WO2007001928A2 - Targets for inhibiting hcv replication - Google Patents

Abstract

Liver expressed proteins involved in HCV replication were identified using a procedure measuring the effect of inhibiting expression of host cell proteins on HCV replicon activity. The identified proteins and encoding nucleic acid provide targets for inhibiting HCV replication and for evaluating the ability of compounds to inhibit HCV replication. Compounds inhibiting HCV replication include compounds targeting identified proteins and compounds targeting nucleic acid encoding the identified protein. Several of the host genes identified as targets for inhibiting HCV replication were also found to be a target for inhibiting HIV replication. The ability to serve as a target for inhibiting both HIV and HCV replication indicates that such an identified gene and encoded protein may be a useful target for inhibiting replication of different types of viruses and not limited to inhibiting replication of a particular virus.

Description

TITLE OF TBE INVENTION

TARGETS FOR INHIBITING HCV REPLICATION

RELATED APPLICATIONS The present application claims priority to U.S. Provisional Application No. 60/692,821, filed June 22, 2005, and U.S. Provisional Application No. 60/710,006, August 19, 2005, each of which are hereby incorporated by reference herein.

BACKGROUND OF THE INVENTION The references cited in the present application are not admitted to be prior art to the claimed invention.

Exposure to HCV results in an overt acute disease in a small percentage of cases, while in most instances the virus establishes a chronic infection causing liver inflammation and slowly progresses into liver failure and cirrhosis. (Iwarson, FEMS Microbiol. Rev., 74:201-204, 1994.) Epidemiological surveys indicate HCV plays an important role in hepatocellular carcinoma pathogenesis. (Kew, FEMS Microbiol. Rev., 14:211-220, 1994, Alter, Blood, §5:1681-1695, 1995.)

The HCV genome consists of a single strand RNA about 9.5 kb in length, encoding a precursor polyprotein about 3000 amino acids. (Choo et al., Science, 244:362-364-, 1989, Choo et al, Science, 244:359-362, 1989, Takamizawa et al. J. Virol, 65:1105-1113, 1991.) The HCV polyprotein contains the viral proteins in the order: C-El-E2-p?-NS2-NS3-NS4A-NS4B-NS5A-NS5B.

Individual viral proteins are produced by proteolysis of the HCV polyprotein. Host cell proteases release the putative structural proteins C, El, E2, and p7, and create the N-terminus of NS2 at amino acid 810. (Mizushima et al, J. Virol, 68: 2731-2734, 1994, Hijikata et al, Proc. Natl. Acad. ScL USA., 90:10773-10777, 1993.) The non-structural proteins NS3, NS4A, NS4B, NS5A and NS5B presumably form the virus replication machinery and are released from the polyprotein. A zinc-dependent protease associated with NS2 and the N-terminus of NS3 is responsible for cleavage between NS2 and NS3. (Grakoui et al, J. Virol, 67:1385-1395, 1993, Hijikata et al, Proc. Natl. Acad. Sci. USA, 90:10773-10777, 1993.)

A distinct serine protease located in the N-terminal domain of NS3 is responsible for proteolytic cleavages at the NS3/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A/NS5B junctions. (Barthenschlager et al, J. Virol, 67:3835-3844, 1993, Grakoui et al, Proc. Natl. Acad. Sci. USA, 90:10583-10587, 1993, Tomei et al, J. Virol. 67:4017-4026, 1993.) RNA stimulated NTPase and helicase activities are located in the C-terminal domain of NS3.

NS4A provides a cofactor for NS3 protease activity. (Failla et al, J. Virol, 68:3753- 3760, 1994, De Francesco et al, U.S. Patent No. 5,739,002.)

NS5A is a highly phosphorylated protein conferring interferon resistance. (Pawlotsky. J. Viral Hepat. SuppL, 1:47-48, 1999.)

NS5B provides an RNA-dependent RNA polymerase. (De Francesco et al, International Publication Number WO 96/37619, published November 28, 1996, Behrens et al, EMBO 15:12-22, 1996, Lohmann et al., Virology 249:108-118, 1998.)

SUMMARY OF THE INVENTION Liver expressed proteins involved in HCV replication were identified using a procedure measuring the effect of inhibiting expression of host cell proteins on HCV replicon activity. The identified proteins and encoding nucleic acid provide targets for inhibiting HCV replication and for evaluating the ability of compounds to inhibit HCV replication. Compounds inhibiting HCV replication include compounds targeting identified proteins and compounds targeting nucleic acid encoding the identified protein. Several of the host genes identified as targets for inhibiting HCV replication were also found to be a target for inhibiting HTV replication. The ability to serve as a target for inhibiting both HTV and HCV replication indicates that such an identified gene and encoded protein may be a useful target for inhibiting replication of different types of viruses and not limited to inhibiting replication of a particular virus. , Thus, a first aspect of the present invention describes a method of identifying a host cell factor involved in viral {e.g., HCV) replication using a short inhibitory RNA (siRNA) library. The method comprises the step of measuring the ability of a siRNA library targeting different cell factors to inhibit viral (e.g.,HCV) replication, wherein the siRNA library comprises at least 10 different siRNA's targeting a different host factor that was. not previously associated with viral (e.g., HCV) replication. A "library" contains a collection of different siRNA that is screened as part of an experiment. The experimental results are obtained at about the same time or over a limited time period. In different embodiments, the limited time period is within about a week or within about a day. Preferably, the members of the library are tested at the same time.

Reference to the library comprising a certain number of siRNA targeting different host cell factors indicates that at least the indicated number of different siRNA are used. In different embodiments the library comprises 100, 500 or 1000 members and/or each of the different members is a kinase or phosphatase. In another embodiment, the library comprises 5000, 10000, or 20000 members and is considered to be a genome scale library.

Another aspect of the present invention describes a method of evaluating the ability of a compound to inhibit replication of a virus. The method involves:

(a) identifying a compound binding to, or inhibiting the activity or expression of, a target protein selected from the group consisting of: AKAP8 (SEQ ID NO: 11), ALK (SEQ ID NO: 67), ATM (SEQ ID NO: 68), C14ORF24 (SEQ ID NO: 69), DGKD (SEQ ID NO: 15), DGKZ (SEQ JD NO: 70), DUSP19 (SEQ ID NO: 49), DUSP22 (SEQ ID NO: 71), DUSP6 (SEQ ID NO: 9), DUT (SEQ ID NO: 51), DYRK2 (SEQ ID NO: 72), DYRK4 (SEQ ID NO: 53), ENPP5 (SEQ ID NO: 73), EPHA2 (SEQ ID NO: 13), FGFR2 (SEQ ID NO: 74), FHIT (SEQ ID NO: 75), FRK (SEQ ID NO: 76), GAK (SEQ ID NO: 55), GCK (SEQ ID NO: 19), MAP2K3 (SEQ ID NO: 77), NME4 (SEQ ID NO: 1), PANKl (SEQ ID NO: 78), PCKl (SEQ ID NO: 3), PFKL (SEQ ID NO: 63), PIK4CA (SEQ ID NO: 21), PRKWNK3 (SEQ ED NO: 5), PRPSlLl (SEQ ED NO: 79), PSKHl (SEQ ED NO: 57), PTK9L (SEQ ID NO: 80), S0CS5 (SEQ ID NO: 59), SRPKl (SEQ ID NO: 17), STK16 (SEQ ID NO: 7), STK35 (SEQ ID NO: 81), TAFl (SEQ ID NO: 82), TBKl (SEQ ED NO: 83), TJP2 (SEQ ID NO: 84), TNKl (SEQ ID NO: 61), TPKl (SEQ ED NO: 85), TRB33 (SEQ ID NO: 86), TRPM7 (SEQ ID NO: 87), VRK (SEQ ED NO: 88), ALPPL2 (SEQ ID NO: 89), AP4M1 (SEQ ID NO: 23), CAPZAl (SEQ ED NO: 25), DNAH5 (SEQ ID NO: 27), DOM3Z (SEQ ID NO: 90), FTCD (SEQ DD NO: 29), PDIA3 (SEQ ID NO: 31), MDM4 (SEQ ID NO: 91), WDR66 (SEQ ED NO: 92), NOP5/NOP58 (SEQ ED NO: 33), NSUN6 (SEQ ED NO: 93), PAFAHlBl (SEQ ED NO: 35), PARVB (SEQ ID NO: 37), PHEX (SEQ ED NO: 39), PKNl (SEQ ED NO: 44), POLR2J2 (SEQ ED NOs: 41 or 65), RAB20 (SEQ ID NO 43), SYNPR (SEQ ID NO: 45), and TRPM5 (SEQ ID NO: 47); or a protein substantially similar to the target protein, wherein the substantially similar protein has a sequence identity of at least 95% to the target protein; and

(b) determining the ability of the compound identified in step (a) to inhibit viral replication.

The initial identification of a compound binding to, or inhibiting the activity or expression of a target protein, can be performed experimentally or based on known information concerning the ability of a compound to bind or inhibit one of the identified targets. Information on the different targets is available in the scientific literature. Preferably, the compound is initially identified as inhibiting activity.

In preferred embodiments the virus is either HTV or HCV. Determining the ability of a compound to inhibit viral replication includes either, or both, (1) the initial identification of a compound as able to bind or inhibit viral replication or (2). determining the extent to which the compound inhibits viral replication. Inhibition of viral replication can be determined with quantitative or qualitative measurements. For example, determining the ability of a compound to inhibit HCV replication includes either, or both, (1) the initial identification of a compound as able to bind or inhibit HCV replication or (2) determining the extent to which the compound inhibits HCV replication.

Sequence identity to a reference protein sequence is determined by aligning the protein sequence with the reference sequence and determining the number of identical amino acids in the corresponding regions. This number is divided by the total number of amino acids in the reference sequence {e.g., SEQ ED NO: 1) and then multiplied by 100 and rounded to the nearest whole number. Another aspect of the present invention describes a method of inhibiting HCV replication. The method employs an effective amount of a compound inhibiting the activity or expression of a target protein selected from the group consisting of: AKAP8 (SEQ ED NO: 11), ALK (SEQ ED NO: 67), ATM (SEQ ED NO: 68), C14ORF24 (SEQ ED NO: 69), DGKD (SEQ ID NO: 15), DGKZ (SEQ ID NO: 70), DUSP19 (SEQ ID NO: 49), DUSP22 (SEQ ED NO: 71), DUSP6 (SEQ ED NO: 9), DUT (SEQ ID NO: 51), DYRK2 (SEQ ED NO: 72), DYRK4 (SEQ ED NO: 53), ENPP5 (SEQ ED NO: 73), EPHA2 (SEQ ID NO: 13), FGFR2 (SEQ ED NO: 74), FHIT (SEQ ID NO: 75, FRK (SEQ ID NO: 76), GAK (SEQ ID NO: 55), GCK (SEQ DD NO: 19), MAP2K3 (SEQ ID NO: 77), NME4 (SEQ ED NO: 1), PANKl (SEQ ED NO: 78), PCKl (SEQ ID NO: 3), PFKL (SEQ ID NO: 63), PIK4CA (SEQ ID NO: 21), PRKWNK3 (SEQ ID NO: 5), PRPSlLl (SEQ ID NO: 79), PSKHl (SEQ ID NO: 57), PTK9L (SEQ ID NO: 80), S0CS5 (SEQ ID NO: 59), SRPKl (SEQ ID NO: 17), STK16 (SEQ ID NO: 7), STK35 (SEQ ID NO: 81), TAFl (SEQ ID NO: 82), TBKl (SEQ ID NO: 83), TJP2 (SEQ ID NO: 84), TNKl (SEQ ID NO: 61), TPKl (SEQ ID NO: 85), TRIB3 (SEQ ID NO: 86), TRPM7 (SEQ ID NO: 87), VRK (SEQ ID NO: 88), ALPPL2 (SEQ ID NO: 89), AP4M1 (SEQ ID NO: 23), CAPZAl (SEQ ID NO: 25), DNAH5 (SEQ ID NO: 27), DOM3Z (SEQ ID NO: 90), FTCD (SEQ ID NO: 29), PDIA3 (SEQ ID NO: 31), MDM4 (SEQ ID NO: 91), WDR66 (SEQ ID NO: 92), NOP5/NOP58 (SEQ ID NO: 33), NSUN6 (SEQ ID NO: 93), PAFAHlBl (SEQ ID NO: 35), PARVB (SEQ ID NO: 37), PHEX (SEQ ID NO: 39), PKNl (SEQ ID NO: 44), POLR2J2 (SEQ ID NOs: 41 or 65), RAB20 (SEQ ID NO 43), SYNPR (SEQ ID NO: 45), and TRPM5 (SEQ ID NO: 47); or a protein substantially similar to the target protein, wherein the substantially similar protein has a sequence identity of at least 95% to the target protein.

Another aspect of the present invention describes a method of inhibiting replication of a virus in a host. The host is provided with an effective amount of a compound able to inhibit the activity or expression of a target protein selected from the group consisting of: AKAP8 (SEQ ID NO: 11), ALK (SEQ ED NO: 67), ATM(SEQ ID NO: 68), C14ORF24 (SEQ ID NO: 69), DGKD (SEQ ID NO: 15),

DGKZ (SEQ ID NO: 70), DUSP19 (SEQ ID NO: 49), DUSP22 (SEQ ID NO: 71), DUSP6 (SEQ ID NO: 9), DUT (SEQ ID NO: 51), DYRK2 (SEQ ID NO: 72), DYRK4 (SEQ ID NO: 53), ENPP5 (SEQ ID NO: 73), EPHA2 (SEQ ID NO: 13), FGFR2 (SEQ ID NO: 74), FHIT (SEQ ID NO: 75), FRK (SEQ ID NO: 76), GAK (SEQ ID NO: 55), GCK (SEQ ID NO: 19), MAP2K3 (SEQ ID NO: 77), NME4 (SEQ ID NO: 1), PANKl (SEQ ID NO: 78), PCKl (SEQ ID NO: 3), PFKL (SEQ ID NO: 63), PIK4CA (SEQ ID NO: 21), PRKWNK3 (SEQ ID NO: 5), PRPSlLl (SEQ ID NO: 79), PSKHl (SEQ ID NO: 57), PTK9L (SEQ ID NO: 80), SOCS5 (SEQ ID NO: 59), SRPKl (SEQ ID NO: 17), STK16 (SEQ ID NO: 7), STK35 (SEQ ID NO: 81), TAFl (SEQ ED NO: 82), TBKl (SEQ ID NO: 83), TJP2 (SEQ ID NO: 84), TNKl (SEQ ED NO: 61), TPKl(SEQ ID NO: 85), TREB3 (SEQ ID NO: 86), TRPM7 (SEQ ID NO: 87), VRK (SEQ ED NO: 88), ALPPL2 (SEQ ID NO: 89), AP4M1 (SEQ ED NO: 23), CAPZAl (SEQ ID NO: 25), DNAH5 (SEQ ID NO: 27), DOM3Z (SEQ ID NO: 90), FTCD (SEQ ED NO: 29), PDIA3 (SEQ ED NO: 31), MDM4 (SEQ ID NO: 91), WDR66 (SEQ ID NO: 92), NOP5/NOP58 (SEQ ID NO: 33), NSUN6 (SEQ ED NO: 93), PAFAHlBl (SEQ ID NO: 35), PARVB (SEQ ED NO: 37), PHEX (SEQ ID NO: 39), PKNl (SEQ ED NO: 44), POLR2J2 (SEQ ID NOs: 41 or 65), RAB20 (SEQ ED NO 43), SYNPR (SEQ ID NO: 45), and TRPM5 (SEQ ID NO: 47); or a protein substantially similar to said target protein, wherein said substantially similar protein has a sequence identity of at least 95% to said target protein.

Reference to "host" indicates a cell, animal, or human that is, or can be, infected with the virus. The compound can be administered prior to viral infection or to a host infected with the virus. Reference to open-ended terms such as "comprises" allows for additional elements or steps. Occasionally phrases such as "one or more" are used with or without open-ended terms to highlight the possibility of additional elements or steps. Unless explicitly stated reference to terms such as "a" or "an" is not limited to one. For example, "a cell" does not exclude "cells". Occasionally phrases such as one or more are used to highlight the possible presence of a plurality.

Other features and advantages of the present invention are apparent from the additional descriptions provided herein including the different examples. The provided examples illustrate different components and methodology useful in practicing the present invention. The examples do not limit the claimed invention. Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present invention.

BRIEFDESCRIPTION OF THE DRAWINGS

Figure 1 provides results illustrating the time course for knockdown of PIK4CA rnRNA levels and HCV RNA levels after transfection of siRNA targeting PIK4CA.

Figure 2A provides results illustrating the production of a rabbit polyclonal antisera that recognizes PIK4CA protein. Figure 2B provides results showing inhibition of HCV replication after transfection of two separate siRNAs targeting PIK4CA, or an siRNA directly targeting HCV but not after transfection of a non-silencing siRNA (NSl). Figure 2C provides results showing that the siRNAs targeting PIK4CA used in figure 2B also knock down PIK4CA protein levels, but the siRNA targeting HCV and the nonsilencing siRNA (NSl) do not affect PHC4CA protein levels.

Figure 3 provides resulting illustrating the ability of siRNA targeting different host genes to inhibit HTV replication. siRNA results for the following genes are provided in Figure 1: 1- CycTl; 2- PFKL; 3- PIK4CA; 4- DYRK4; 5- SYNPR; 6- GAK; 7- DUSP19; 8- DUT; 9- PSKHl; 10- LUC; 11- S0CS5; 12- PARVB; and 13- TRPM5.

DETAILED DESCRIPTION OF THE INVENTION Different host cell protein targets for inhibiting HCV replication were identified using a procedure measuring the effect of inhibiting expression of host cell protein on HCV replicon activity and taking into account liver expression of targeted proteins. The proteins identified as involved in HCV replication and the encoding nucleic acid provide targets for inhibiting HCV replication and for evaluating the ability of compounds to inhibit HCV replication. Several of the host genes identified as targets for inhibiting HCV replication were also found to inhibit HIV replication. The ability to serve as a target for both HTV and HCV indicates that such a target may be a useful target for inhibiting replication different types of viruses and not limited to a inhibiting replication of particular virus.

Inhibiting viral replication, such as HIV and HCV, has research and therapeutic implications. Research applications include providing tools to study viral replication and expression, for example, HCV or HTV replication and expression. Therapeutic applications include using those compounds having appropriate pharmacological properties such as efficacy and lack of unacceptable toxicity to treat or inhibit onset of viral infection in a patient {e.g., HCV or HTV infection). Identified Targets

The targets for inhibiting viral replication, such as HCV replication, identified herein are host cell factors. Nucleic acid sequences encoding the identified host cell factors, host cell factors, and substantially similar nucleic acid or protein can be used as a target for inhibiting viral replication, such as HCV replication. Table 1 provides information on the identified host cell factors for inhibiting HCV replication, and in some cases HTV replication.

The targets provide in Table 1 may also be useful for inhibiting viral replication beyond HCV. A subset of the HCV targets were tested for inhibiting HIV replication. As described in Example 13, infra inhibition of PIK4CA, SYNPR, DYRK4, PFKL, GAK, DUSP19, and DUT also inhibited HEV replication. The ability to inhibit both HCV and HTV replication indicates a role for such target in the replication of different types of viruses.

TABLE l

TABLE 1

TABLE l

Nucleic acid and protein substantially similar to a particular identified sequence provide sequences with a small number of changes to the particular identified sequence. Substantially similar sequences include sequences containing one or more naturally occurring polymorphisms or artificial changes.

A substantially similar protein sequence is at least 95% identical to a reference sequence. The substantially similar protein sequence should also not have significantly less activity than the reference sequence. Significantly less activity is less than about 80% activity of the identified protein.

In different embodiments, the substantially similar protein sequence differs from the reference sequence by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid alterations. Each amino acid alteration is independently an addition, deletion or substitution. Preferred substantially similar sequences are naturally occurring variants.

A substantially similar nucleic acid is at least 95% identical to a reference sequence. The substantially similar nucleic acid sequence should encode a protein that does not have significantly less activity than the protein encoded by the reference sequence. Significantly less activity is less than about 80% activity of the identified protein.

Sequence identity to a reference nucleic acid sequence is determined by aligning the nucleic acid sequence with the reference sequence and determining the number of identical nucleotides in the corresponding regions. This number is divided by the total number of nucleotides in the reference sequence (e.g., SEQ ID NO: 2) and then multiplied by 100 and rounded to the nearest whole number.

In different embodiments, the substantially similar nucleic acid sequence differs from the reference sequence by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotide alterations. Each nucleic acid alteration is independently an addition, deletion or substitution. Preferred substantially similar sequences are naturally occurring variants.

Protein Production

Proteins can be produced using techniques well known in the art including those involving chemical synthesis and those involving purification from a cell producing the protein. Techniques for chemical synthesis of proteins are well known in the art. (See e.g., Vincent, Peptide and Protein Drug Delivery, New York, N. Y., Decker, 1990.) Techniques for recombinant protein production and purification are also well known in the art. (See for example, Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-2002.) Obtaining a protein from a cell is facilitated using recombinant nucleic acid techniques to produce the protein. Recombinant nucleic acid techniques for producing a protein involve introducing, or producing, a recombinant gene encoding the protein in a cell and expressing the protein.

A recombinant gene contains nucleic acid encoding a protein along with regulatory elements for protein expression. The recombinant gene can be present in a cellular genome or can be part of an expression vector.

The regulatory elements that may be present as part of a recombinant gene include those naturally associated with the protein encoding sequence and exogenous regulatory elements not naturally associated with the protein encoding sequence. Exogenous regulatory elements such as an exogenous promoter can be useful for expressing a recombinant gene in a particular host or increasing the level of expression. Generally, the regulatory elements that are present in a recombinant gene include a transcriptional promoter, a ribosome binding site, a terminator, and an optionally present operator. A preferred element for processing in eukaryotic cells is a polyadenylation signal.

Expression of a recombinant gene in a cell is facilitated through the use of an expression vector. Preferably, an expression vector in addition to a recombinant gene also contains an origin of replication for autonomous replication in a host cell, a selectable marker, a limited number of useful restriction enzyme sites, and a potential for high copy number. Examples of expression vectors are cloning vectors, modified cloning vectors, specifically designed plasmids and viruses.

Due to the degeneracy of the genetic code, a large number of different encoding nucleic acid sequences can be used to code for a particular protein. The degeneracy of the genetic code arises because almost all amino acids are encoded by different combinations of nucleotide triplets or "codons".

Amino acids are encoded by codons as follows:

A=Ala=Alanine: codons GCA, GCC, GCG, GCU

C=Cys=Cysteine: codons UGC, UGU D=Asp=Aspartic acid: codons GAC, GAU

E=Glu=Glutamic acid: codons GAA, GAG

F=Phe=Phenylalanine: codons UUC, UUU

G=Gly=Glycine: codons GGA, GGC, GGG, GGU

H=His=Histidine: codons CAC, CAU I=De=Isoleucine: codons AUA, AUC, AUU

K=Lys=Lysine: codons AAA, AAG

L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU

M=Met=Methionine: codon AUG

N=Asn=Asparagine: codons AAC, AAU P=Pro=Proline: codons CCA, CCC, CCG, CCU

Q=Gln=Glutamine: codons CAA, CAG

R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU

S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU T=Thr=Threonine: codons ACA, ACC, ACG, ACU V=Val=Valine: codons GUA, GUC, GUG, GUU W=Trp=Tryptophan: codon UGG Y=Tyr=Tyrosine: codons UAC, UAU Techniques for recombinant gene production, introduction into a cell, and recombinant gene expression are well known in the art. Examples of such techniques are provided in references such as Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-2002, and Sambrook et al, Molecular Cloning, A Laboratory Manual, 2^nd Edition, Cold Spring Harbor Laboratory Press, 1989.

If desired, expression in a particular host can be enhanced through codon optimization. Codon optimization includes use of more preferred codons. Techniques for codon optimization in different hosts are well known in the art.

Identifying a Compound Inhibiting Binding to, or Activity or Expression of. a Target Protein

The initial identification of a compound inhibiting binding to, or activity or expression of, a target protein can be determined experimental or based on available information concerning a target. Compounds binding to, or inhibiting protein activity, are directed at the protein. Compounds inhibiting protein expression are directed at nucleic acid encoding the protein or having a regulatory function.

The ability of a compound to bind to a protein can be determined using techniques such as competitive and non-competitive binding assays. Such assays can be performed, for example, using a labeled compound to directly measure binding ox measuring binding using a detectable reagent that binds to compound.

Techniques for assaying kinase and phosphatase activity are well known in the art. For example, references describing techniques that can be used for measuring activity of NME4, PCKl, STK16, DUSP6, PIK4CA, PKWNK3, AKAP8, GCK, SRPKl, DGKD, DUT, GAK, PFKL, PSKHl, SOCS5, TNKl, DUSP19, and EPHA2 include: Kowluru et al., Arch. Biochem. Biophys., 398(2): 160-169, 2002 (NME4); Mahoud et al., Biochemica Biophysica Acta, 753(5:549-556, 1997 (PCKl); Ohta et al, Biochem J., 350 (Pt 2):395-404, 2000 (STK16); Kim et al, Biochemistry, 42(51): 15197-207, 2003 (DUSP6); Varsanyi et al, Eur. J. Biochem., 179: 473-479, 1989 (PIK4CA); Xu et al, J. Biol. Chem. 275: 16795-16801, 2000, (PRKWNK3); Akileswaran et al, J. Biol. Chem., 18:276(20): 17448-54, 2001 (AKAP8); Van Schaftingen et al, Eur. J. Biochem., 179:119 -184, 1989 (GCK); Aubol et al, Proc. Natl. Acad. ScL, U SA., 100(22):12601-6, 2003 (SRPKl); Imai et al, J. Biol. Chem. 277(38 ):35323-32, 2002 (DGKD); Climie et al, Protein Express Purif. 5(3):252-25S (1993) (DUT); Greener et al, J. Biol. Chem. 275(2): 1365-1370, 2000 and Kimura et al, Genomics 44:179-187 (1997) (GAK); Furuya and Uyeda, J. Biol. Chem. 256(14):7109-7112, 1981, Hirada et al, Biosci. Biotechnol. Biochem. 64(70): 2047-2052, 2000 (PFKL); Brede et al, Genomics 70(1): 82-92. 2000, Brede et al, Nucl. Acid Res. 30(23):5301- 5309, 2002 (PSKHl); Kario et al, J. Biol Chem., 280 fS):7038-7048, 2005 (SOCS5); Felschow et al, Biochem. Biophys. Res. Comm. 273:294-301, 2000 (TNKl); Zama et al, J. Biol. Chem. 277(26):23909- 23918, 2002; and Pratt et al,Oncogene 21(50):! 690-9, 2002 (EPHA2). Inhibitors of different kinases and phosphatases are well known in art. Examples of such inhibitors include: 2-morpholin-4-yl-6-thianthren-l-yl-pyran-4-one (KU-55933), which inhibits ATM (Hickson et al., Cancer Res., 64(24):9152-9, 2004); (5R,6S)-5-Azido-6-benzoyloxy-2-cyclohexen-l-one, which inhibits PIK4CA (Pelyvas et al., J. Med. Chem., 44: 627-632, 2001); l-allyl-3-butyl-8- methylxanthine, which inhibits PCKl (Foley et al, Bioorg. Med. Chem. Lett, 13(20):3607-10, 2003); 5- mercuri-dUTP and other UTP analogues that inhibit DUT (Climie et al, Protein Express Purif 5(3 ):252- 258, 1993, Beck et al, Adv. Exp. Med. Biol, 195 Pt B (97-104), 1986; and N-bromoaceylethanolamine phosphate, which inhibits PFKL (Hirada et al., Biosci. Biotechnol. Biochem. 64(10):2047-2052, 2000).

Techniques that can be used for assaying the enzymatic activity and/or compound binding for the non-kinase target proteins described in Table 1 are also known. Examples of references describing techniques for activity assays that can be used for measuring TRPM5, PHEX, MDM4, PDIA3, APPL2, and PARVB include: Prawitt et al, Proc Natl Acad Sci USA, 700:15166-15171, 2003 (TRPM5); Boileau et al, Biochem J. 355:707-713, 2001 (PHEX); Badciong and Haas, J. Biol. Chem., 277:49668- 49775, 2002 (MDM4); Frickel et al, J. Biol Chem., 279: 18277-18287, 2004, (PDIA3); Hoylaerts et al, Biochem. J. 277:49808-49814, 1992 (ALPPL2); Olski et al, J. Cell Sci. 114:525-538 (2000) (PARVB). Although PAFAHlBl is the catalytically inactive subunit of platelet activating factor acetylhydrolase, assay conditions for the full acetylhydrolase enzyme complex can be found in Hattori and Inoue, J. Biol. Chem. 268: 18748-18753, 1993 (PAFAHlBl).

The encoding nucleic acid sequence of an identified protein provides a target for compounds able to hybridize to the nucleic acid. Examples of compounds able to hybridize to a nucleic acid sequence include siRNA, ribozymes, and antisense nucleic acid. The mechanism of inhibition varies depending upon the type of compound. Techniques for producing and using sRNAi, ribozymes, and antisense nucleic acid are well known in the art. (E.g., Probst, Methods 22:271-281, 2000; Zhang et al, Methods in Molecular Medicine Vol. 106: Antisense Therapeutics 2^nd Edition, p. 11-34, Edited by I. Philips, Humana Press Inc., Totowa, NJ, 2005.) In addition, the examples provided below illustrate the use of siRNA.

Vector for delivering nucleic acid based compounds include plasmid and viral based vectors. Preferred vectors for therapeutic applications are retroviral and adenovirus based vectors. (Devroe et al, Expert Opin. Biol. Ητer. 4(3)319-327, 2004, Zhang et al, Virology 320:135-143, 2004.)

Measuring HCV Inhibitory Activity

The ability of a compound to inhibit HCV replication can be measured in vitro or using animal models. (Pietschmann et al., CUn Liver Dis. 7(1 j:23-43, 2003.) In vitro techniques for measuring the ability of a compound to inhibit HCV replication involve using HCV or an HCV replicon. Because HCV is difficult to grow in culture, preferred in vitro techniques employ an HCV replicon.

An HCV replicon is an RNA molecule able to autonomously replicate in a cultured cell, such as Huh7. The HCV replicon expresses the HCV derived components of the replication machinery and contains cis-elements required for replication in a cultured cell. The production and use of HCV replicons are described in different references. (See, for example, Lohmann et al, Science, 285:110-113, 1999; Blight et al, Science, 290:1912-191 A, 2000; Lohmann et al, Journal of Virology, 75:1437-1449, 2001; Pietschmann et ah, Journal of Virology, 75:1252-1264, 2001; Grobler et al, J. Biol. Chem., 278: 16741-16746, 2003; Murray et al, J. Virol, 77(5):2928-2935, 2003; Zuck et al, Anal Briochetn.334(2):344-355, 2004; Ludmerer et al, Antimicrob. Agents Chemother,. 49(5):2059-69, 2005; Rice et al, International Publication Number WO 01/89364, published November 29, 2001; Bichko, International Publication Number WO 02/238793, published May 16, 2002; Kukolj et al, International Publication Number WO 02/052015, published July 4, 2002; De Francesco et al, International Publication Number WO 02/059321, published August 1, 2002; Glober et al, International Publication Number WO 04/074507, published September 2, 2004; and BartenschlagerU.S. Patent No. 6,630,343.)

The ability of a compound to inhibit HCV replication can be measured in naturally occurring or artificial animal models susceptible to HCV infection. Only a few animals such as humans and chimpanzees are susceptible to HCV infection. Chimpanzees have been used as animal models for determining the effect of a compound on HCV infection.

Artificial animal models susceptible to HCV infection have been produced by transplanting human livers cells into a mouse. (Pietschmann et al, Clin Liver Dis., 7(1 ):23-43, 2003.) The use of transgenic mice with chimeric mouse-human livers provides for a small animal model.

Measuring HIV Inhibitory Activity

The ability of a compound to inhibit HTV replication can be measured in vitro or using animal models. In vitro techniques for measuring the ability of a compound to inhibit HTV replication include, for example, techniques measuring early steps in the viral life cycle (entry through integration) or involve using HIV infection of T-cell lines, or peripheral blood mononuclear cells to follow a spreading viral infection. (E.g., Joyce et al, J. Biol. Chem. 277(48):45Sl l-20, 2002; Nunberg et al, J. Virol 65(9): 4887-4892, 1991; Goldman et al, Antimicrob Agents Chemother. 36(5): 1019-1023, 1992.)

The ability of a compound to inhibit HTV replication can be measured in non-human primate models susceptible to infection with either HTV; or a chimeric virus created by combining fragments of the HTV and SIV (simian immunodeficiency virus) genome, termed a SHIV (simian-human immunodeficiency virus). Only a few animals such as humans and chimpanzees are susceptible to HTV infection. Chimpanzees have been used as animal models for determining the effect of a compound on HTV infection. (Grob et al, Nat Med. 3(6):665-610, 1997.) More frequently, determining the effect of a compound on HIV infection in non-human primates is carried out in rhesus macaques infected with SHIV. (Hazuda et al, Science 305( 5683 ):52&-32, 2004.)

Administration

Guidelines for pharmaceutical administration of a therapeutic compound in general are provided in, for example, Remington's Pharmaceutical Sciences 20^th Edition, Ed. Gennaro, Mack Publishing, 2000; and Modern Pharmaceutics 2^nd Edition, Eds. Banker and Rhodes, Marcel Dekker, Inc., 1990.

Compounds active at a therapeutic target having appropriate functional groups can be prepared as acidic or base salts. Pharmaceutically acceptable salts (in the form of water- or oil-soluble or dispersible products) include conventional non-toxic salts or the quaternary ammonium salts that are formed, e.g., from inorganic or organic acids or bases. Examples of such salts include acid addition salts such as acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylρropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate; and base salts such as ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D- glucamine, and salts with amino acids such as arginine and lysine.

Compounds can be administered using different routes including oral, nasal, by injection, transdermal, and transmucosally. Active ingredients to be administered orally as a suspension can be prepared according to techniques well known in the art of pharmaceutical formulation and may contain macrocrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners/flavoring agents. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants.

When administered by nasal aerosol, or inhalation, compositions can be prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents.

The compounds may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. When administered by injection, the injectable solutions or suspensions may be formulated using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.

When rectally administered in the form of suppositories, compositions may be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidify and/or dissolve in the rectal cavity to release the drug. Suitable dosing regimens for the therapeutic applications of the present invention are selected taking into account factors well known in the art including age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound employed. Guidelines for pharmaceutical administration and pharmaceutical compositions are provided in, for example,

Remington's Pharmaceutical Sciences 20^th Edition, supra, and Modern Pharmaceutics 2^nd Edition, supra.

Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves, a consideration of the distribution, equilibrium, and elimination of a drug. The daily dose for a patient is expected to be between 0.01 and 1,000 mg per adult patient per day.

Examples

Examples are provided below further illustrating different features of the present invention. The examples also illustrate useful methodology for practicing the invention. The examples do not limit the claimed invention.

Example 1: Screening the Protein Kinase and Phosphatase siRNA library

Potential host cell factor targets for inhibiting HCV replicon replication were identified using an HCV Conl-lb β-lactamase-expressing replicon expressed in Huh-7 cells and a siRNA library targeting host siRNA. The procedure was performed as follows:

Day 1: Plate HuH-7 cells expressing a tissue culture-adapted HCV Conl-lb β-lactamase-expressing replicon (Zuck et al, Anal. Biochem., 334(2)-344-355, 2004), at 2000 cells/well in black, clear-bottom 96-well plates (Costar, cat # 3603). Plate cells in 100 μL DMEM (Invitrogen, 21063-029), 10% Fetal bovine serum (Invitrogen 16140-071), IX Non-essential amino acids (Invitrogen, 11140-050), IX Glutamax (Invitrogen, 35050-061). Allow cells to attach and grow overnight at 37⁰C, 5% CO2-

Day 2: Transfect plated HuH-7/replicon cells as follows:

1. Resuspend siRNA by adding 200 μL of IX buffer (Dharmacon) to each well, producing a final siRNA concentration of 10 μM.

Control siRNAs: (+ control) HCV siRNA (Randall et al, Virus Res. 102(1 ):19-25, 2004)

AACCUCAAAGAAAAACCAACdTdT (SEQ ED NO: 95)

(+ control) HVAP33 siRNA (TGI) CUGUUCCACUGAAUGCAUCdTdT (SEQ ID NO: 96) (TGI) AUCGGCACCUGAGAGAUGAdTdT (SEQ ID NO: 96) (Zhang et aL, Virology. 320(1): 135-143, 2004) GAUGGACCUAUGCCAAAACdTdT (SEQ ID NO: 97)

(- control) NS 1 siRNA (Dharmacon)

Use controls at 10 μM as well. The final concentration of siRNA added to the cells will be 50 nM.

2. Dispense 33 μL of Optimem/well into a sterile 96-well plate, leaving the 12^th column empty.

3. Transfer 1 μL of siRNA from each well of the siRNA stock plate into the Optimem-containing plates such that the siRNA from well A3 of the mother plate is transferred into well A2 of the daughter plate (1 μL of siRNA from each well is transferred into the corresponding plate into the same row position and the N-I column position).

4. Mix by pipetting up and down.

5. In a tube, add 100 μL Oligofectamine (Invitrogen, 12252-011), 1210 μL Optimem Reduced Serum Media (Invitrogen, 11058-021). Incubate 5 minutes at room temperature.

6. Dispense 6 μL of the Oligofectamine to each well and mix by pipetting up and down. Incubate the plate at room temperature for 15 minutes.

7. Remove media from the cells and replace with 80 μL (DMEM (phenol red free), IX Nonessential amino acids, IX Glutamax). Add 20 μL of the siRNA/Oligofectamine mixture to each corresponding well.

8. Incubate the plate at 37⁰C, 5% CO2 for 4 hours. 9. Add 50 μL of DMEM (phenol red free) + 30% Fetal bovine serum, IX Non-essential amino acids, IX Glutamax. Incubate the cells at 37⁰C, 5% CO2 for 48 hours.

10. Remove media from cells and replace with 100 μL/ well DMEM (phenol red free), 10% FBS, IX non-essential amino acids, IX Glutamax and 0.5 μM Clavulanic acid. Incubate cells at 37⁰C, 5% CO2 for 24 hours. 11. Remove media from cells and replace with 50 μL of the following solution: 12 mL DMEM

(phenol red-free), 24 μL CCF4 (1 mM solution in DMSO), 2 mL Solution C (Aurora), 120 μL Solution B (Pluronic Acid, Aurora).

12. Incubate cells in the dark at room temperature for 1.5 hours.

13. Read the plates at 460 nm and 530 nm using a plate reader capable of reading fluorescence intensity.

14. Data analysis: Determine an average background for both A460 and A538 (average of counts from column 12). Subtract background from each well. Divide A460 by A538 after background subtraction to determine blue/green ratio for each cell. Calculate average and standard deviation for each transfection. This analysis was carried out in duplicate for each plate of the Dharmacon Kinase and Phosphatase siRNA libraries. Hits were considered to be those siRNA that suppressed replication of the replicon by 40% or more, as measured by β-lactamase activity (A460/A530 ratio compared with NSl siRNA-transfected control) and did not inhibit cell viability by more than 20% as measured by the A530 reading compared with NSl siRNA-transfected control.

Results:

Eighty-nine hits were identified in the primary screen: AKAP8, ALK, ASP, ATM, C14ORF24, CSNKlE, CSNK2A1, CSNK2B, CXCLlO, DDRl, DGKD, DGKG, DGKZ, DLG2, DUSP18, DUSP19, DUSP22, DUSP5, DUSP6, DUT, DYRK2, DYRK4, EGFR, ENPP5, EPHA2,

EPHA3, FBP2, FGFR2, FGFR4, FHIT, FLJ20442, FLJ35107, FN3K, FRK, GAK, GCK, HIPK3, HK3, I- 4, IGBPl, IMPK, LOC339221, LPPR4, MAP2K3, MAP2K6, MAP3K1, MAP3K7IP1, MGCl 136, MTMRl, MTMR?, NME4, PANKl, PCKl, PCK2, PCTKl, PFKL, PK4CA, PHKA2, PHKG2, PPAP2C, PPPlRIl, PPP1R2, PRKCL2, PRKD2, PRKWNK3, PRKY, PRPSl, PRPSlLl, PRPSAPl, PSKHl, PTK2B, PTK9L, PTP4A3, PTPN2, RIPK2, SOCS5, SRPKl, STK16, STK3, STK33, STK35, STK38, TAFl, TBKl, TJP2, TNKl, TPKl, TRPM7, TXK, ULK2, and VRKl.

Example 2: Counter Screening the Hits for Liver Expression using the Body Atlas

Since HCV replicates in the liver, one requirement for host HCV targets is that the gene is expressed in the liver. The gene expression for the 89 genes described in Example 1 in different tissues were evaluated using a previously generated Body Atlas. The Body Atlas provides the relative gene expression for different genes in different tissues compared to expression of an mRNA pool from multiple tissues.

Genes were considered to have liver high expression if the relative expression level was greater than 1.5 relative intensity, medium expression if the gene was between 0.5 and 1.5 relative intensity, and low expression if the gene was between 0.05 and 0.5 relative intensity. Genes with less than 0.05 relative intensity were not considered to be expressed in the liver.

Genes eliminated from further consideration as targets included those not expressed in liver and those with low level liver expression that were only identified as hits in one of the two replicate screens. The genes that were not expressed in liver included: ASP, FBP2, 1-4, LPPR4, MTMR7,

PRP4A3, and STK33. These genes were considered to be false positives or genes that were essential for maintenance of the replicon in cultured cells only.

Genes with moderate to high levels of expression within the liver were selected for further analysis. Genes with low level liver expression were only selected for further analysis if they inhibited HCV replication in each of the two times the library was screened. Genes with low level liver expression that were not chosen for further analysis included CSNKlE, DGKG, DLG2, DUSP5,

MAP3K1, MAP3K7P1, PCTKl, PTK2B, RIPK2, and STK38. Table 2 provides a list of the targeted genes chosen for further analysis and an indication of their relative level of expression in liver:

Example 3: Confirmation of siRNA Hits

From the primary screen of the kinase and phosphatase library, 80 siRNA pools were selected that both suppressed the HCV replicon and also were expressed in liver. siRNA pools were designed to have four siRNAs directed against the same gene in each well of the pool plate. To confirm the hits, each of the four siRNAs in the pool were tested individually in an 8-point, 2-fold titration. Confirmed hits were expected to demonstrate titrateable inhibition with at least two of the individual siRNAs present in the pool (since it is highly unlikely that two distinct siRNA sequences would have the same off-target effect).

DAY 1:

Reagents and Consumables: CM.10 Cells Trypsin (Invitrogen)

DMEM Plating Media: DMEM (Invitrogen), 10% FBS (Invitrogen), IX GlutaMax (Invitrogen), IX Non-Essential Amino Acids (Invitrogen) Trypan Blue (Invitrogen) 96-well Cell Plate (Corning)

Procedure: 1. Trypsinize a flask of CM.10 cells.

2. Inactivate the Trypsin by adding DMEM Plating Media to the flask.

3. Remove a 10 ul aliquot of the cell suspension and dilute it 1:2 in Trypan Blue for viability staining.

4. Count the cells using a hemocytometer. 5. Dilute the cells in DMEM Plating Media to a final concentration of 20,000 cells/ml.

6. Plate the cells at a density of 2000 cells/well by adding 100 ul of the cell suspension [20,000 cells/ml] to columns 1-11 of a 96-well Cell Plate.

7. Incubate the 96-well Cell Plate under normal growth conditions (37 ⁰C, 5% CO₂) for 18-20 hours.

DAY 2

Reagents and Consumables: siRNA Master Plate Oligofectamine (Invitrogen) Optimem-I (Invitrogen)

DMEM Transfection Media: DMEM (Invitrogen), IX GlutaMax (Invitrogen), IX Non-Essential Amino

Acids (Invitrogen)

96-well Titration Plate (Corning)

Procedure:

1. Add Optimem-I to the 96-well Titration Plate (Duplicate eight times). The Titration plate can be set up in a grid with columns numbered 1-12 and rows A-H. Column 1, rows A-H gets 33 ul:

Columns 2-11 row A gets 66 ul; columns 2-11, rows B-H gets 34 ul; and column 12 is kept empty. 2. A 96-well Master Plate is set up in a grid with columns numbered 1-12 and rows A-H. Column

1, rows A-H is a control; Columns 2-11 rows A-H are sample; and column 11 is empty.

3. Transfer 2 uL of siRNA [10 uM] (Samples) from the wells in Row A, Columns 2-11 of the Master Plate to the wells in Row A, Columns 2-11 of the 96-well Titration Plate.

4. Transfer 1 uL of siRNA [ 10 uM] (Controls) from the wells in Column 1 of the Master Plate to the wells in Column 1 of the 96-well Titration Plate.

5. Mix the samples by pipetting up and down gently.

6. Perform an 8-point, 2-fold serial dilution of the siRNA (Sample) by transferring and mixing 34 uL of sample from the wells in Row A, Columns 2-11 to the wells in Row B, Columns 2-11. Continue the titration by transferring and mixing 34 uL of sample from Row C to D, D to E, E to F, etc. The final concentration of siRNA at each titration point will be 50 nM, 25 nM, 12.5 nM,

6.25 nM, 3.125 nM, 1.5625 nM, 0.78125 nM and 0.390625 nM. Repeat the plate setup and titration setup eight times to account for all eight rows of samples from the Master plate.

7. In a 15 rriL conical tube, add 5.50 mL of Optimem-I and 0.50 mL of Oligofectamine. Mix gently.

8. Incubate the mixture for 5 minutes at room temperature. 9. Add 6 uL of the Oligofectamine mixture to each well of the 96-well Titration Plate, containing siRNA. Mix gently. 10. Incubate the siRNA:Oligofectamine mixtures for 15 minutes at room temperature to form the transfection complexes.

11. During the siRNA:Oligofectamine incubation, remove the media from the cells in the 96-well Cell Plate with a multi-channel pipettor. 12. Replace the media with 50 uL of DMEM Transfection Media.

13. After the 15 minutes needed for the siRNA:Oligofectamine complex formation has expired, transfer 20 uL of complex from the 96-well Titration Plate to the matching wells on the 96-well Cell Plate. The final concentration of siRNA at each titration point will be 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, 0.78125 nM and 0.390625 nM. 14. Incubate the 96-well Cell Plate under normal growth conditions (37°C, 5% CO₂) for 4 hours.

15. After the 4-hour incubation time has expired, add 30 uL of DMEM FBS Media to each of the wells containing cells and transfection mixes on the 96-well Cell Plate.

16. Incubate the 96-well Cell Plate under normal growth conditions (37°C, 5% CO₂) for 48 hours.

DAY 4

Reagents and Consumables: Clavulanic Acid

DMEM Plating Media: DMEM (Invitrogen), 10% FBS (Invitrogen), IX GlutaMax (Invitrogen), IX Non- Essential Amino Acids (Invitrogen)

Procedure:

1. Remove the media from the wells on the 96-well Cell Plates.

2. Add 100 uL of DMEM Plating Media; 0.5 uM Clavulanic Acid.

3. Incubate the 96-well Cell Plate under normal growth conditions (37°C, 5% CO₂) for 24 hours. DAY 5

Reagents and Consumables:

DMEM Staining Media: DMEM (Invitrogen)

1 M HEPES

CCF4 Solution B (Aurora, CET338P41), Solution C (Aurora, CET338P39),

Procedure:

1. Prepare the staining solution as follows (sufficient amount for eight plates): a. 72 mL of DMEM Staining Media; 25 mM HEPES b. 0.144 mL of CCF4 [I mM] c. 0.720 mL of Solution B d. 12 ml of Solution C

2. Remove the media from the wells on the 96-well Cell Plates.

3. Add 50 uL of staining solution to all of the wells on the 96-well Cell Plates, including the wells in Column 12.

4. Incubate the plates at room temperature in the dark for 1.5 hours.

5. Read fluorescence intensity at A460 and A530

Data Analysis: Subtract background (no cell wells) from each sample reading. Calculate the

A460/A530 ratio. Normalize as a percentage of Nonsilencing control siRNA. Graph the data for each siRNA as % of control A460/A530 vs. Concentration of siRNA transfected.

For an siRNA to be considered validated, at least two of the four siRNAs tested inhibited HCV replication by at least 40% to 60% of control levels and the inhibition should titrate with decreasing amounts of transfected siRNA.

Genes that were validated as essential for maintenance of the HCV replicon using the above criteria were then categorized into four groups, with priority 1 having the most desirable criteria for a HCV target. Gene classification was determined as follows:

Priority 1: The most potent siRNA resulted in > 70% inhibition of HCV replicon replication. Moderate to high expression in Liver. These genes and encoded proteins are the preferred targets for inhibiting HCV infection. Priority 2: The most potent siRNA resulted in > 60% inhibition of HCV replicon replication. Any level liver expression.

Priority 3: The most potent siRNA resulted in between 60 and 50% inhibition of HCV replicon replication.

Priority 4: The most potent siRNA resulted in between 50 and 40% inhibition of HCV replicon replication.

Genes essential for HCV replicon replication and their designated categories are shown in Table 3.

TABLE 3

Priority Gene

1 NME4, PCKl, PRKWNK3, STK16, DUSP6

SOCS5 , SRPKl, DGKD, DGKZ, ENPP5, EPHA2,

2 GAK, GCK, PANKl, PFKL, PIK4CA, PTK9L , STK35, TAFl, FHIT, AKAP8

Example 4: Screening of a Genome-Scale siRNA library

Screening of a library of over 22,000 siRNA pools was carried out essentially using the methods described in example 1. Briefly, HuH-7 cells containing the HCV conl-lb replicon expressing beta-lactamase (CM.10) were plated onto 384-well plates. The following day the cells were transfected with an array of siRNA pools including control siRNAs using Oligofectamine according to the manufacturer's directions. 72 hours after transfection, the cells were stained for beta-lactamase expression using CCF4, according to the manufacturer's instructions. Unlike in example 1, clavulanic acid was not added to the cells. 640 siRNA pools that affected beta-lactamase expression were then assayed an additional three times to confirm their effect on HCV replication.

39 siRNA pools were then selected for further confirmation. Six individual siRNAs targeting each of the genes targeted by the 39 siRNA pools were transfected separately into CM.10 cells and assayed as above. Effective inhibition of HCV replication by a minimum of two siRNAs targeting one of the tested genes was considered to be confirmation of the importance of the host factor for HCV conl Ib replication.

Results:

Nineteen genes were confirmed in this screen, including: ALPPL2, AP4M1, CAPZAl, DNAH5, D0M3Z, FTCD, PDIA3, MDM4, WDR66, NOP5/NOP58, NSUN6, PAFAHlBl, PARVB,

PHEX, PKN, POLR2J2, RAB20, SYNPR, and TRPM5. All of the confirmed siRNA pools targeted genes that had at least low levels of liver expression.

Example 5: Screening of Hits Using a Chimeric BK^bCNSSb^') Replicon An experiment was performed to determine whether a chimeric BK:2b(NS5b) replicon is sensitive to knock-down of the same genes as the Conl Ib replicon. The employed procedure is described in Examples 1 and 4, except a BK replicon containing a genotype 2b NS5b sequence was used.

The BK:2b(NS5b) replicon is described by Grobler et al, J. Biol. Chem., 275:16741-16746, 2003.

Data analysis was carried for each confirmed siRNA hit. The effects of siRNA transfection on the BK:2b(NS5b) replicon are shown in Table 4: TABLE 4

Example 6: Activity against HCV Genotype Ia, Con-lb and 2a Replicons

A quantitative, multiplex TaqMan assay was established to measure the relative quantities of HCV Replicon RNA in three HCV Replicon cell lines, Con-lb, Ia and 2a. Briefly, total RNA was isolated from replicon cells transfected with siRNAs using the RNeasy 96-well Kit (Qiagen, #74182). TaqMan reactions utilized the TaqMan EZ RT-PCR Kit (Applied Biosystems, #403028), TaqMan PDAR Control Reagent Human Cyclophilin A (Applied Biosystems, #431O883E) as well as a probe and primer set targeting the neomycin resistance gene of the HCV Replicon genome (Neo fwd: SEQ ID NO: 98; Neo rev: SEQ ID NO: 99; and Neo probe 5' FAM-SEQ ID NO: 100-TAMRA 3')- The final concentration of each component in the reaction mixture was as follow: IX TaqMan EZ Buffer, 3 mM Mn(OaC)₂, 0.3 mM dATP, 0.3 mM dCTP, 0.3 mM dGTP, 0.6 mM dUTP, 0.2 mM Forward Primer, 0.2 mM Reverse Primer, 0.1 mM Probe, IX PDAR Cyclophilin A Mix, 0.1 Unit/μl xTth DNA Polymerase, 0.01 Unit/μl AmpErase UNG, 10 μl of total RNA and H₂O to 50 μl. The 96-tube optical plate (Applied Biosystems #N801-0560) was covered with an optical adhesive cover (Applied Biosystems, #4311971) and mixed by inverting several times. The samples were placed in an ABI 7700 (Applied Biosystems) for multiplex TaqMan analysis by setting the entire plate to the FAM dye layer for "unknowns" (HCV) and to the VIC dye layer for the Endogenous control, Cyclophilin A. The cycling parameters were set to 50⁰C, 2 min.; 60⁰C, 30 min.; 95°C, 5 min.; (94⁰C, 20 sec; 55⁰C, 1 min.) 40 cycles, utilizing spectral compensation and an exposure time of 10 milliseconds. To calculate the relative quantities of each target RNA in the samples, a standard curve was generated for the HCV Neo primer and probe sets as well as the endogenous control, Cyclophilin A Pre-Developed Assay Reagent (PDAR) in each of the three HCV Replicon cell lines. Total RNA was isolated from the Replicons using the RNeasy Mini Kit (Qiagen, # 74104) following the manufacturer's protocol. The TaqMan Cycle Threshold (Ct) values was determined for a 7-point, five-fold titration of the total RNA. The natural log of the mass (ng) of total RNA was determined and plotted over the Ct values. The equation of the line for each probe and primer set in each of the HCV replicon cell lines was calculated to derive the relative quantity of the HCV RNA and the endogenous control, Cyclophilin A mRNA, from their respective Ct values. HCV RNA levels were normalized to the endogenous control, Cyclophilin A, mRNA levels.

Results Priority 1 targets: siRNAs inhibited replication of HCV genotype Ia, Con- Ib , and 2a replicons by greater than 30%: DUSP19, DUT, DYRK4, GAK, PARVB, PFKL, PK4CA, PSKHl, SOCS5, SYNPR, and TRPM5.

Priority 2 targets: siRNAs inhibited replication of two HCV genotypes by at least 50%: PAFAHlBl, STK16, and TNKl. Priority 3 targets: siRNAs inhibited replication of two HCV genotypes by at least 30%:

AKAP8, ALK, AP4M1, CAPZAl, DGKD, DNAH5, DYRK2, EPHA2, FGFR2, FRK, FTCD, GCK, NOP5/NOP58, PDIA3, PHEX, POLR2J2, PRKWNK3, RAB20, STK35, TJP2, TPKl, TREB3, and TRPM7.

Priority 4 targets: siRNAs inhibited replication of one HCV genotype by at least 30%: ALPPL2, CSNK2A1, CSNK2B, DDRl, DGKZ, D0M3Z, DUSP22, DUSP6, MAP2K6, NME4, PCKl, PRPSlLl, PTK9L, SRPKl, TAFl, TBKl, VRKl, and WDR66.

Example 7: Inhibition of HCV Ib Subgenomic Replication in HeLa Cells

The ability of Priority 1 targets to inhibit HCV Ib subgenomic replication in a replicon clone engineered to replicate in HeLa cells was tested. Inhibition of subgenomic replication in HeLa cells serves as evidence that the requirement for the target gene is not an artifact of the HuH-7 cell line.

The HCV HeLa replication system was as described in Zhu et at, J. Virol, 77(17 ):9204-9210, 2003. siRNA transfection and quantification of subgenomic replication were carried out as described in

Example 6.

Results:

The priority 1 targets DUT, GAK, PFKL, PIK4CA, PSKHl, and SYNPR inhibited HCV con Ib subgenomic replication in HeLa cells by at least 30%. PARVB, SOCS5, and TRPM5 siRNAs were toxic in these cells and effects on HCV replication could not be assessed. DUSP19 and DYRK 4 siRNAs were ineffective against HCV subgenomic replication in HeLa cells.

Example 8: Inhibition of HCV Replication with Wortrnannin

PIK4CA has two reported isoforms. Isoform 1 (mRNA: NM_002650, protein:

NP_002641) is shorter and lacks much of the N-terminal portion of the Isoform 2 (mRNA: NM_058004, protein: NP_477352) protein. PIK4CA isoform 1 has been characterized as a type II phosphatidylinositol 4-kinase, which is sensitive to adenosine and insensitive to wortmannin (Wong and Cantley, J. Biol. Chem., 2(59:28878-28884, 1994), while PIK4CA isoform 2 has been characterized as a type m phosphatidylinositol 4-kinase, which is sensitive to wortmannin and insensitive to adenosine

(Gehrmann et al., Biochim. Biophys. Acta, 1437:341-356, 1999).

To ascertain whether isoform 2 was the relevant isoform for HCV replication, we tested the effect of the kinase inhibitor, wortmannin, which inhibits type IH phosphatidylinositol 4-kinases in cells at micromolar levels, on HCV replication. Briefly, the beta-lactamase expressing, HCV Conl-b

CMlO cell line was plated at 7500 cells/well in 50 ul of cell Complete Media (DMEM, 10% FBS, Ix

NEAA, Ix Glutamax, (-) Pen/Strep) on 96-well Black Tissue Culture Treated Plate. A 10 mM DMSO stock of wortmannin (Sigma, #W1628) was diluted to 100 μM in 200 μl of Complete Media and titrated over a seven point, 2.5-fold dilution series. Fifty (50) μl from each of the dilution points was transferred to the assay plate containing the HCV Conl-b CMlO cells to produce the following concentrations of wortmannin [μM]: 50, 20, 8, 3.2, 1.28, 0.512 and 0.2048. The cells were incubated at 37⁰C, 5% CO₂ for

24 hours.

To inhibit endogenous β-lactamase activity in the HCV Conl-b CMlO cell line, a 1 mM

DMSO stock of Clavulanic acid (US Pharmocopeia, #1134426) was diluted to 5.5 μM in Complete Media of which 10 μl was added to the cells to produce a final concentration of 0.5 μM. The cells were incubated at 37⁰C, 5%CO₂ for 24 hours.

To measure the β-lactamase activity in the HCV Conl-b CMlO cell line, the GeneBlazer β-lactamase (hrvitrogen, #K1085) stain mixture was prepared based on the manufacturer's protocol. The cell culture/compound media was removed from the cells and replaced with 50 μl of the GeneBlazer β- lactamase stain mixture. The cells were incubated in the dark at room temperature for 1.5-2.0 hours.

The A460 and A530 fluorescence intensities at were measured on an LJL Analyst.

Results:

Wortmannin inhibited HCV replication with an IC₅₀ of 7.1 μM. The data is consistent with HCV replication having a requirement for PIK4CA function.

Example 9: siRNAs Specifically Targeting PIK4CA Isoform 2 Disrupt HCV Replication

To demonstrate the requirement for PIK4CA isoform 2 in HCV replication, siRNAs targeting isoform 2 mRNA only (NM_058004) were tested for inhibition of HCV replication. The following siRNAs were transfected into CM.10 cells and tested for inhibition of HCV subgenomic replication as described in Example 1:

PK4CA2-1: sense 5' UCAACGGUUCACAUAUAAdTdT 3' (SEQ ID NO: 101)

Antisense 5' UUAUAUGUGACACCGUUGAdTdT 3' (SEQ ID NO: 102)

PIK4CA2-2: sense 5' GGUCCGUCCUCCAGUAUAAdTdT 3' (SEQ ID NO: 103) A Annttiisseennssee 5' UUAUACUGGAGGACGGACCdTdT 3' (SEQ ID NO: 104)

PK4CA2-3 sense 5' CAGACCGGAUCCACAAUGAdTdT 3' (SEQ ID NO: 105)

Antisense 5' UCAUUGUGGAUCCGGUCUGdTdT 3' (SEQ ID NO: 106)

PIK4CA2-4 sense 5' GGAGUACUCAUUCCUGUAAdTdT 3' (SEQ ID NO: 107) Antisense 5' UUACAGGAAUGAGUACUCCdTdT 3' (SEQ ID NO: 108)

PK4CA2-5 sense 5' UGAUUGCAGUCGCGGACAAdTdT 3' (SEQ ID NO: 109)

Antisense 5' UUGUCCGCGACUGCAAUCAdTdT 3' (SEQ ID NO: 110)

PIK4CA2-6 sense 5' AAAGACUACUCCAACUUCAdTdT 3' (SEQ ID NO: 111) Antisense 5' UGAAGUUGGAGUAGUCUUUdTdT 3' (SEQ ID NO: 112)

Result:

AU six siRNAs targeting PIK4CA inhibited HCV subgenomic replication, confirming that isoform 2 of PIK4CA is the relevant isoform for HCV replication.

Example 10: siRNAs Targeting PIK4CA Knock Down PIK4CA mRNA Levels Prior to Disrupting HCV

Replication

If HCV replication is dependent upon PIK4CA expression or activity, the decrease in

HCV replication occurring after transfection of PIK4CA-targeting siRNA should occur subsequent to loss of PIK4CA expression. To verify this, HuH-7 cells containing the HCV Conl-lb replicon were transfected with siRNA targeting PIK4CA. At 0, 12, 24, 36, 48, 60 and 72 h following transfection, total

RNA was isolated from the cells and PIK4CA and HCV RNA levels were determined as described in

Example 6.

Results As shown in Figure 1, siRNAs targeting PIK4CA lead to maximal inhibition of PIK4CA mRNA levels at between 12 and 24 h post-transfection. In contrast, HCV RNA levels begin to decrease between 36 and 48 h post-transfection and continue to decrease through the 72 h time point.

Example 11: Validation of knock down of PIK4CA Protein Levels Using a Polyclonal PDC4CA antibody Antibodies were raised against antigens IP1240 SEQ ID NO: 113 (aa 893-904) and

IP1241 SEQ ID NO: 114 (aa 696-707). Antisera from two of the rabbits (D3792 and D3793) was sensitive enough to detect endogenous levels of PIK4CA when assayed by western blot. V5-tagged PIK4CA or empty pCDNA 3.1 vectors were over-expressed in HCV HBl Con Ib cells. The cell lysates were harvested in RIPA buffer and loaded on a 4% Tris-Glycine SDS-PAGE Gel. The custom antibodies (lmg/ml) were diluted 1:1000 for western blot analysis using the Licor Odyssey Imaging system.

To determine the gel migration characteristics of PIK4CA, we performed a dual-probe western blot using the V5 and D3972 antibodies. The dual color western blot demonstrates that the less intense, lower band seen in the D3972 blot migrates at the same size as the V5-tagged protein (Figure 2A).

To determine if the PIK4CA protein is reduced in the presence of PIK4CA siRNA, we transfected HCV HBl Conlb cells with control siRNAs as well as PIK4CA siRNAs targeting the ORF or the 3'UTR of the PIK4CA mRNA. The results demonstrate that HCV RNA levels are reduced by PIK4CA siRNAs as well as the positive control, HCV siRNA (Figure 2B). In addition, the PIK4CA siRNAs targeting both the ORF and 3'UTR of the PIK4CA mRNA reduce PDC4CA protein to undetectable levels by western blot (as noted above, the lower, less intense band represents PIK4CA, Figure 2C). These data both validate the knockdown of PIK4CA protein levels by the siRNAs used and also confirm the identity of the PIK4CA band recognized by the D3972 antibody.

Example 12: Additional Information on Some Targets

Additional information on some of the targets is provided below:

DUSP 19 (Dual Specificity Phosphatase 19)

DUSP 19 is a member of a family of dual specificity mitogen-activated protein kinase phosphatases. The protein sequence and encoding cDNA sequence are provided by SEQ TD NOs: 49 and 50. DUSP 19 polymorphisms are shown in Table 5.

TABLE 5

DUT (dUTP Pyrophosphatase)

DUT maintains dUTP at low levels to prevent misincorporation into DNA during replication, mediates resistance to 5-ftuorouracil, and may regulate peroxisome proliferation. The protein sequence and encoding cDNA sequences are provided by SEQ ID NOs: 51 and 52. DUT polymorphisms are shown in Table 6.

TABLE 6

DYRK4 (Dual Specificity Tyrosine-Regulated Kinase 4)

DYRK4 is a member of the DYRK family of protein tyrosine kinases. The protein sequence and encoding cDNA sequences are provided by SEQ ID NOs: 53 and 54. DYRK4 polymorphisms are shown in Table 7.

TABLE 7 mRNA position NM. 003845 seq Polymorphism NP_003836 seq Amino Acid change

284 C T Leu42 No Change

341 G A Alaόl Thr

GAK (Cy din G Associated Kinase)

GAK is a putative serine/threonine protein kinase that shares homology with tensin and auxilin, and may play a role in cell cycle regulation. The protein sequence and encoding cDNA sequence for GAK are provided by SEQ ID NOs: 55 and 56. GAK nucleotide polymorphisms are shown in Table 8

TABLE 8

PAR VB (Parvin beta) Isoform A: PARVB is a focal adhesion protein containing two calponin homology domains that binds integrin-linked kinase and is likely involved in integrin-ILK signaling to establish cell-substrate adhesion. The protein sequence and encoding cDNA sequence for PARVB are provided by SEQ ID NOs: 37 and 38. PARVB nucleotide polymorphisms are shown in Table 9.

TABLE 9

PFKL (Phosphofructokina.se, liver)

Liver phosphofructokinase catalyses the phosphorylation of fructose-6-phosphate to fructose-l,6-bisphosphate in glycolysis. Deficiency is linked to glycogenosis type VII while overexpression may lead to the cognitive disabilities of Down's syndrome. The protein and encoding cDNA sequence for PFKL are provided by SEQ ID NOs: 63 and 64.

PIK4CA

PIK4CA is a type HI phosphatidylinositol-4 kinase. It catalyzes the first step in the formation of phosphatidylinositol 4,5-bisphosphate and its activity is inhibited by high concentrations of wortmannin. The protein and encoding cDNA sequence for PIK4CA are provided by SEQ ID NOs: 21 and 22. PIK4CA nucleotide polymorphisms are shown in Table 10.

TABLE 10

PSKHl (Protein Serine Kinase Hl)

PSKHl is a protein serine kinase that undergoes calcium-dependent autophosphorylation. Overexpression of PSKHl leads to nuclear reorganization of splicing factors SFRSl and SFRS2 and stimulates RNA splicing. The protein and encoding cDNA sequence for PSKHl are provided by SEQ TD NOs: 57 and 58. SOCS5 (Suppressor of Cytokine Signaling 5)

SOCS5 is a cytokine-inducible protein containing an SH2 domain and a SOCS box. It negatively regulates cytokine signaling via the JAK-STAT pathways. The protein and encoding cDNA sequence for SOCS5 are provided by SEQ ID NOs: 59 and 60. Nucleotide polymorphisms identified for S0CS5 are shown in Table 11.

TABLE I l

SYNPR (Synaptoporin)

Synaptoporin is a protein with high homology to rat synaptophysin, an integral- membrane synaptic vesicle protein involved in targeting of synaptic vesicles. It contains a membrane- associating domain, often found in lipid-associating proteins. The protein and encoding cDNA sequence for SYNPR are provided by SEQ ID NOs: 45 and 46.

TRPM5 (Transient Receptor Potential Cation Channel, Subfamily M, Member 5) TRPM5 is related to the transient receptor potential family of cation channels. It has six predicted transmembrane domains. The protein and encoding cDNA sequence for TRPM5 are provided by SEQ ID NOs: 47 and 48. Nucleotide polymorphisms identified for TRPM5 are presented in Table 12.

TABLE 12

PAFAHlBl (Platelet-Activating Factor Acetylhydrolase (koform Ib) Alpha Subunit (45JcD)

PAFAHlB 1 is the noncatalytic subunit of a heterotrimeric enzyme that inactivates platelet-activating factor. The protein and encoding cDNA sequence for PAFAHlBl are provided by SEQ ID NOs: 35 and 36. Nucleotide polymorphisms for PAFAHlBl are shown in Table 13.

TABLE 13 mRNA position NM. 000430 seq Polymorphism NP. .000421 seq Amino Acid change

1248 A T Thr 231 No change

1614 T C Be 353 No change

STK16 (Serine/Threonine Kinase 16)

Serine/threonine kinase 16 is a myristoylated and palmitoylated protein kinase that may regulate transcription in response to signaling by transforming growth factor beta. The protein and encoding cDNA sequence for STK16 are provided by SEQ ID NOs: 7 and 8.

TNKl (Tyrosine Kinase,Non-recβptor 1)

TNKl is a kinase that interacts with phospholipase C gamma 1 (PLCGl). It may regulate phospholipid signaling pathways during fetal development and in adult cells of the lymphohematopoietic system. The protein and encoding cDNA sequence for TNKl are provided by SEQ ID NOs: 61 and 62.

Example 13: siRNA hits that also block HTV Infection

High priority siRNA pools were tested for their ability to disrupt HIV infection in HeLa cells. The procedure was performed as follows: Day 1 : Plate HeLa (P4/R5) cells at 2000 cells per well in 4x96-well plates. Day 2: Transfect HeLa (P4/R5) cells with siRNA pools as follows:

1. siRNAs were transfected at a final concentration of 100 nM using Oligofectamine™ reagent (Invitrogen) at a final concentration of 0.5%. Positive and negative control siRNAs are included as follows: Cyclin Tl (positive control): purchased from Santa Cruz Biotechnology (Cat. No. sc-35144)

Luciferase (negative control): CGUACGCGGAAUACUUCGAdTdT (SEQ ID NO: 115) siRNAs tested in duplicate included a pool of 3 siRNAs targeting: DUSP19, DUT, DYRK4 GAK, PARVB₁ PFKL, PIK4CA, PSKHl, SOCS5, SYNPR and TRPM5

2. Dispensed 66 μL of OptiMEM/ well into a sterile 96-well plate, leaving the 12^th column empty.

3. Transferred 2 μL of each siRNA (resuspended at 10 μM) into the OptiMEM-containing plate.

4. Mixed by pipetting up and down. 5. In a tube, added 240 μL Oligofectamine, 1210 μL OptiMEM. Incubated 5 minutes at room temperature.

6. Dispensed 12 μL of the Oligofectamine to each well and mixed by pipetting up and down. Incubated the plate at room temperature for 15 minutes. 7. Added 20 μL of the siRNA-Oligofectamine complex to each well of the HeLa (P4/R5) cells.

Day 3: Transfected HeLa (P4/R5) cells were infected with HXB2 HTV as follows:

1. Media was removed from the cells .

2. 80 μL fresh media was added to each well.

3. HXB2 HTV was diluted with media. 40 μL of diluted HXB2 was added to each well. 4. Viral infection was allowed to proceed for 96 hours.

Day 5: Beta-galactosidase activity, an indication of viral infection, was measured as follows:

1. Media was removed from the cells.

2. Cells were washed with 200 μL PBS per well.

3. 20 μL lysis buffer (Galacto-Light Plus, Tropix, Cat. No. BLlOOP) containing DTT was added to each well, and the plates were shaken for 10 minutes.

4. 80 μL of substrate was then added to each well and the plates were incubated at room temperature in the dark for 1 hour.

5. 100 μL of enhancing solution was added to each well and the plates were read using a Dynex luminometer.

Data was analyzed in the following manner. Readings for each plate were normalized to the reading for the luciferase negative control and expressed as "percent of Luciferase Control". Preferred targets were considered to be those siRNA pools that suppressed beta-galactosidase activity by an average of 40% or more.

Results: siRNAs targeting PIK4CA, SYNPR, DYRK4, and PFKL inhibited HTV replication by greater than 40% (see Figure 3). Thus, these genes are essential for the replication of both HTV and HCV in human cell lines. Between 30 and 40% inhibition of HTV replication was also observed with siRNAs targeting GAK, DUSP19, and DUT, indicating these genes may also be targets for HTV infection.

As in Example 7, toxicity associated with SOCS5, PARVB, and TRPM5 siRNAs in HeLa cells precludes any understanding of the role these genes play in viral infection in these cells. No effect on HTV replication was observed with PSKHl siRNAs.

Other embodiments are within the following claims. While several embodiments have been shown and described, various modifications may be made without departing from the spirit and scope of the present invention.

Claims

WHAT IS CLAIMED IS:

1. A method of identifying a host cell factor involved in replication of a virus comprising the step of measuring the ability of a siRNA library targeting different cell factors to inhibit replication of said virus, wherein said siRNA library comprises at least 10 different siRNA's targeting a different host factor that was not previously associated with replication of said virus.

2. The method of claim 1, wherein said virus is a hepatitis C virus (HCV) and where said siRNA library comprises at least 100 different siRNA's targeting a different host factor that was not previously associated with HCV replication.

3. The method of claim 2, wherein each of said at least 100 different siRNA' s is either a kinase or phosphatase.

4. A method of screening for a virus inhibitory compound comprising the steps of:

(a) identifying a compound binding to, or inhibiting the activity or expression of, a target protein selected from the group consisting of: AKAP8 (SEQ ID NO: 11), ALK (SEQ ID NO: 67),

ATM (SEQ ID NO: 68), C14ORF24 (SEQ ID NO: 69), DGKD (SEQ ID NO: 15), DGKZ (SEQ ED NO:

70), DUSP19 (SEQ ID NO: 49), DUSP22 (SEQ ID NO: 71), DUSP6 (SEQ ID NO: 9), DUT (SEQ ID NO: 51), DYRK2 (SEQ ID NO: 72), DYRK4 (SEQ ID NO: 53), ENPP5 (SEQ ID NO: 73), EPHA2 (SEQ ID NO: 13), FGFR2 (SEQ ID NO: 74), FHIT (SEQ ID NO: 75), FRK (SEQ ID NO: 76), GAK (SEQ ID NO: 55), GCK (SEQ ID NO: 19), MAP2K3 (SEQ ID NO: 77), NME4 (SEQ ID NO: 1), PANKl (SEQ ID NO: 78), PCKl (SEQ ID NO: 3), PFKL (SEQ ID NO: 63), PK4CA (SEQ ID NO: 21), PRKWNK3 (SEQ ID NO: 5), PRPSlLl (SEQ ID NO: 79), PSKHl (SEQ ID NO: 57), PTK9L (SEQ ID NO: 80), SOCS5 (SEQ ID NO: 59), SRPKl (SEQ ID NO: 17), STK16 (SEQ ID NO: ?), STK35 (SEQ ID NO: 81), TAFl (SEQ ED NO: 82), TBKl (SEQ ID NO: 83), TJP2 (SEQ ID NO: 84), TNKl (SEQ ID NO: 61), TPKl (SEQ ID NO: 85), TRIB3 (SEQ ID NO: 86), TRPM7 (SEQ ID NO: 87), VRK (SEQ ID NO: 88), ALPPL2 (SEQ ID NO: 89), AP4M1 (SEQ ID NO: 23), CAPZAl (SEQ ID NO: 25), DNAH5 (SEQ ID NO: 27), DOM3Z (SEQ ID NO: 90), FTCD (SEQ ID NO: 29), PDIA3 (SEQ ID NO: 31), MDM4 (SEQ ID NO: 91), WDR66 (SEQ ID NO: 92), NOP5/NOP58 (SEQ ID NO: 33), NSUN6 (SEQ ID NO: 93), PAFAHlBl (SEQ ID NO: 35), PARVB (SEQ ID NO: 37), PHEX (SEQ ID NO: 39), PKNl (SEQ ID NO: 44), POLR2J2 (SEQ ID NOs: 41 or 65), RAB20 (SEQ ID NO 43), SYNPR (SEQ ID NO: 45), and TRPM5 (SEQ ID NO: 47); or a protein substantially similar to said target protein, wherein said substantially similar protein has a sequence identity of at least 95% to said target protein; and (b) determining the ability of said compound identified in said step (a) to inhibit replication of said virus.

5. The method of claim 4, wherein said target protein is either SEQ ID NO: 21, 45, 47, 49, 51, 53, 55, or 63, or a substantially similar protein having a sequence identity of at least 95% to said target protein.

6. The method of claim 5, wherein said target protein is either SEQ ID NO: 21, 45,

47, 53, or 63.

7. The method of claim 4, wherein said virus is either hepatitis C virus (HCV) or human immunodeficiency virus (HIV).

8. The method of claim 5, wherein said compound identified in said step (a) inhibits the activity of said target protein.

9. The method of claim 8, wherein said virus is HCV and said target protein is either SEQ ID NO: 7, 21, 35, 37, 45, 47, 49, 51, 53, 55, 57, 59, 61, or 63, or a substantially similar protein having a sequence identity of at least 95% to said target protein.

10. The method of claim 9, wherein said target protein is either SEQ ID NO: 7, 21, 35, 37, 45, 47, 49, 51, 53, 55, 57, 59, 61, or 63.

11. The method of claim 10, wherein said step (b) is performed using a cultured cell containing an HCV replicon.

12. The method of claim 8, wherein said virus is HTV and said target protein is either SEQ ID NO: 21, 45, 47, 49, 51, 53, 55, or 63, or a substantially similar protein having a sequence identity of at least 95% to said target protein.

13. The method of claim 12, wherein said target protein is either SEQ ID NO: 21, 45, 47, 53, or 63.

14. A method of inhibiting hepatitis C virus (HCV) replication in a cell containing an HCV replicon or infected with HCV comprising the step of providing to said cell an effective amount of a compound able to inhibit the activity or expression of a target protein selected from the group consisting of: AKAP8 (SEQ ID NO: 11), ALK (SEQ ID NO: 67), ATM (SEQ ID NO: 68), C14ORF24 (SEQ ID NO: 69), DGKD (SEQ ID NO: 15), DGKZ (SEQ ID NO: 70), DUSP19 (SEQ ID NO: 49), DUSP22 (SEQ ID NO: 71), DUSP6 (SEQ ID NO: 9), DUT (SEQ ID NO: 51), DYRK2 (SEQ ID NO: 72), DYRK4 (SEQ ID NO: 53), ENPP5 (SEQ ID NO: 73), EPHA2 (SEQ ID NO: 13), FGFR2 (SEQ ED NO: 74), FHTT (SEQ ID NO: 75), FRK (SEQ ID NO: 76), GAK (SEQ ID NO: 55), GCK (SEQ ID NO: 19), MAP2K3 (SEQ ED NO: 77), NME4 (SEQ TD NO: 1), PANKl (SEQ ID NO: 78), PCKl (SEQ TD NO: 3), PFKL (SEQ E) NO: 63), PIK4CA (SEQ E⁾ NO: 21), PRKWNK3 (SEQ E) NO: 5), PRPSlLl (SEQ E) NO: 79), PSKHl (SEQ E) NO: 57), PTK9L (SEQ E) NO: 80), SOCS5 (SEQ E) NO: 59), SRPKl (SEQ E) NO: 17), STK16 (SEQ E) NO: 7), STK35 (SEQ E) NO: 81), TAFl (SEQ E) NO: 82), TBKl (SEQ E) NO: 83), TJP2 , TNKl (SEQ E) NO: 61), TPKl (SEQ E⁾ NO: 85), TRIB3 (SEQ E) NO: 86), TRPM7 (SEQ E⁾ NO: 87), VRK (SEQ E) NO: 88), ALPPL2 (SEQ E) NO: 89), AP4M1 (SEQ E) NO: 23), CAPZAl (SEQ E) NO: 25), DNAH5 (SEQ E) NO: 27), DOM3Z (SEQ E) NO: 90), FTCD (SEQ E) NO: 29), PDIA3 (SEQ E) NO: 31), MDM4 (SEQ E) NO: 91), WDR66 (SEQ E) NO: 92), NOP5/NOP58 (SEQ E) NO: 33), NSUN6 (SEQ E) NO: 93), PAFAHlBl (SEQ E) NO: 35), PARVB (SEQ E) NO: 37), PHEX (SEQ E) NO: 39), PKNl (SEQ E) NO: 44), POLR2J2 (SEQ E) NOs: 41 or 65), RAB20 (SEQ E) NO 43), SYNPR (SEQ E) NO: 45), and TRPM5 (SEQ E) NO: 47); or a protein substantially similar to said target protein, wherein said substantially similar protein has a sequence identity of at least 95% to said target protein.

15. The method of claim 14, wherein said target protein is either SEQ E) NO: 7, 21,

35, 37, 45, 47, 49, 51, 53, 55, 57, 59, 61, or 63, or a substantially similar protein having a sequence identity of at least 95% to said target protein.

16. The method of claim 15, wherein said method is used to inhibit HCV replication in a cultured cell infected with an HCV replicon.

17. A method of inhibiting replication of a virus in a host comprising the step of providing to said host cell an effective amount of a compound able to inhibit the activity or expression of a target protein selected from the group consisting of: AKAP8 (SEQ E) NO: 11), ALK (SEQ E) NO: 67), ATM (SEQ E) NO: 68), C14ORF24 (SEQ E) NO: 69), DGKD (SEQ E) NO: 15), DGKZ (SEQ E) NO: 70), DUSP19 (SEQ E) NO: 49), DUSP22 (SEQ E) NO: 71), DUSP6 (SEQ E) NO: 9), DUT (SEQ E) NO: 51), DYRK2 (SEQ E) NO: 72), DYRK4 (SEQ E) NO: 53), ENPP5 (SEQ E) NO: 73), EPHA2 (SEQ E⁾ NO: 13), FGFR2 (SEQ E) NO: 74), FHIT (SEQ E) NO: 75), FRK (SEQ E) NO: 76), GAK (SEQ E⁾ NO: 55), GCK (SEQ E) NO: 19), MAP2K3 (SEQ E) NO: 77), NME4 (SEQ E) NO: 1), PANKl (SEQ E) NO: 78), PCKl (SEQ E) NO: 3), PFKL (SEQ E) NO: 63), PIK4CA (SEQ E) NO: 21), PRKWNK3 (SEQ E) NO: 5), PRPSlLl (SEQ E) NO: 79), PSKHl (SEQ E) NO: 57), PTK9L (SEQ E⁾ NO: 80), S0CS5 (SEQ E) NO: 59), SRPKl (SEQ E) NO: 17), STK16 (SEQ E) NO: 7), STK35 (SEQ E) NO: 81), TAFl (SEQ E) NO: 82), TBKl (SEQ E) NO: 83), TJP2 (SEQ E) NO: 84), TNKl (SEQ E⁾ NO: 61), TPKl (SEQ E⁾ NO: 85), TRffi3 (SEQ E) NO: 86), TRPM7 (SEQ TD NO: 87), VRK (SEQ TD NO: 88), ALPPL2 (SEQ TD NO: 89), AP4M1 (SEQ E) NO: 23), CAPZAl (SEQ TD NO: 25), DNAH5 (SEQ TD NO: 27), DOM3Z (SEQ TD NO: 90), FTCD (SEQ TD NO: 29), PDIA3 (SEQ TD NO: 31), MDM4 (SEQ TD NO: 91), WDR66 (SEQ E) NO: 92), NOP5/NOP58 (SEQ E) NO: 33), NSUN6 (SEQ TD NO: 93), PAFAHlBl (SEQ TD NO: 35), PARVB (SEQ TD NO: 37), PHEX (SEQ TD NO: 39), PKNl (SEQ ID NO: 44), POLR2J2 (SEQ ID NOs: 41 or 65), RAB20 (SEQ ID NO 43), SYNPR (SEQ ID NO: 45), and TRPM5 (SEQ ID NO: 47); or a protein substantially similar to said target protein, wherein said substantially similar protein has a sequence identity of at least 95% to said target protein.

18. The method of claim 17, wherein said target protein is either SEQ ID NO: 7, 21,

35, 37, 45, 47, 49, 51, 53, 55, 57, 59, 61, or 63.

19. The method of claim 17, wherein said virus is hepatitis C virus (HCV) or human immunodeficiency virus (HTV).

20. The method of claim 19, wherein said target protein is either SEQ ID NO: 21, 45, 47, 53, or 63.