses (Brass (2008) Science 319, 921-926.). Rab9p40 functions in the movement of late endosomes to the trans Golgi network (TGN), and along with its interaction partners, Rab9 and PIKfyve, is required for the particle assembly of multiple enveloped viruses (HFV, Marburg, Ebola and measles viruses), though the mechanism remains poorly understood (Murray (2008) Nat Rev Microbiol 6, 699-708; Murray (2005) J Virol 79, 11742-11751; Shisheva (2008) Cell Biol Int 32, 591-604.). This study, and that of Tai et al., now add HCV to the list of Rab9p40-dependent viruses.

Example 2

This Example demonstrates the identification points in the HCV life-cycle affected by knock-down of particular host-factor genes. siRNA Library and Functional Genomic Screens: Genes identified by the genome- wide siRNA screen in Example 1 served as a starting point for defining the comprehensive interactions between HCV and host. The life cycle of HCV infection broadly encompasses viral entry, intracellular trafficking, viral RNA translation and replication, and virion assembly and release. Each of these steps engages in a complex set of host cell machineries. To functionally validate the HCV host dependencies discovered from the siRNA screen, and to study how these host factors are relevant for the various steps of HCV life cycle, 199 of the most significant primary screen hits and 33 other cellular genes designated as putative false-negative hits by hypothesis-driven follow-up studies were selected for further analysis. These genes are listed in Table 5.

High-throughput screens with various in vitro HCV model systems (HCVcc, HCVpp, and subgenomic replicons) were undertaken using a custom-designed arrayed library targeting these 232 genes (Dharmacon ON-TARGETplus SMARTpool siRNA library). ON- TARGETplus pools were used to significantly reduce off-target effects while maintaining siRNA potency, and hence provide a highly effective tool for validation and functional stratification following primary screen with the siGENOME SMARTpools.

A. Identification of host factors that affect the early stages of the HCV life-cycle

Core Staining: siRNAs were transiently transfected into the Huh7.5.1 cells at a final concentration of 50 nM, using a transfection protocol employing 0.44% Oligofectamine (Invitrogen) in a 96-well format. Briefly, this protocol is as follows: Oligofectamine was diluted in Opti-MEM (Invitrogen) and incubated for 10 min before being distributed into wells using a Wellmate liquid handling robot (Thermo Fisher Matrix). Following distribution, the plates were spun down at 1,000 RPM and the arrayed siRNAs added. The plates were incubated for 20 minutes, after which 4,500 Huh 7.5.1 cells, in 60 μL Dulbecco's modified minimal essential media (DMEM, Invitrogen) supplemented with 15% FBS (FBS, Invitrogen), were added to each well. The plates were spun at 1,000 RPM and then placed in a humidified tissue culture incubator at 37°C and 5% CO₂. After 72 h of incubation, to allow for siRNA-mediated gene knockdown, the medium was removed and the cells were infected with the JFH-I genotype 2a virus, at an MOI of 0.5 in 100 niL DMEM with 10% FBS. At 48 h post-infection (when silencing is still operative), 80 mL media was removed and used in "late-stage" experiments, described below. The cells were then fixed with 4% formalin, permeabilized with 0.3% Triton- XlOO (Sigma) in Dulbecco's phosphate buffered saline (D-PBS, Invitrogen) containing 3% FBS and 3% BSA Fraction V (BSA, Sigma Aldrich), then stained for HCV core, using purified anti-HCV core monoclonal antibody (produced from the anti-HCV core 6G7 hybridoma cells) diluted in D-PBS with 1% BSA. The cells were then incubated with an Alexa Fluor 488 goat anti-mouse secondary (A-11001, Invitrogen) at 1:1,000 in D-PBS with 1% BSA, and stained for DNA with Hoechst 33342 at 1:5,000-10,000 (Invitrogen). Each step was followed by two washes with D-PBS. The cells were then imaged on an automated Image Express Micro (IXM) microscope (Molecular Devices) at 4x magnification, using two wavelengths: 488 nm to detect HCV infected cells expressing core, and 350 nm for nuclear DNA bound by Hoechst 33342. Images were then analyzed using the Metamorph Cell Scoring software program (Molecular Devices Inc.) to determine the total cells per well, and the percentage of core positive cells in each well (percent infected).

B. Identification of host factors affecting late-stages of the HCV life-cycle

The 80 ml of medium removed from the infected cells in part A were replica plated onto a new 96-well plate, which contained 10,000 Huh 7.5.1 cells per well plated out 24 h before viral supernatant addition. At 48 h post-infection, the cells were fixed, stained, and imaged using the staining and imaging protocol described in part A.

The experiment described in parts A and B above was performed in triplicate. siRNA pools were classified as hits if the average of the triplicate plates showed that the percentages of core positive cells were decreased (proviral) or increased (antiviral) for more than 1.5 -fold comparing with that of non-targeting control siRNA. The results of these experiments are shown in Table 6.

Table 6. Identification of Host Factors Affecting Early and Late Stages of the HCV Life-Cycle

C. Identification of host factors affecting HCV RNA levels

HCV RNA Quantification: Infected cells were prepared as described above in parts A and B. The level of extracellular and intracellular HCV RNA was determined as follows: Total RNA was extracted with QIAamp Viral RNA Mini Kit (Qiagen) from 140 mL culture medium, or with RNeasy Mini Kit (Qiagen) from whole-cell lysate. Copy numbers of HCV RNA were determined by quantitative PCR with the probe and primers listed below, using the TaqMan EZ RT-PCR CORE REAGENTS (Applied Biosystems) on an ABI 7500 Real Time PCR System (Applied Biosystems). PCR parameters consisted of 1 cycle of 50°C for 2 min, then 60°C for 30 min, and 95°C for 3:30 min, followed by 50 cycles of PCR at 95°C for 20 s, and 62°C for 1 min. The relative amount of HCV RNA was normalized to the internal control Human 18S rRNA (Applied Biosystems). HCV TaqMan probe used was 6-FAM

CTGCGGAACCGGTGAGTACACTAMRA (IDT). HCV primer sequences were 5' CGGGAGAGCCATAGTGG, and 3' AGTACCAC AAGGCCTTTCG. siRNA pools were classified as proviral or antiviral hits if the average normalized HCV RNA levels decreased or increased by more than 1.5 -fold comparing with non-targeting control siRNA and the P value was less than 0.05. The results of these experiments are shown in Table 7.

Table 7. HCV RNA Quantification

D. Identification of host factors affecting viral entry

The effect of various host factors on entry of virus into cells was determined using a virus entry assay. Briefly, HCV pseudoparticles (HCVpp), defective lentiviral or retroviral particles that display the HCV glycoproteins, were used to study HCV entry. The vesicular stomatitis virus pseudoparticles (VSVpp), which enters cells likely via a different mechanism, was employed as a control. Transfected and infected cells were prepare as described above in part A. Virus entry was assessed two days after infection by measuring the firefly luciferase reporter activities in cell lysate using the Bright GIo Luciferase assay system (Promega. This screen identified eight HCV-specific entry factors and 15 common factors for the entry of HCV and VSV. The results of these experiments are shown in Table 8 & 11.

Table 8. Identification of Host Factors Involved in Entry

Table 11. Host Factors Modulating HCV Entry

E. Identification of host factors affecting HCV replication

The effect of various host factors on HCV replication was measured using a HCV subgenomic replicon assay. Huh 7.5.1 cells were transfected with siRNA using the protocol described in part A. After 48 hours, SGR-Luc- JFH-I subgenomic replicon RNA, which contains the luciferase gene, was transiently transfected into the Huh7.5.1 cells. After 72 hours, cells were lysed directly in 96-well plates and Renilla luciferase reporter activities were measured using Renilla Luciferase assay system (Promega) to determine levels of HCV RNA replication. siRNA pools were classified as proviral or ICT antiviral hits if the average normalized HCV subgenomic replicon activities were decreased or increased for more than 1.5 -fold comparing with non-targeting control siRNA and the P value was less than 0.05. 52 human genes were identified to be important for HCV RNA replication. These genes are listed in Table 9. As shown in Table 13, 17 genes restrict HCV infection at the RNA replication level.

15 Table 9. Host Factors Affecting Replication

Table 13. Host Factors Modulating HCV RNA Replication

F. Identification of host factors affecting IRES -mediated translation

An HCV IRES-luciferase assay was used to identify host factors associated with HCV IRES-mediated translation. Briefly, Huh7.5.1 cells were transfected with siRNA as described in part A. After 48 hours, the transfected cells were transiently transfected with pHCV-CLX-CMV WT RNA. Cell lysate was prepared at 24 h following HCV RNA transfection, and assayed for firefly luciferase reporter activities using Luciferase assay system (Promega). siRNA pools were classified as pro viral or antiviral hits if the average normalized firefly luciferase activities were decreased or increased for more than 1.5 -fold comparing with non-targeting control siRNA and the P value was less than 0.05. These results are shown in Table 10. Table 10. Host Factors Modulating HCV Translation

Twelve of these host factors are specifically involved in HCV IRES-mediated translation, and nine host factors are antiviral by inhibiting HCV IRES-mediated translation (Table 12).

Table 12. Host Factors Modulating HCV Translation

G. Identification of Host Factors Modulating HCV Trafficking, Assembly, and Release

A conjugative analysis of virological assays with HCV cell culture system (HCVcc), including intracytoplasmic HCV RNA quantification, infectious viral production assays, and confocal microscopic imaging of viral and cellular factors, validated that 77 host proteins are required for HCV endocytic trafficking, virion assembly, and particle release. These host factors are listed in Table 14.

Table 14. Host Factors Modulating Trafficking, Assembly and release

With stringent criteria for selection, 62 genes selected for this study were shown to have an undetectable affect (i.e., no detectable activity) on HCV infection. These genes are listed in Table 15. Table 15. Host Factors Having no Effect on the HCV Life-Cycle

A major concern of RNAi-based mammalian genetics is false negatives, stemming in part from inefficient targeting or nonspecific toxicity. To preserve new connections and minimize bias, we have performed bioinformatics meta-analyses after the selection of our validated gene set, which integrated several HCV host factor data sets and disclosed statistically enriched complexes and pathways. By performing hypothesis-driven follow- up studies of these highlighted pathways, we were able to identify 22 additional host factors that were not previously identified by our primary screen. These host factors are listed in Table 16.

Example 3.

Among the host factors identified in the primary screen described in Example 1, IKKα was a strongly positive hit as a proviral factor. IKKα is an IKB kinase that plays a predominant role in non-canonical NFKB activation pathway. Innate immunity mediated by activation of NF-κB is a fundamental early signaling step of host antiviral defense. This Example demonstrates an NF-κB-independent function of IKKα in hepatitis C virus infection and propagation.

A. To demonstrate the effect of Ikkα on the HCV life-cycle, an siRNA screen was performed as described in Example 1. Briefly, siRNA's for the IKKα gene were transiently transfected into Huh 7.5.1 cells and after 72 hours, the cells infected with hepatitis C virus. AT 48 hours post-infection, 80 ml of media were removed and used for "late-stage" experiments. After removal of the media, the infected cells were fixed and stained with anti-HCV core 6G7 monoclonal antibody, as described in Example 1. The results of this experiment are shown in Figure 5(A). The data show that siRNA silencing of IKKα production resulted in a marked decrease of HCV production in Huh7.5 cells.

The 80 ml of medium removed from the infected cells were replica plated onto a new 96-well plate, which contained 10,000 Huh 7.5.1 cells per well plated out 24 h before viral supernatant addition. At 48 h post-infection, the cells were fixed, stained, and imaged using the staining and imaging protocol described in Example 1. The results of these "late stage: experiments are shown in Figure 5(B).

B. To study the functional effects of various NF-κB pathway host factors on HCV infection, Huh7.5.1 cells were transfected with siRNA's targeting these specific factors, and then infected with HCV, as described in Example 1. Intracellular and extracellular HCV RNA levels were then determined at 48 hours post-infection, using the protocol described in Example 1. The results of this experiment, which are shown in Figure 6, demonstrate that siRNA silencing of IKKα, and two IKKα upstream activators, NF-κB inducing kinase (NIK) and lymphotoxin-β (LTB), resulted in a marked decrease of HCV RNA production in Huh 7.5. cells. Likewise, overexpression of the dominant negative IKKα mutant (kinase-defective) suppressed HCV infection (Figure 7). In contrast, as shown in Figure 6, siRNA knocking-down of two other key IKB kinases, IKKβ and IKKα/NEMO, and components of NF-κB transcription factors, enhanced HCV replication, confirming the role of NF-κB in innate antiviral defense. Using various virologic assays including HCVcc, HCVpp and HCV replicon, silencing of IKKα specifically blocked HCV assembly. Confocal microscopy showed a marked diminution of core-associated lipid droplets, presumably site of viral assembly, in IKKα-silenced cells infected with HCV. We further showed by confocal microscopy and co- immunoprecipitation that NS5B but not other HCV proteins interacts specifically with and activates IKKα. The activated IKKα translocates to the nucleus and induces a CREB- binding protein (CBP)-mediated transcriptional program that includes SREBPl, a critical transcriptional factor controlling lipid metabolism and biogenesis of lipid droplets.

SREBPl was identified as a proviral factor in the genome- wide siRNA screen and siRNA knock-down of SREBPl displayed the same phenotype as IKKα silencing in HCV infection. Our study demonstrates that HCV has evolved a mechanism to exploit the host innate defense for its own advantage.

C. Effect of IKKα silencing on HCV RNA levels. To measure the effect of silencing of IKKα expression on HCV RNA levels,

Huh7.5.1 cells were transiently transfected with a pool of siRNAs, or individual siRNAs, against IKKα, and then infected with HCV. At 48 hours post-infection, the levels of intracellular and extracellular HCV RNA were measured using quantitative PCR, as described in Example 2(C). CD81, a major receptor of HCV, was used as a positive control for the early stage of the HCV lifecycle. ApoE, which is required for infectious viral particle formation, was the positive control for the late stage. The results are shown in Figure 8.

D. Effect of IKKα silencing on viral entry and RNA replication

HCV pseudovirus and replicon assays, described in Example 2, parts D and E, respectively, were used to measure the effect of IKKα silencing on viral entry and replication. Positive siRNA controls of CD81 and PIK4CA were used for the HCVpp and HCV replicon assays, respectively. These assays, the results of which are shown in Figure 9, demonstrate that IKKα is not involved in HCV entry (panel A) or HCV replication (panel B). Example 4

This Example demonstrates the ability of certain compounds to inhibit HCV entry.

Several proviral host factors identified in the initial siRNA screen suggest that a special process of HCV entry involves macropinocytosis. Thus, we tested various compounds that have been shown to target various host factors associated with

macropinocytosis of viral entry. Huh7.5.1 cells were infected with HCVpp in increasing concentrations of compounds as indicated, and assayed for luciferase activities 48 h later. The results, which are shown in Figure 10, show that HCV entry was inhibited by these compounds, i.e., rotterlin, colchicines, EIPA, wormannin, cytochalasin, and UO 126. To further confirm the HCVpp data, Huh7.5.1 cells were infected with HCVcc in the presence of varying concentrations of the Rottlerin and EIPA. After 4 h of infection, the culture medium was removed, the cells washed to remove the inoculating virus, fresh medium added and the cells incubated for 48 hours. At the end of the incubation period, HCV RNA levels in the culture supernatant were determined as described in Example 2(C). The results, which are shown in Figure 11, indicate that both drugs inhibited HCV infection.

HCV replication involves formation of membranous webs, which are sites of RNA replication. The present work identified several ADP-ribosylation factors and related proteins as proviral factors. Brefeldin A and golgicide A are compounds that have been shown to interfere with the functions of these factors in membrane-associated mechanisms of secretory and endocytic pathways. To test the effects of these drugs on HCV infection, Huh7.5.1 cells were infected with HCVcc in the presence of increasing concentrations of the compounds for 4 h and the culture supernatant was then removed with washing to remove the inoculating virus. Infected cells were treated with the same concentrations of the compounds for an additional 48 h and the culture supernatant was harvested for HCV RNA determination. The results, which are shown in Figure 12, indicate that both drugs inhibit HCV infection.

The present work enabled the reconstruction of an in-depth network map of cellular pathways and machineries that are associated with the complete life cycle of HCV (summarized in Table 1). A comprehensive investigation of HCV-host interactions yields critical insights into HCV pathogenesis and valuable targets for prophylactic and therapeutic interventions.

While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims.

Claims

1. A method to inhibit viral infection, said method comprising contacting a cell with an inhibitory compound that interacts with a host factor encoded by a nucleic acid molecule listed in Table 5 or with a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1 , thereby inhibiting viral infection.

2. A method to inhibit viral infection, said method comprising contacting a cell with an inhibitory compound that modulates a pathway comprising a host factor encoded by a nucleic acid molecule listed in Table 5 or a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1, thereby inhibiting viral infection.

3. A method to protect a patient from hepatitis C virus infection comprising administering an inhibitory compound that interacts with a host factor encoded by a nucleic acid molecule listed in Table 5 or with a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1.

4. A method to protect a patient from hepatitis C virus infection comprising administering an inhibitory compound that modulates a pathway comprising a host factor encoded by a nucleic acid molecule listed in Table 5 or a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1.

5. A method to enhance viral infection, said method comprising contacting a cell with an enhancing compound that interacts with a host factor encoded by a nucleic acid molecule listed in Table 5 or with a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1, thereby enhancing viral infection.

6. A method to enhance viral infection, said method comprising contacting a cell with an enhancing compound that modulates a pathway comprising a host factor encoded by a nucleic acid molecule listed in Table 5 or a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1, thereby enhancing viral infection.

7. The method of any of Claims 3-4 wherein said protection is prophylactic.

8. The method of any of Claims 3-4 wherein said protection is therapeutic.

9. The method of any of Claims 1 -6, wherein said host factor is encoded by a nucleic acid molecule listed in Table 2.

10. The method of any of Claims 1, 2, 5 or 6, wherein said virus is a Flavivirus.

11. The method of any of Claims 1 , 2, 5 or 6, wherein said virus is hepatitis C virus.

12. The method of any of Claims 1-2, wherein said method comprises contacting a patient with said inhibitory compound.

13. The method of Claim 12, wherein said patient is infected with said virus.

14. The method of any of Claims 1 -4, wherein said inhibitory compound is selected from one or more of the compounds listed in Table 3.

15. The method of any of Claims 1 -4, wherein said inhibitory compound is selected from colchicine, cytochalasin, EIPA₃ rottlerin, UO 126, wortmannin or a mixture of two or more of said compounds.

16. The method of any of Claims 1 -4, wherein said inhibitory compound is selected from brefeldin A₃ golgicide A or a mixture thereof.

17. The method of any of Claims 1-4, wherein said inhibitory compound comprises a small interfering RNA (siRNA).

18. The method of Claim 17, wherein said siRNA is selected from one or more of the siRNAs listed in Table 4.

19. The method of any of Claims 1 -6, wherein said host factor is encoded by a nucleic acid molecule listed in Table 11.

20. The method of any of Claims 1 -6, wherein said host factor is encoded by a nucleic acid molecule listed in Table 12.

21. The method of any of Claims 1 -6, wherein said host factor is encoded by a nucleic acid molecule listed in Table 13

22. The method of any of Claims 1 -6, wherein said host factor is encoded by a nucleic acid molecule listed in Table 14.

23. The method of any of Claims 1 -4, wherein said host factor is encoded by a nucleic acid molecule comprising SEQ ID NO: 79.

24. The method of any of Claims 1-4, wherein said host factor comprises amino acid sequence SEQ ID NO:80.

25. An inhibitory compound that interacts with a host factor encoded by a nucleic acid molecule listed in Table 5 or with a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1, thereby inhibiting viral infection.

26. An inhibitory compound that modulates a pathway comprising a host factor encoded by a nucleic acid molecule listed in Table 5 or a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1, thereby inhibiting viral infection.

27. An enhancing compound that interacts with a host factor encoded by a nucleic acid molecule listed in Table 5 or with a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1, thereby enhancing viral infection.

28. An enhancing compound that modulates a pathway comprising a host factor encoded by a nucleic acid molecule listed in Table 5 or a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1, thereby enhancing viral infection.

29. The compound of any of Claims 25-28, wherein said host factor is encoded by a nucleic acid molecule listed in Table 2.

30. The compound of any of Claims 25-28, wherein said host factor is encoded by a nucleic acid molecule listed in Table 11, Table 12, Table 13 or Table 14.

31. The inhibitory compound of any of Claims 25-26, wherein said host factor is encoded by a nucleic acid molecule comprising SEQ ID NO:79.

32. The inhibitory compound of any of Claims 25-26, wherein said host factor comprises amino acid sequence SEQ ID NO:80.

33. The compound of any of Claims 25-28, wherein said virus is hepatitis C virus.

34. A method to identify a compound that inhibits viral infection, said method comprising assaying a candidate compound for the ability to interact with a host factor in such a manner as to inhibit said infection, wherein said host factor is encoded by a nucleic acid molecule listed in Table 5 or is encoded by a nucleic acid molecule having a SEQ ID NO. listed in Table 1.

35. A method to identify a compound that inhibits viral infection, said method comprising:

(a) combining a candidate compound with a host factor encoded by a nucleic acid molecule listed in Table 5 or with a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1 under conditions suitable for said candidate compound to interact with said host factor;

(b) assaying for the presence of said interaction; and

(c) determining if said interaction results in a decrease in the amount of virus obtained upon infection of a cell with said virus; wherein a decrease in the amount of virus indicates that said compound inhibits viral infection.

36. A method to identify a compound that inhibits viral infection, said method comprising assaying a candidate compound for the ability to modulate a pathway comprising a host factor encoded by a nucleic acid molecule listed in Table 5 or a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1, wherein the ability to modulate said pathway identifies a compound that inhibits viral infection.

37. A method to identify a compound that enhances viral infection, said method comprising assaying a candidate compound for the ability to interact with a host factor in such a manner as to enhance said infection, wherein said host factor is encoded by a nucleic acid molecule listed in Table 5 or is encoded by a nucleic acid molecule having a SEQ ID NO. listed in Table 1.

38. A method to identify a compound that enhances viral infection, said method comprising: (a) combining a candidate compound with a host factor encoded by a nucleic acid molecule listed in Table 5 or with a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1 under conditions suitable for said candidate compound to interact with said host factor;

(b) assaying for the presence of said interaction; and (c) determining if said interaction results in an increase in the amount of virus obtained upon infection of a cell with said virus; wherein an increase in the amount of virus indicates that said compound enhances viral infection.

39. A method to identify a compound that enhances viral infection, said method comprising assaying a candidate compound for the ability to modulate a pathway comprising a host factor encoded by a nucleic acid molecule listed in Table 5 or a host factor encoded by a nucleic acid molecule having a SEQ ID NO: listed in Table 1, wherein the ability to modulate said pathway identifies a compound that enhances viral infection.

40. The method of any of Claims 34-39, wherein said host factor is encoded by a nucleic acid molecule listed in Table 2.

41. The method of any of Claims 34-39, wherein said virus is a Flavivirus.

42. The method of any of Claims 34-39, wherein said virus is hepatitis C virus.

43. The method of Claim 34, wherein said assay comprises:

(a) contacting said candidate compound with said host factor under conditions suitable for formation of a complex between said compound and said host factor; and

(b) detecting the presence of a complex, if present; wherein detection of a complex indicates that the candidate compound inhibits viral infection.

44. The method of Claim 34, wherein said assay comprises:

(a) contacting said candidate compound with said host factor under conditions suitable for allowing an interaction between said compound and said host factor; and

(b) determining if said interaction results in modification of said host factor; wherein modification of said host factor indicates that the candidate compound inhibits viral infection.

45. The method of Claim 35 or Claim 38, wherein said interaction is selected from the group consisting of (a) formation of a complex between said candidate compound and said host factor and (b) modification of said host factor.

46. The method of Claim 37, wherein said assay comprises: (a) contacting said candidate compound with said host factor under conditions suitable for formation of a complex between said compound and said host factor; and

(b) detecting the presence of a complex, if present; wherein detection of a complex indicates that the candidate compound enhances viral infection.

47. The method of Claim 37, wherein said assay comprises:

(a) contacting said candidate compound with said host factor under conditions suitable for allowing an interaction between said compound and said host factor and said host factor; and

(b) determining if said interaction results in modification of said host factor; wherein modification of said host factor indicates that the candidate compound enhances viral infection.

48. The method of any of Claims 34-39, wherein said host factor is encoded by a nucleic acid molecule listed in Table 11, Table 12, Table 13 or Table 14.

49. An inhibitory compound identified by the method of any of Claims 34-36.

50. An enhancing compound identified by the method of any of Claims 37-39.