Patent application title: Inhibitors of hepatitis C virus
Inventors:
Robert F. Garry (New Orleans, LA, US)
Robert F. Garry (New Orleans, LA, US)
Jane A. Mckeating (Birmingham, GB)
IPC8 Class: AA61K39395FI
USPC Class:
4241391
Class name: Drug, bio-affecting and body treating compositions immunoglobulin, antiserum, antibody, or antibody fragment, except conjugate or complex of the same with nonimmunoglobulin material binds antigen or epitope whose amino acid sequence is disclosed in whole or in part (e.g., binds specifically-identified amino acid sequence, etc.)
Publication date: 2008-10-02
Patent application number: 20080241156
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Patent application title: Inhibitors of hepatitis C virus
Inventors:
Robert F. Garry
Jane A. McKeating
Agents:
HOWREY LLP-HN
Assignees:
Origin: FALLS CHURCH, VA US
IPC8 Class: AA61K39395FI
USPC Class:
4241391
Abstract:
The present invention relates to methods that employ peptides or peptide
derivatives to inhibit hepatitis C virus infection. The present invention
is based in part on the discovery that E2 envelope glycoprotein of
hepatitis C virus has previously undescribed domains that are important
for interactions with cellular or viral proteins that are necessary for
early steps in HCV infection. The present invention provides peptides and
methods of treatment and prophylaxis of diseases induced by hepatitis C
virus and related viruses.Claims:
1. A pharmaceutical composition comprising one or more peptides selected
from the group consisting of:a) a peptide having the sequence of any of
SEQ ID NO:1 to SEQ ID NO:6;b) a peptide homologous to any one of SEQ ID
NO:1 to SEQ ID NO:6 from another hepatitis C virus strain or from
hepatitis GB virus; andc) a peptide functionally equivalent to any one of
SEQ ID NO:1 to SEQ ID NO:6,wherein the functionally equivalent peptide is
identical to at least one of SEQ ID NO:1 to SEQ ID NO:6 except that one
or more amino acid residues has been substituted with a homologous amino
acid, resulting in a functionally silent change, or one or more amino
acids has been deleted.
2. A pharmaceutical composition comprising at least one peptide selected from one or more of the following:a) a peptide having the amino acid sequence one or more of SEQ ID NO:1 to SEQ ID NO:6, wherein the N-terminal moiety is an amino group and the C-terminal moiety is a carboxyl group;b) a peptide having the sequence of any of SEQ ID NO:1 to SEQ ID NO:6, wherein the N-terminal moiety is not an amino group and/or the C-terminal moiety is not a carboxyl group, wherein in place of an amino moiety the N-terminal moiety is selected from the group consisting of: an acetyl group, a hydrophobic group, carbobenzoxyl group, dansyl group, a t-butyloxycarbonyl group, or a macromolecular carrier group, and/or wherein in place of a carboxyl moiety the C-terminal moiety is selected from the group consisting of an amido group, a hydrophobic group, t-butyloxycarbonyl group or a macromolecular group;c) a peptide having the sequence of any of SEQ ID NO:1 to SEQ ID NO:6, wherein at least one bond linking adjacent amino acid residues is a non-peptide bond;d) a peptide having the sequence of any of SEQ ID NO:1 to SEQ ID NO:6, wherein at least one amino acid residue is in the D-isomer configuration;e) a peptide as in part "a)" or "b)" except that at least one amino acid has been substituted for by a different amino acid; orf) a functional fragment of a peptide as set out in any of parts "a)" to "e)", having at least 3 contiguous nucleotides of any one of SEQ ID NO:1 to SEQ ID NO:42.
3. The composition of claim 2 wherein at least one peptide has an amino acid sequence is selected from one or more of the group consisting of SEQ ID NO: 1, 2, 3, 4, 5 and 6.
4. The composition of claim 3 wherein the terminal moiety on the N-terminal amino acid is an acetyl group, a hydrophobic group a carbobenzoxyl group, a dansyl group, a t-butyloxycarbonyl group, or a macromolecular carrier group; and/or the terminal moiety on C-terminal amino acid is a hydrophobic group, a t-butyloxycarbonyl group or a macromolecular group.
5. The composition of claim 3 wherein the terminal moiety on the N-terminal amino acid is a macromolecular carrier group selected from a lipid conjugate, polyethylene glycol, or a carbohydrate; and/or the terminal moiety on the C-terminal amino acid is a macromolecular carrier group selected from a lipid conjugate, polyethylene glycol, or a carbohydrate.
6. The composition of claim 3 wherein at least one bond is a non-peptide bond selected from the group consisting of an imido bond, an ester bond, a hydrazine bond, a semicarbazoide bond and an azo bond.
7. The composition of 3 wherein at least one amino acid is a D-isomer amino acid.
8. The composition of claim 3 wherein terminal moiety on the N-terminal amino acid group is an amino group and the terminal moiety on the C-terminal amino acid is a carboxyl group.
9. The composition of claim 8 wherein the peptide is selected from one or more of the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, and 6.
10. The composition of claim 1 wherein at least one peptide is selected from the group of peptides having a sequence selected from the sequences of SEQ ID NO:7-42.
11. A method of treating or preventing hepatitis C virus infection comprising administering to the patient an effective amount of a pharmaceutical composition according to any of claims 1-10.
12. A substantially purified antibody specific for a peptide as described in any of claims 1-10.
13. A method of treating or preventing hepatitis C virus infection comprising administering to a patient one or more peptides according to any of claims 1-9 and/or an antibody according to claim 11 and/or one or more peptides and/or one or more antibodies targeted to inhibiting the membrane fusion step mediated by hepatitis C virus envelope protein 1.
14. The method of claim 13, wherein the combination of the compounds from claims 1-9 and/or 11 with the peptides and/or antibodies that inhibit fusion mediated by hepatitis C virus envelope protein 1 is synergistic in preventing or inhibiting HCV infection.
15. A peptide having the sequence of any one of SEQ ID NO:1-6 or an inhibitory portion thereof.
16. A peptide consisting of the sequence of any one of SEQ ID NO: 1-6.
17. A peptide having the sequence of any one of SEQ ID NO:7-42 or an inhibitory portion thereof.
18. A peptide consisting of the sequence of any one of SEQ ID NO:7-42.
Description:
CROSS-REFERENCE TO RELATED APPLICATION
[0001]This application claims priority to U.S. provisional patent application Ser. No. 60/614,280, entitled "Inhibitors of Hepatitis C Virus," by Robert F. Garry, Jr. and Jane A. McKeating, filed Sep. 29, 2004, which is incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0002]The present invention relates to peptides and methods of inhibiting virus-cell binding and entry of hepatitis C virus. Specific embodiments of the invention are drawn to the inhibition of infection by hepatitis C virus (HCV).
BACKGROUND OF THE INVENTION
[0003]Viruses must infect host cells to replicate, produce a spreading infection, and cause disease. Infection by enveloped viruses requires binding of the virion to one or more structures on the cell surface (Flint and McKeating, 2000). The initial step may be a low affinity non-specific binding (Barth et al., 2003). Subsequently, the virus binds with high affinity to primary receptors, and then in some cases to secondary receptors or co-receptors (Bartosch et al., 2003; Hsu et al., 2003; Roccasecca et al., 2003; Cormier et al., 2003; _Pohlmann et al., 2003; Zhang et al., 2004). The cell surface binding steps can be associated with a variety of structural rearrangements of the virion surface proteins and changes in protein-protein interactions between the viral surface proteins (Jardetsky and Lamb, 2004; Modis et al., 2004; Bressanelli et al., 2004; Gibbons et al, 2004). The latter steps can expose the fusion peptide, a hydrophobic domain of a viral glycoprotein that is able to interact with cell membranes (Flint et al., 1999; Allison et al., 2001). In some cases, the binding of the virus to the cell surface receptors triggers uptake of the virus through endocytic, or similar vesicular pathways (Garry and Dash, 2003; Jardetsky and Lamb, 2004). Exposure to more acidic conditions in the vesicles can trigger conformational changes in the viral surface proteins, including those that expose the fusion peptide (Kuhn et al., 2002; Lescar et al., 2001). For most viruses, binding to the cellular receptor is primarily the function of one viral surface protein, whereas fusion of the viral and cellular membranes is primarily the function of another viral surface protein. An example of a virus with separate receptor binding and fusion protein is HIV. The receptor binding protein of HIV is the surface glycoprotein (SU; gp120) and the fusion protein is the transmembrane glycoprotein (TM; gp41) (Kwong et al., 1998; Gallaher et al., 1987; 1989). Most viruses with class I fusion proteins in which the fusion peptide is located at or near the amino terminus, for example retroviruses, orthomyxoviruses, paramyxoviruses arenaviruses and coronaviruses, use one protein for receptor binding and another for fusion (Wilson et al., 1981; Gallaher et al, 1996; 2001). Alphaviruses, which have a class II fusion protein with an internal fusion peptide, also use one protein principally for receptor binding and another for fusion of the viral and cellular membranes (Straus and Straus, 1994). The envelope (E) protein encoded by members of the flavivirus genus of the Flaviviridae, has an internal fusion peptide, but serves both receptor binding and fusion function (Allison et al., 2001).
[0004]Hepatitis C virus encodes two envelope glycoproteins, E1 (gp35) and E2 (gp70), both with C-terminal transmembrane anchor domains (Flint and McKeating, 2000). E2 interacts with several cell surface proteins (CD81, SR-BI and L-SIGN) suggesting that it is the receptor binding protein of HCV (McKeating, 2004). The function of E1 is less clear and may act to chaperone E2 (Flint et al., 1999; Garry and Dash, 2003). Synthetic peptides corresponding to hepatitis C virus E2 can block infection mediated by hepatitis C virus. Structural determinations of the hepatitis C virus E2 allow the identification of several heretofore unknown features of hepatitis C virus E2 for drug and vaccine development.
[0005]The Flavivirus family includes a variety of important human and animal pathogens. Hepatitis C virus (HCV) is the leading viral cause of chronic hepatitis, cirrhosis, liver failure, and hepatocellular carcinoma (Poynard et al., 2003). In the United States alone, an estimated 4 million people are infected with HCV. This is approximately four times the number infected by HIV. Each year in the US, 30-50,000 new HCV infections occur, and about 15-20,000 people die. Moreover, these numbers are expected to increase dramatically given that a substantial portion of HCV infected individuals show little or no response to the only currently approved therapeutics (i.e. treatment with interferons and/or ribavirin). HCV infection is spread primarily through needle sharing among drug users, although there is some risk from accidental needle sticks, blood products before 1992, chronic blood dialysis, and frequent sexual contact. Current treatments for HCV using ribavirin and interferon cost ˜$8,000 to $20,000 per year, and are only partially successful in about half of patients treated. Overall, about 80% of HCV carriers suffer chronic liver inflammation and cirrhosis, of these 25% will develop end stage liver disease or hepatocellular carcinoma (HCC) (Colombo, 2000). End stage HCV disease is the most frequent indication for liver transplants and this costs $250,000 to $300,000. Better drugs to treat HCV infection and an effective vaccine to prevent HCV infection are urgently needed.
SUMMARY OF THE INVENTION
[0006]The present invention relates to the compositions comprising peptides or peptide derivatives and methods that employ these compositions to treat, prevent or inhibit infection by hepatitis C virus (HCV) and related viruses. The present invention is made possible by the inventors' discovery that that HCV encoded E2 glycoprotein (and the analog(s) from related viruses) has previously undescribed domains that are important for interaction(s) and rearrangements of E2 with E2 and/or E1, for high affinity interactions with cellular receptors, or for E2 and E1-E2 protein-protein interactions that occur prior to virion:cell membrane fusion. Thus, the present invention provides peptides and methods for treatment and prophylaxis of diseases induced by HCV and related viruses.
[0007]The instant invention teaches that HCV envelope glycoprotein E2 has several domains that can be targeted by synthetic peptides to block infection and pathogenesis. The regions of HCV E2 are important for the binding of HCV to its low or high affinity cellular receptors, for rearrangements of E2 or for protein-protein interactions of E2 that occur prior to virion:cell membrane fusion. This invention also teaches and provides synthetic peptides that can inhibit receptor binding and other pre-fusion steps mediated by HCV E2.
[0008]Features of hepatitis C virus envelope glycoprotein 2 identified herein provide surprising guidance for the development of vaccines and/or drugs to prevent or treat hepatitis C virus infections. According to one embodiment of the invention, the target for the peptides is E2, the receptor binding protein of HCV. Although proteins, such as soluble CD4, chemokines and antibodies have been developed that block infection by targeting virion receptor binding interactions, peptide mimics of viral surface proteins that block this or other pre-fusion steps have not be described. Prior to the availability of X-ray structural data (Qureshi et al., 1990; Wild, et al., 1993; 1994), several potent HIV-1 inhibitors were developed based on the Gallaher HIV-1 TM fusion protein model (Gallaher et al., 1989). One of these inhibitors, FUZEON® (aka enfuvirtide, DP178; T20) peptide has been shown to substantially reduce HIV-1 load in AIDS patients in clinical trials (Lalezari, et al., 2003). The peptide drugs, which are the subject of the instant invention, were also developed without benefit of X-ray structural data. FUZEON® targets HIV fusion protein and the steps in HIV entry involving fusion between the viral and cellular membrane. Certain hepatitis C virus E2 inhibitory peptides target different steps in the viral replication cycle than are targeted by FUZEON® and other known viral peptide inhibitors. E2-based peptide drugs should be relatively easy to develop, based on our identification of E2 domains that can be targeted by synthetic peptides to block infection and pathogenesis. Once an effective peptide inhibitor is described a non-peptide drug can be developed.
[0009]More specifically, the present invention provides for methods of inhibiting viral infection and pathogenesis by hepatitis C virus. The invention is related to the discovery, as described herein, of hepatitis C virus E2 domains that can be targeted by synthetic peptides to block infection and pathogenesis. Various embodiments of the invention provide methods that employ peptides or peptide derivatives to inhibit hepatitis C virus receptor binding, E2 structural rearrangements or protein-protein interactions, or other pre-fusion steps. The present invention provides for methods of treatment and prophylaxis of diseases induced by hepatitis C virus.
[0010]Various embodiments of the instant invention provide for pharmaceutical compositions comprising one or more peptides selected from one or more of the following groups. [0011]A) Peptides having the sequence of any of SEQ ID NO: 1 to SEQ ID NO:6; [0012]B) Peptides homologous to any one of SEQ ID NO:1 to SEQ ID NO:6, except that they are from a different strain of hepatitis C virus. [0013]C) Peptides homologous to any one of SEQ ID NO: 1 to SEQ ID NO:6, except that they are from hepatitis GB virus. [0014]D) Peptides that are functionally equivalent to any one of SEQ ID NO: 1 to SEQ ID NO:6, wherein the functionally equivalent peptide is identical to at least one of SEQ ID NO:1 to SEQ ID NO:6 except that one or more amino acid residues has been substituted with a homologous amino acid, resulting in a functionally silent change, or one or more amino acids has been deleted. An homologous amino acid is an amino acid with chemical or functional similarity to another amino acid. Sets of homologous amino acids are: nonpolar amino acids: alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine; polar neutral amino acids: glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; hydrophobic amino acids: leucine, isoleucine, valine, methionine, alanine, phenylalanine; basic amino acids: lysine, arginine, histidine; acidic amino acids and their amides: aspartic acid, asparginine; glutamic acid, glutamine; aromatic amino acids: tyrosine, tryptophan, phenylalanine, histidine; amino acid alcohols: serine, threonine; and small amino acids: glycine, proline. For example, and not by way of limitation, such peptides may also comprise one or more D-amino acids.
[0015]Various aspects of this embodiment of the invention provide for compositions that comprise one or more peptides having one or more of the following traits: [0016]A) Peptides having the amino acid sequence of one or more of SEQ ID NO:1 to SEQ ID NO:42, wherein the N-terminal end of the peptide terminates in an amino group and the C-terminal end of the peptide terminates in a carboxyl group. [0017]B) Peptides having the sequence of any of SEQ ID NO: 1 to SEQ ID NO:42, wherein the peptide's N-terminal end does not terminate in an amino group and/or the peptide's C-terminal end does not a terminate in carboxyl group, wherein the peptide's N-terminal end terminates in a moiety selected from the group consisting of: an acetyl group, a hydrophobic group, carbobenzoxyl group, dansyl group, a t-butyloxycarbonyl group, or a macromolecular carrier group, and/or wherein the peptide's C-terminal end terminates in a moiety selected from the group consisting of an amido group, a hydrophobic group, t-butyloxycarbonyl group or a macromolecular group. [0018]C) Peptides having the sequence of any of SEQ ID NO: 1 to SEQ ID NO:42 except that at least one bond linking adjacent amino acid residues is a non-peptide bond. [0019]D) Peptides having the sequence of any of SEQ ID NO:1 to SEQ ID NO:42, except that at least one amino acid residue is in the D-isomer configuration. [0020]E) Peptides as in groups "A)" or "B)" except that at least one amino acid has been substituted for by a different amino acid (whether a conservative or non-conservative change). Preferably the peptide comprises 1, 2, 3, 4, 5, or more conservative or non-conservative changes. As used herein the term "a conservative change" is preferably defined as substitution in the peptide sequence of an amino acid by a homologous amino acids (for example, a substitution of a leucine for another hydrophobic amino acid such as isoleucine). A non-conservative change is defined as substitution in the peptide sequence of an amino acid by a non-homologous amino acids (for example, a substitution of the acidic aspartic acid for the basic amino acid arginine). [0021]F) Peptides that are a functional fragment of a peptide as set out in any of groups "A)" to "E)", above, where the peptides have at least 3 contiguous nucleotides of any one of SEQ ID NO:1 to SEQ ID NO:42. As used herein, the term "a functional fragment of an inhibitory peptide" is preferably defined is a peptide with a shorter sequence consisting of a subset of the amino acids of the inhibitory peptide, which fragment retains the inhibitory properties. For example, "DEFGHKL" could represent a functional fragment of inhibitory peptide "ABCDEFGHIJKLMNOP" if it was inhibitory. And,
[0022]G) Peptides that combine the modification of two or more of part "A"-"F". For example, a peptide that comprises non-peptide bonds linking two or more of the constituent amino acid residues and has an N-terminal moiety that is not an amino group.
[0023]The instant invention also provides for substantially purified antibodies that specifically react with one or more of the peptides described above.
[0024]The instant invention also provides for methods for treating or preventing HCV infections where the method comprises administering one or more of the peptides and/or antibodies as described above.
[0025]The instant invention also provides for methods for treating or preventing hepatitis C virus infections where the method comprises administering to a patient in need of such treatment a composition comprising one or more of the peptides and/or antibodies as described above in combination with peptides and/or antibodies or peptides targeting the fusion step mediated at least in part by hepatitis C virus envelope protein 1 as described in international application PCT/US2003/035666 which is herein incorporated by reference. The HCV E2 and E1 peptides and/or antibodies may work synergistically (i.e. they may be active at lower concentrations in combination that when used separately) or they may act in a complimentary or additive fashion.
ABBREVIATIONS
[0026]HCV--hepatitis C virus [0027]HSA--human serum albumen
BRIEF DESCRIPTION OF THE DRAWINGS
[0028]FIG. 1: Alignments of protein E2 peptide sequences from two strains of hepatitis C virus showing locations of active peptides. E2 sequences from H77, a genotype 1a strain of HCV, and J4 a genotype 1b strain of HCV were aligned. A ":" indicates identical amino acid and a "." indicates a chemically similar amino acid in the two sequences. Bars above or below the peptide sequence indicate the locations of peptides, numbered as in Tables 7 and 8, that inhibit infection by an HCV pseudotype.
[0029]FIG. 2. Specificity of HCV E2 inhibitory peptides. E2 peptides, numbered as in Tables 7 and 8, were added to pseudotypes containing the core proteins of HIV and HCV E1, E2, murine leukemia virus surface and transmembrane glycoproteins (SU and TM), or vesicular stomatitis virus glycoprotein (G). Supernatants were also treated with DMSO vehicle alone or with a Mab (monoclonal antibody) to HCV E2 known to neutralize pseudotype infectivity. Peptide treated and control pseudotypes were added to cells, which were incubated at 37° C. for 72 h. Cell lysates were then tested for luciferase activity as described (Hsu et al., 2003).
[0030]FIG. 3: Structures of hepacivirus E2 glycoprotein showing location of active peptides, numbered as in Tables 7 and 8. A two dimensional model of HCV envelope protein 2 was constructed using proteomics computational tool and comparisons to receptor binding proteins of other RNA viruses. Sequences that resulted in greater than 70% reduction in HCV pseudotype infectivity are indicated.
[0031]FIG. 4. Model depicting pre-fusion site of action of HCV E2 peptides. Panel A. HCV E2 peptides disruption of E1-E2 interactions or E2-E2 interactions in HCV virions. Panel B: HCV E2 disruption of HCV virion-receptor interactions.
DETAILED DESCRIPTION OF THE INVENTION
[0032]The present invention relates to compositions for and methods of preventing, treating, or inhibiting Flavivirus infection. The currently disclosed compositions and methods are theorized to operate by inhibiting the fusion between the virion envelope and a cell membrane, the process that delivers the viral genome into the cell cytoplasm.
[0033]Various embodiments of the invention, provide the identification and sequence of HCV inhibitory peptides representing specific portions of the HCV E2 glycoprotein. These inhibitory peptides proteins are believed to function by interfering with and blocking receptor binding. These peptides include those represented by SEQ ID NOs 142 and derivatives thereof as described below.
[0034]In particular aspects of this embodiment of the invention compositions comprising peptides corresponding to the HCV envelope glycoprotein 2 are useful at a broad range of doses (as shown in Example 1 these peptides are effective at inhibiting HCV fusion with the cells).
[0035]For purposes of clarity of disclosure, and not by way of limitation, the description of the present invention will be divided into the following subsections:
(i) Peptides of the invention(ii) Utilities of the invention (including compositions and methods for employing the peptides)
TABLE-US-00001 TABLE 1 HCV E2 inhibitory peptide 1 PROTEIN SEQUENCE* HCV E2 a X-LVGLLTPGAKQNIQLINTNGSWHIN (SEQ ID NO:1) S-Z HCV E2 b X-FTSLFSSGASQKIQLVNTNGSWHIN (SEQ ID NO:7) R-Z HCV E2 a X-LAGLFTSGAKQNIQLINTNGSWHIN (SEQ ID NO:8) R-Z HCV E2 b X-FTSFFTRGPSQNLQLVNSNGSWHIN (SEQ ID NO:9) S-Z HCV E2 a X-LANLFSSGSKQNLQLINSNGSWHIN (SEQ ID NO:10) R-Z HCV E2 a X-LTSFFNPGPQRQLQFVNTNGSWHIN (SEQ ID NO:11) S-Z HCV E2 a X-FASLLTPGAKQNIQLINTNGSWHIN (SEQ ID NO:12) R-Z
TABLE-US-00002 TABLE 2 HCV E2 inhibitory peptide 2 PROTEIN SEQUENCE* HCV E2 1a X-CNESLNTGWLAGLFYQH-Z (SEQ ID NO:2) HCV E2 1b X-CNDSLHTGFLAALFYTH-Z (SEQ ID NO:13) HCV E2 2a X-CNDSLNTGFIASLFYTY-Z (SEQ ID NO:14) HCV E2 3b X-CNDSLNTGFIAGLFYYH-Z (SEQ ID NO:15) HCV E2 4a X-CNDSLNTGFLASLFYTH-Z (SEQ ID NO:16) HCV E2 5a X-CNDSLQTGFIAGLMYAH-Z (SEQ ID NO:17) HCV E2 6a X-CNDSLQTGFLASLFYTH-Z (SEQ ID NO:18)
TABLE-US-00003 TABLE 3 HCV E2 inhibitory peptide 3 PROTEIN SEQUENCE* HCV E2 1a X-YSWGANDTDVFVLNNTRPPLGNWF (SEQ ID NO:3) GCTWMNSTGF-Z HCV E2 1b X-YSWGENETDVMLLNNTRPPQGNWF (SEQ ID NO:19) GCTWMNSTGF-Z HCV E2 2a X-YTWGENETDVFILNSTRPPGGSWF (SEQ ID NO:20) GCTWMNSTGF-Z HCV E2 3b X-YRFGVNESDVFLLTSLRPPQGRWF (SEQ ID NO:21) GCVWMNSTGF-Z HCV E2 4a X-YTWGENETDVFLLNSTRPPHGAWF (SEQ ID NO:22) GCVWMNSTGF-Z HCV E2 5a X-YNWGSNETDILLLNNIRPPAGNWF (SEQ ID NO:23) GCTWMNSTGF-Z HCV E2 6a X-YTWGENETDVFMLESLRPPTGGWF (SEQ ID NO:24) GCTWMNSTGF-Z
TABLE-US-00004 TABLE 4 HCV E2 inhibitory peptide 4 PROTEIN SEQUENCE* HCV E2 1a X-DYPYRLWHYPCTINYTIFKVRMYV (SEQ ID NO:4) GGV-Z HCV E2 1b X-DYPYRLWHYPCTLNFSIFKVRMYV (SEQ ID NO:25) GGV-Z HCV E2 2a X-DYPYRLWHYPCTINYTIFKIRMYV (SEQ ID NO:26) GGV-Z HCV E2 3b X-DYPYRLWHYPCTVNFSIFKVRMFV (SEQ ID NO:27) GGH-Z HCV E2 4a X-DYPYRLWHFPCTANFSVFNIRTFV (SEQ ID NO:28) GGI-Z HCV E2 5a X-HYPYRLWHYPCTVNYTIFKVRMFI (SEQ ID NO:29) GGL-Z HCV E2 6a X-DYAYRLWHYPCTVNFTLHKVRMFV (SEQ ID NO:30) GGT-Z
TABLE-US-00005 TABLE 5 HCV E2 inhibitory peptide 5 PROTEIN SEQUENCE* HCV E2 1a X-ALSTGLIHLHQNIVDVQYLYGVGS (SEQ ID NO:5) SIASWAIKWEY-Z HCV E2 1b X-ALSTGLIHLHQNIVDVQYLYGVGS (SEQ ID NO:31) AFVSFAIKWEY-Z HCV E2 2a X-ALSTGLLHLHQNIVDVQYMYGLSP (SEQ ID NO:32) ALTKYIVRWEW-Z HCV E2 3b X-RLSTGLIHLHQNIVDVQYLYGVGS (SEQ ID NO:33) AVVGWALKWEF-Z HCV E2 4a X-ALSTGLIHLHQNIVDVQYLYGVGS (SEQ ID NO:34) AVVSWALKWEY-Z HCV E2 5a X-ALSTGLIHLHQNIVDTQYLYGLSS (SEQ ID NO:35) SIVSWAVKWEY-Z HCV E2 6a X-ALSTGLIHLHQNIVDVQYLYGVST (SEQ ID NO:36) NVTSWVVKWEY-Z
TABLE-US-00006 TABLE 6 HCV E2 inhibitory peptide 6 PROTEIN SEQUENCE* HCV E2 1a X-VVLLFLLLADARVCSCLWMMLLIS (SEQ ID NO:6) QAEA-Z HCV E2 1b X-ILLLFLLLADARVCACLWMMLLIA (SEQ ID NO:37) QAEA-Z HCV E2 2a X-VVLLFLLLADARVCACLWMLILLG (SEQ ID NO:38) QAEA-Z HCV E2 3b X-VVLVFLLLADARVCVALWMMLLIS (SEQ ID NO:39) QAEA-Z HCV E2 4a X-VVLAFLLLADARVSAYLWMMFMVS (SEQ ID NO:40) QVEA-Z HCV E2 5a X-IMLVFLLLADARICTCLLILLLIC (SEQ ID NO:41) QAEA-Z HCV E2 6a X-IVLMFLVLADARICTCLWLMLLIS (SEQ ID NO:42) TVEA-Z *In tables 1-6 the "X" and "Z" on each peptide respectively represent the N- and C-terminal moiety. As described above the N-terminal moiety may be either an amino group or it may be selected from the group consisting of an acetyl group, a hydrophobic group, carbobenzoxyl group, dansyl group, a t-butyloxycarbonyl group, or a macromolecular carrier group and/or the peptide's C-terminal moiety may be a carboxy group or it may be a moiety selected from the group consisting of: an amido group, a hydrophobic group, t-butyloxycarbonyl group or a macromolecular group.
Peptides of the Invention
[0036]Any peptide or protein which inhibits the fusion between the Hepatitis C virus E2 virion envelope and a cell membrane, including those of Hepatitis C virus E2 which infect human as well as nonhuman hosts, may be used according to the invention. In various embodiments of the invention, these inhibitors may include, but are not limited to peptides related to several membrane-interactive domains of Hepatitis C virus E2.
[0037]Hepatitis C virus E2 inhibitory peptides are, according to the instant invention, identical or homologous to the amino acid sequences
[0038]As used herein the term "conservative substitution" preferably refers to substitution in the peptide sequence of an amino acid by a homologous amino acid.
[0039]As used herein the term "peptide derivative" preferably refers to: a peptide modified by the addition of one or more groups, including, but not limited to a carbobenzoxyl group, a dansyl group, t-butyloxycarbonyl group, a lipid conjugate, a polyethylene glycol group, or a carbohydrate
[0040]As used herein the term "similar peptides" refers to those peptides having at least 70% identical or chemically similar amino acids. More preferably, it refers to peptides having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or greater identical and/or chemically equivalent amino acid resides.
[0041]As used herein the terms "portion thereof" refers to the peptide resulting from the removal of from one or more amino acids from either or both ends of the listed peptide, i.e. a truncated peptide. The number of amino acids removed may vary from 1-10 so long as the remaining fragment is "functional". As defined herein the term "functional fragment" refers to a fragment capable of inhibiting virus:cell fusion, inhibiting viral infectivity, capable of eliciting an antibody capable of recognizing and specifically binding to non-truncated peptide and/or interfering with hepatitis C virus envelope protein 2-mediated cell infection.
[0042]According to the instant invention peptides related to the Hepatitis C virus E2 inhibitory peptides (E2IP) preferably comprise at least three contiguous residues of the E2IP peptides, or a homologous peptide, more preferably they comprise 4, 5, 6, or 7 contiguous residues. Even more preferably they comprise at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more contiguous residues (up to the maximum number of residues in the peptide), and most preferably all residues of these sequences. As used herein the term Hepatitis C virus E2 inhibitory peptides preferably means peptides having a sequence identical to the corresponding portion of the Hepatitis C virus E2 inhibitory protein and peptides in which one or more amino acids are substituted by functionally equivalent amino acids (see infra). The term also refers to derivatives of these peptides, including but not limited to benzylated derivatives, glycosylated derivatives, and peptides which include enantiomers of naturally occurring amino acids. In other embodiments of the invention, the Hepatitis C virus E2 inhibitory peptides, related peptides or derivatives are linked to a carrier molecule such as a protein. Proteins contemplated as being useful according to this embodiment of the invention, include but are not limited to, (human serum albumen). Hepatitis C virus E2 inhibitory peptide-related peptides comprising additional amino acids are also contemplated as useful according to the invention.
[0043]Peptides may be produced from naturally occurring or recombinant viral proteins, or may be produced using standard recombinant DNA techniques (e.g. the expression of peptide by a microorganism which contains recombinant nucleic acid molecule encoding the desired peptide, under the control of a suitable transcriptional promoter, and the harvesting of desired peptide from said microorganism). Preferably, the peptides of the invention may be synthesized using any methodology known in the art, including but not limited to, Merrifield solid phase synthesis (Clark-Lewis et al., 1986).
[0044]The E2IP, or fragments or derivatives thereof, of the invention include, but are not limited to, those containing, as a primary amino acid sequences the amino acid sequence hepatitis C virus E2 Inhibitory peptide 1: LVGLLTPGAKQNIQLINTNGSWHINS SEQ ID NO:1; HCV E2 Inhibitory peptide 2 CNESLNTGWLAGLFYQH SEQ ID NO:2; HCV E2 Inhibitory peptide 3, YSWGANDTDVFVLNNTRPPLGNWFGCTWMNSTGF SEQ ID NO:3; or HCV E2 Inhibitory peptide 4, DYPYRLWHYPCTINYTIFKVRMYVGGV SEQ ID NO:4; HCV E2 Inhibitory peptide 5, X-ALSTGLIHLHQNIVDVQYLYGVGSSIASWAIKWEY SEQ ID NO:5; E2 Inhibitory peptide 6, VVLLFLLLADARVCSCLWMMLLISQAEA, SEQ ID NO:6 or a functional portion or functional portions thereof.
[0045]Also contemplated are altered sequences analogous to any of SEQ ID NOs 142; more preferably analogous to any of SEQ ID NO:1-6 (i.e. altered from any of the sequences referred to herein) in which functionally equivalent amino acid residues are substituted for residues within the sequence, resulting in a functionally silent change. For example, one or more amino acid residues within the sequence can be substituted by replacing the original amino acid with another amino acid, of a similar polarity, that acts as a functional equivalent, resulting in a functionally silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, classes of homologous amino acids are: nonpolar amino acids: alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine, polar neutral amino acids: glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine, hydrophobic amino acids: leucine, isoleucine, valine, methionine, alanine, phenylalanine; basic amino acids: lysine, arginine, histidine; acidic amino acids and their amides: aspartic acid, asparginine; glutamic acid, glutamine; aromatic amino acids, tyrosine, tryptophan, phenylalanine, histidine; amino acid alcohols: serine, threonine and small amino acids: glycine, proline. For example, and not by way of limitation, such peptides may also comprise one or more D-amino acids. Furthermore, in any of the embodiments of the instant invention the peptide may comprise an inefficient carrier protein, or no carrier protein at all.
[0046]Further, as noted supra, in any embodiment of the invention the peptide's N-terminal moiety may be either an amino group (as is typically found in naturally occurring proteins/peptides) or it may be selected from the group consisting of an acetyl group, a hydrophobic group, carbobenzoxyl group, dansyl group, a t-butyloxycarbonyl group, or a macromolecular carrier group and/or the peptide's C-terminal moiety may be a carboxy group (as is typically found in naturally occurring proteins/peptides) or it may be a moiety selected from the group consisting of: an amido group, a hydrophobic group, t-butyloxycarbonyl group or a macromolecular group.
UTILITY OF THE INVENTION
[0047]The hepatitis C virus E2 inhibitory peptides of the instant invention may be utilized to inhibit hepatitis C virus infection and may, accordingly, be used in the treatment of hepatitis C virus infection and also in prophylaxis against hepatitis C virus infection. The peptides of the invention may be administered to patients in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. Methods for administering peptides to patients are well known to those of skill in the art; they include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, and intranasal. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection.
[0048]The instant invention provides for compositions, especially pharmaceutical compositions, comprising hepatitis C virus E2 inhibitory peptides, peptide fragments, or derivatives (as described supra) administered via liposomes, microparticles, or microcapsules. Various embodiments of the invention, contemplate the use of such compositions to achieve sustained release of hepatitis C virus E2 inhibitory peptides. Other embodiments contemplate the administration of the FIP or derivatives thereof, linked to a molecular carrier (e.g. HSA).
[0049]Various embodiments of the instant invention provide for administration of the hepatitis C virus E2 inhibitory peptides and/or antibodies specific for these peptides to human subjects who suffer from hepatitis C virus infection. In any embodiment the peptides and/or antibodies are typically substantially purified. As used herein the term "substantially purified" refers to a peptide, peptide analog, or antibody that is greater than about 80% pure. More preferably, "substantially purified" refers to a peptide, peptide analog, or antibody that is greater than about 90% or greater than about 95% pure. Most preferably it refers to a peptide, peptide analog, or antibody that is greater than 99% pure. Functionally, "substantially purified" means that it is free from contaminants to a degree that that makes it suitable for the purposes provided herein. Other embodiments provide for the prophylactic administration of the peptides to those at risk for hepatitis C virus infection.
[0050]Other embodiments of the instant invention provide for methods for identifying the structure of truncated hepatitis C virus E2 proteins which involved hepatitis C virus receptor binding, E2 structural rearrangements or protein-protein interactions, or other pre-fusion steps by members of the Flaviviridae family and for the structures themselves.
[0051]Other embodiments of the invention provide for a peptide having a formula selected from one or more of the following.
[0052]The E2IP, or fragments or derivatives thereof, of the invention include, but are not limited to, those containing, as a primary amino acid sequences the amino acid sequence hepatitis C virus E2 Inhibitory peptide 1: LVGLLTPGAKQNIQLINTNGSWHINS (SEQ ID NO:1); HCV E2 Inhibitory peptide 2: CNESLNTGWLAGLFYQH (SEQ ID NO:2); HCV E2 Inhibitory peptide 3: YSWGANDTDVFVLNNTRPPLGNWFGCTWMNSTGF (SEQ ID NO:3); or HCV E2 Inhibitory peptide 4: X-DYPYRLWHYPCTINYTIFKVRMYVGGV (SEQ ID NO:4); HCV E2 Inhibitory peptide 5 ALSTGLIHLHQNIVDVQYLYGVGSSIASWAIKWEY (SEQ ID NO:5); HCV inhibitory peptide 6: VVLLFLLLADARVCSCLWMMLLISQAEA (SEQ ID NO:6); or a functional portion or functional portions thereof of any of these peptides.
[0053]According to various embodiments of the instant invention, any of the peptides described herein may comprise an amino group at the amino-terminal end or may be modified to comprise any of the following: an acetyl group, a hydrophobic group or a macromolecular carrier group. Similarly, the carboxy-terminus of any of the peptides may comprise a carboxyl group or may be modified to comprise any of the following groups: an amido group a hydrophobic group or a macromolecular carrier group. In other aspects of this embodiment of the invention, the amino terminal group is a hydrophobic group, a carbobenzoxyl group, a dansyl group, t-butyloxycarbonyl group, a lipid conjugate, a polyethylene glycol group, or a carbohydrate. In any aspect of this embodiment the carboxy terminal group may be a t-butyloxycarbonyl group, a lipid conjugate, a polyethylene glycol group, or a carbohydrate.
[0054]Moreover, aspects of this embodiment also include peptides wherein at least one bond linking adjacent amino acids residues is a non-peptide bond. In particularly preferred aspects of this embodiment the non-peptide bond is an imido, ester, hydrazine, semicarbazoide or azo bond . . . .
[0055]Other aspects of this embodiment provide for peptides wherein at least one amino acid is a D-isomer amino acid.
[0056]Additional aspects of this embodiment of the invention provide for peptides wherein compromising at least one amino acid substitution has been made so that a first amino acid residue is substituted for a second, different amino acid residue. These substitutions may be conservative or non-conservative, as long as the peptide is still functional according to the instant invention.
[0057]Other aspects of this embodiment of the invention provide for peptides wherein at least one amino acid has been deleted. As noted, supra, the peptides according to this embodiment of the invention must comprise at least 3 contiguous amino acids of one of the SEQ ID NOs indicated above and must be a functional segment.
[0058]Other embodiments of the invention provide for compositions comprising one or more of the peptides and/or antibodies described herein, either alone, or with a carrier compound. Preferably, the carrier is a pharmaceutically acceptable excipient.
[0059]Other embodiments provide for use of the methods described herein in combination with one or more other treatment regimens. For example, one or more peptides of the current invention and/or one or more antibodies specific for peptides of the current invention may be used in combination with or one or more peptides or antibodies targeted to inhibiting the membrane fusion step mediated by hepatitis C virus envelope protein 1. Such peptides are described in international application PCT/US2003/035666 which is herein incorporated by reference in its entirety. The HCV E2 and E1 peptides and/or antibodies may work synergistically (i.e. they may be active at lower concentrations in combination that when used separately) or they may act in a complimentary or additive fashion.
[0060]It is noted that any combination of the modifications listed above is considered as part of the instant invention.
EXAMPLES
Example 1
Identification of Hepatitis Virus E2 Peptides that Inhibit Infectivity Mediated by HCV Envelope Proteins
[0061]The selective association of a virus with a target cell is usually determined by an interaction between the viral surface glycoproteins and specific receptor or receptors on the cell surface. Receptor-binding is an essential step in the initiation of infection, and precedes other steps such as fusion between the virus and cellular membranes. Virion:receptor interaction(s) can define the host range and cellular or tissue tropism of a virus and may determine pathogenicity. HCV encodes two putative surface glycoproteins, E1 and E2, which are both believed to have carboxyl terminal transmembrane domains that anchor them in the virion envelope. In vitro expression studies have shown that E1 and E2 associate to form heterodimers, which accumulate in the endoplasmic reticulum (ER), the proposed site for HCV assembly and budding (Flint et al., 2004). Several lines of evidence indicate that E2 is the receptor binding protein (Flint and McKeating, 2000). It has been suggested that E1 is the HCV fusion protein (Flint et al., 1999; Garry and Dash, 2003). Other studies, however, indicate that E2, has a class II viral fusion protein structure and represented the fusion protein of HCV (Yagnik et al., 2000), and it is possible that both HCV E1 and E2 have a role in membrane fusion. The lack of in vitro systems for HCV propagation has hampered biological and physiochemical studies on the virion and its mechanism(s) of cell entry, and the cellular receptors remain unknown. HCV purified from plasma has been reported to exist in association with plasma lipoproteins, suggesting that the virus may use the low-density lipoprotein receptor (LDLR) to gain entry into cells (Agnello et al., 1999. Truncated soluble versions of E2 have been reported to bind specifically to human cells and were used to identify interactions with CD81 (Pileri et al., 1998; Roccasecca et al., 2003; Cormier et al., 2004), scavenger receptor class B type 1 (SR-BI) (Scarselli et al., 2002), and dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintegrin (DC-SIGN) (Pohlmann et al., 2003). The results suggest that E2 may represent a target to develop peptide drugs against hepatitis C virus infection.
Materials And Methods
[0062]To overcome the lack of a conventional cell culture system for the propagation of HCV, infectious pseudotype viruses expressing HCV envelope glycoproteins have been generated (Hsu et al., 2003). Pseudotypes with HIV core proteins and HCV envelope proteins were generated by cotransfection of 293-T cells with equal amounts of plasmids expressing HCV E1 and E2 of strain H77 and the HIV envelope-defective proviral genome, pNL4.3.Luc.R-E- (Pohlmann et al., 2003). Peptides from an 18mer peptide set, overlapping by 7-10 amino acids and representing the entire amino acid sequence of E2 of HCV strain H77 (genotype 1a) and the entire amino acid sequence of HCV strain J4 (genotype 1b) were solubilized in 20% DMSO and diluted (final DMSO concentration <2%). Peptides were incubated at 37° C. with p24 antigen-normalized HCV pseudotype viral supematants. The average concentration of peptides was ˜25 μM, however, actual concentrations of some peptides in solution were 10 μM or less due to low solubility in DMSO. Supernatants were also treated with DMSO vehicle alone or with a Mab (monoclonal antibody) to HCV E2 known to neutralize pseudotype infectivity. Peptide treated and control HCV pseudotypes were added to cells, incubated for 16 hrs, virus removed and cells incubated at 37° C. for 72 h. Cell lysates were then tested for luciferase activity as described (Hsu et al., 2003).
Results and Discussion
[0063]Fifty HCV H77 1a E2 peptides were tested in the HCV infectivity assay, and nine demonstrated greater than 70% inhibition of infectivity, with several demonstrating approximately 95% inhibition (Table 7). Of 46 HCV J4 1b E2 peptides tested four demonstrated greater than 70% inhibition of infectivity (Table 8). Several of the inhibitory peptides were overlapping in the E2 sequence, for example HCV H77 1a E2 peptides 4 and 5, 32 and 33, and 43, 44, 45 and 46 (FIG. 1). This result suggests that any of several peptides targets to a particular region may be inhibitory. A comparison of the two sets of E2 peptides, H77 and J4, reveals that while similar peptides can be inhibitory (i.e., peptides 32, 33 and 108, 109), while other peptides with closely related sequences are not inhibitory (i.e., peptides 4, 5, and 99).
[0064]Selected E2 inhibitory peptides were added to pseudotypes containing the core proteins of HIV and murine leukemia, virus surface, and transmembrane glycoproteins (MuLV SU and TM), or vesicular stomatitis virus glycoprotein (VSV G). These peptides inhibited pseudotypes with HCV E1 and E2 (FIG. 2A), but failed to significantly inhibit the MuLV or VSV pseudotypes (FIGS. 2B and 2C). The results indicate that these HCV E2 inhibitory peptides are specific for inhibition of the infectivity of HCV. These results also demonstrate the potential of E2 peptides as antiHCV drugs.
TABLE-US-00007 TABLE 7 Identification of HCV E2 inhibitory peptides (strain H77) that inhibit HCV infectivity. HCV H77 1a peptide # Luciferase units† Percent inhibition 1 250,376 44.1 2 447,336 0.2 3 447,906 0.05 4 10,620 97.6 5 48,000 89.3 6 503,446 -12.3 7 381,340 14.9 8 113,650 74.6 9 501,126 -11.8 10 334,196 25.4 11 360,410 19.6 12 417,706 6.8 13 313,323 31.1 14 279,626 37.6 15 253,410 43.5 16 403,430 10.0 17 254,516 43.2 18 435,026 2.9 19 301,406 32.7 20 231,373 48.4 21 242,223 45.9 22 245,900 45.2 23 367,916 17.9 24 391,886 19.7 25 480,280 7.2 26 216,706 51.6 27 575,206 -28.4 28 394,780 11.9 29 297,353 33.6 30 655,040 -46.2 31 419,263 6.4 32 85,086 81.0 33 22,406 95.0 34 354,696 21.8 35 153,553 66.7 36 535,016 -19.5 37 585,553 -30.7 38 345,110 23.0 39 400,756 11.6 40 442,346 1.3 41 434,743 3.0 42 353,516 19.1 43 32,283 92.8 44 91,266 79.6 45 24,703 94.5 46 103,040 77.0 47 195,320 56.4 48 290,786 35.1 49 307,310 31.4 50 58,790 87.9 VIRUS ALONE 448,123 Virus plus anti- 10,309 97.6 E2 2/69a Virus plus anti- 3,567 99.2 E2 9/27 †The numbers represent the number of luciferase units (lumens) produced after infection by either the HCV or the MLV pseudotype in the presence of the peptide at a concentration of ~25 μM. Results above 70% inhibition are indicated in bold-face type.
TABLE-US-00008 TABLE 8 Identification of HCV E2 inhibitory peptides (strain J4) that inhibit HCV infectivity. HCV J4 1b peptide # Luciferase units† Percent inhibition 54 372,393 17.9 81 480,623 -7.3 82 173,156 61.4 83 518,993 -15.8 84 392,023 12.5 85 112,260 74.9 51 237,086 47.1 86 398,110 11.2 87 399,700 10.8 88 412,776 7.9 89 449,293 -0.3 90 423,326 5.5 91 160,883 69.1 92 372,400 16.9 93 409,220 9.7 94 311,736 30.4 95 538,110 -20.1 96 544,596 -21.5 97 218,673 51.2 98 467,636 -4.4 99 111,043 75.2 100 518,190 -15.6 101 502,096 -12.0 102 377,216 15.8 103 305,690 31.8 104 419,876 6.3 105 552,170 -23.2 106 193,533 60.4 107 402,976 11.1 108 40,853 90.9 109 96,893 79.4 110 602,506 -34.5 111 632,613 -39.2 112 527,950 -17.8 113 570,553 -27.3 114 270,190 39.7 115 475,713 -6.2 116 394,096 12.1 117 359,236 19.8 119 69,220 84.6 120 463,243 -3.4 121 338,200 24.5 †The numbers represent the number of luciferase units (lumens) produced after infection by either the HCV or the MLV pseudotype in the presence of the peptide at a concentration of ~25 μM. Samples were compared to controls described in Table 7. Results above 70% inhibition are indicated in bold-face type.
TABLE-US-00009 TABLE 9 Sequence and location of peptides shown in Table 7. HCV 1a H77 Peptide HCV E2 peptide loca- IP # tion* Amino acid Sequence overlap 1 379-396 AGVDAETHVTGGSAGRTT (SEQ ID NO 43) 2 386-403 HVTGGSAGRTTAGLVGLL (SEQ ID NO 44) 3 393-410 GRTTAGLVGLLTPGAKQN (SEQ ID NO 45) HCV E2IP 1 4 399-417 VGLLTPGAKQNIQLINTN (SEQ ID NO 46) HCV E2IP 1 5 407-424 AKQNIQLINTNGSWHINS (SEQ ID NO 47) HCV E2IP 1 6 414-431 INTNGSWHINSTALNCNE (SEQ ID NO 48) HCV E2IP 1 7 421-438 HINSTALNCNESLNTGWL (SEQ ID NO 49) HCV E2IP 1/2 8 428-445 NCNESLNTGWLAGLFYQH (SEQ ID NO 50) HCV E2IP 2 9 442-459 FYQHKFNSSGCPERLASC (SEQ ID NO 51) HCV E2IP 2 10 449-466 SSGCPERLASCRRLTDFA (SEQ ID NO 52) 11 456-473 LASCRRLTDFAQGWGPIS (SEQ ID NO 53) 12 463-480 TDFAQGWGPISYANGSGL (SEQ ID NO 54) 13 470-487 GPISYANGSGLDERPYCW (SEQ ID NO 55) 14 477-494 GSGLDERPYCWHYPPRPC (SEQ ID NO 56) 15 486-501 PYCWHYPPRPCGIVPAKS (SEQ ID NO 57) 16 491-508 PRPCGIVPAKSVCGPVYC (SEQ ID NO 58) 17 501-515 PAKSVCGPVYCFTPSPVV (SEQ ID NO 59) 18 505-522 PVYCFTPSPVVVGTTDRS (SEQ ID NO 60) 19 512-529 SPVVVGTTDRSGAPTYSW (SEQ ID NO 61) HCV E2IP 3 20 526-543 TYSWGANDTDVFVLNNTR (SEQ ID NO 62) HCV E2IP 3 21 533-550 DTDVFVLNNTRPPLGNWF (SEQ ID NO 63) HCV E2IP 3 22 540-557 NNTRPPLGNWFGCTWMNS (SEQ ID NO 64) HCV E2IP 3 23 547-564 GNWFGCTWMNSTGFTKVC (SEQ ID NO 65) HCV E2IP 3 24 554-571 WMNSTGFTKVCGAPPCVI (SEQ ID NO 66) HCV E2IP 3 25 561-578 TKVCGAPPCVIGGVGNNT (SEQ ID NO 67) 26 568-585 PCVIGGVGNNTLLCPTDC (SEQ ID NO 68) 27 575-592 GNNTLLCPTDCFRKHPEA (SEQ ID NO 69) 28 582-599 PTDCFRKHPEATYSRCGS (SEQ ID NO 70) 29 589-606 HPEATYSRCGSGPWITPR (SEQ ID NO 71) 30 596-613 RCGSGPWITPRCMVDYPY (SEQ ID NO 72) HCV E2IP 4 31 603-620 ITPRCMVDYPYRLWHYPC (SEQ ID NO 73) HCV E2IP 4 32 610-627 DYPYRLWHYPCTINYTIF (SEQ ID NO 74) HCV E2IP 4 33 617-634 HYPCTINYTIFKVRMYVG (SEQ ID NO 75) HCV E2IP 4 34 624-641 YTIFKVRMYVGGVEHRLE (SEQ ID NO 76) HCV E2IP 4 35 631-648 MYVGGVEHRLEAACNWTR (SEQ ID NO 77) HCV E2IP 4 36 638-655 HRLEAACNWTRGERCDLE (SEQ ID NO 78) 37 645-662 NWTRGERCDLEDRDRSEL (SEQ ID NO 79) 38 652-669 CDLEDRDRSELSPLLLST (SEQ ID NO 80) 39 659-676 RSELSPLLLSTTQWQVLP (SEQ ID NO 81) 40 666-683 LLSTTQWQVLPCSFTTLP (SEQ ID NO 82) 41 673-690 QVLPCSFTTLPALSTGLI (SEQ ID NO 83) HCV E2IP 5 42 680-697 TTLPALSTGLIHLHQNIV (SEQ ID NO 84) HCV E2IP 5 43 687-704 TGLIHLHQNIVDVQYLYG (SEQ ID NO 85) HCV E2IP 5 44 694-711 QNIVDVQYLYGVGSSIAS (SEQ ID NO 86) HCV E2IP 5 45 701-718 YLYGVGSSIASWAIKWEY (SEQ ID NO 87) HCV:E2IP 5 46 708-725 SIASWAIKWEYVVLLFLL (SEQ ID NO 88) HCV E2IP 5/6 47 715-732 KWEYVVLLFLLLADARVC (SEQ ID NO 89) HCV E2IP 5/6 48 722-739 LFLLLADARVCSCLWMML (SEQ ID NO 90) HCV E2IP 6 49 729-746 ARVCSCLWMMLLISQAEA (SEQ ID NO 91) HCV E21P 6 50 756-773 WMMLLISQAEAALENLVI (SEQ ID NO 92) HCV E2IP 6 *Numbering refers to the numbering provided at Genbank Accession NP_671491
TABLE-US-00010 TABLE 10 Sequence and location of peptides shown in Table 8. HCV 1b J4 Peptide HCV E2 peptide loca- IP # tion* Amino acid sequence overlap 54 (379-396) AGVDGETHTTGRVAGHTT (SEQ ID NO 93) 80 (386-403) HTTGRVAGHTTSGFTSLF (SEQ ID NO 94) HCV E2IP 1 81 (393-410) GHTTSGFTSLFSSGASQK (SEQ ID NO 95) HCV E2IP 1 82 (400-417) TSLFSSGASQKIQLVNTN (SEQ ID NO 96) HCV E2IP 1 83 (407-424) ASQKIQLVNTNGSWHINR (SEQ ID NO 97) HCV E2IP 1 84 (421-438) HINRTALNCNDSLQTGFF (SEQ ID NO 98) HCV E2IP 1/2 85 (428-445) NCNDSLQTGFFAALFYAH (SEQ ID NO 99) HCV E2IP 2 51 (435-452) TGFFAALFYAHKFNSSGC (SEQ ID NO 100) HCV E2IP 2 86 (442-459) FYAHKFNSSGCPERMASC (SEQ ID NO 101) HCV E2IP 2 87 (449-466) SSGCPERMASCRPIDWFA (SEQ ID NO 102) 88 (456-473) MASCRPIDWFAQGWGPIT (SEQ ID NO 103) 89 (463-480) DWFAQGWGPITYTKPNSS (SEQ ID NO 104) 90 (477-494) PNSSDQRPYCWHYAPRPC (SEQ ID NO 105) 91 (484-501) PYCWHYAPRPCGVVPASQ (SEQ ID NO 106) 92 (491-508) PRPCGVVPASQVCGPVYC (SEQ ID NO 107) 93 (498-515) PASQVCGPVYCFTPSPVV (SEQ ID NO 108) 94 (505-522) PVYCFTPSPVVVGTTDRS (SEQ ID NO 109) 95 (512-529) SPVVVGTTDRSGVPTYSW (SEQ ID NO 110) HCV E2IP 3 96 (519-536) TDRSGVPTYSWGENETDV (SEQ ID NO 111) HCV E2IP 3 97 (526-543) TYSWGENETDVMLLNNTR (SEQ ID NO 112) HCV E2IP 3 98 (533-550) ETDVMLLNNTRPPQGNWF (SEQ ID NO 113) HCV E2IP 3 99 (540-557) NNTRPPQGNWFGCTWMNS (SEQ ID NO 114) HCV E2IP 3 100 (554-571) WMNSTGFTKTCGGPPCNI (SEQ ID NO 115) HCV E2IP 3 101 (561-578) TKTCGGPPCNIGGVGNRT (SEQ ID NO 116) 102 (568-585) PCNIGGVGNRTLICPTDC (SEQ ID NO 117) 103 (575-592) GNRTLICPTDCFRKHPEA (SEQ ID NO 118) 104 (582-599) PTDCFRKHPEATYTKCGS (SEQ ID NO 119) 105 (589-606) HPEATYTKCGSGPWLTPR (SEQ ID NO 120) 106 (596-613) KCGSGPWLTPRCLVDYPY (SEQ ID NO 121) HCV E2IP 4 107 (603-620) LTPRCLVDYPYRLWHYPC (SEQ ID NO 122) HCV E2IP 4 108 (610-627) DYPYRLWHYPCTLNFSIF (SEQ ID NO 123) HCV E2IP 4 109 (617-634) HYPCTLNFSIFKVRMYVG (SEQ ID NO 124) HCV E2IP 4 110 (631-648) MYVGGVEHRLNAACNWTR (SEQ ID NO 125) HCV E2IP 4 111 (638-655) HRLNAACNWTRGERCNLE (SEQ ID NO 126) 112 (645-662) NWTRGERCNLEDRDRSEL (SEQ ID NO 127) 113 (652-669) CNLEDRDRSELSPLLLST (SEQ ID NO 128) 114 (659-676) RSELSPLLLSTTEWQILP (SEQ ID NO 129) 115 (666-683) LLSTTEWQILPCAFTTLP (SEQ ID NO 130) 116 (673-690) QILPCAFTTLPALSTGLI (SEQ ID NO 131) HCV E2IP 5 117 (680-697) TTLPALSTGLIHLHQNIV (SEQ ID NO 132) HCV E2IP 5 118 (694-711) QNIVDVQYLYGVGSAFVS (SEQ ID NO 133) HCV E2IP 5 119 (708-725) AFVSFAIKWEYILLLFLL (SEQ ID NO 134) HCV E2IP 5/6 120 (722-739) LFLLLADARVCACLWMML (SEQ ID NO 135) HCV E2IP 6 121 (729-746) ARVCACLWMMLLIAQAEA (SEQ ID NO 136) HCV E2IP 6 *Numbering refers to the numbering provided at Genbank Accession BAA01583
[0065]The peptides of Tables 7-10 are an overlapping set of peptides representing E2 of two strains of HCV (H77 and J4). SEQ ID NO's 1-6 are longer versions of the "hits" highlighted in yellow in Tables 7-10. The rationale for the design of SEQ ID NOs:1-6 is that the optimum inhibitory peptide may be include flanking sequences), and that the ultimate optimum peptide may be a fragment of this longer peptide. SEQ ID NOs:7-42 are variants of SEQ ID NOs: 1-6 representing analogous sequences from the E2 proteins of the other major genotypes of HCV.
Example 2
Proteomic Computational Model of HCV E2
[0066]Because HCV cannot be propagated in cell culture, insufficient numbers of virions are available to perform structural analyses. Thus, the molecular structure of HCV E2 has not been determined and is currently unknown. In the absence of an X-ray crystallographic structure of HCV E2, it is possible to derive useful structural information using newly developed computational analyses supplemented by comparisons to other viral glycoproteins of known structure. Such a model of HCV E2 can be useful in defining the potential mechanisms of action of E2 inhibitory peptides.
Materials and Methods
[0067]Representatives of the most common subtypes of hepatitis C virus were used for sequence and structural comparisons. The strains examined a human prototype HCV strain H77 (subtype 1a, Genbank Accession NP--751921), strain HC-J4 (subtype 1b, Genbank Accession BAA01583), strain NDM59 (subtype 2a, Genbank Accession AF169005), strain TrKj (subtypes 3b, Genbank Accession D49374), strain ED43 (subtype 4a, Genbank Accession Y11604), strain EUH1480 (subtype 5a, Genbank Accession Y13184), strain euhk2 (subtype 6a, Genbank Accession Y12083).
[0068]Methods to derive general models of surface glycoproteins have been described previously (Gallaher et al., 1989). PRSS3, a program derived from rdf2 (Pearson and Lipman, 1988), which uses the Smith-Waterman sequence alignment algorithm (Smith and Waterman, 1981), was used to determine the significance of protein alignments. PRSS3 is part of the FASTA package of sequence analysis programs available by anonymous ftp from ftp.virginia.edu. Default settings for PRSS3 were used, including the blosum50 scoring matrix, gap opening penalty of 12, and gap extension penalty of 2. MacMolly (Soft Gene GmbH, Berlin) was used to locate areas of limited sequence similarity and to perform Chou-Fasman and Robson-Garnier analyses (Biou et al., 1988; Chou and Fasman, 1974). PHDsec (Columbia University Bioinformatics Center, http://cubic.bioc.columbia.edu/predictprotein/) was the preferred method of secondary structure prediction (Rost and Liu, 2003). PHDsec predicts secondary structure from multiple sequence alignments by a system of neural networks, and is rated at an expected average accuracy of 72% for three states, helix, strand and loop. Domains with significant propensity to form transmembrane helices were identified with TMpred (ExPASy, Swiss Institute of Bioinformatics, http://www.ch.embnet.org/software/TMPRED_form.html). TMpred is based on a statistical analysis of TMbase, a database of naturally occurring transmembrane glycoproteins (Hofmann and Stoffel, 1993). Sequences with a propensity to partition into the lipid bilayer were identified with Membrane Protein eXplorer version 2.2a from the Stephen White laboratory using default settings (White et al., 2003).
Results and Discussion
[0069]A two-dimensional model of HCV E2 was developed based on the application of proteomics computational analyses and comparison to the known structures of other receptor-binding viral envelope proteins (FIG. 3). The HCV E2 secondary structure structures drawn in FIG. 3 conform to the PHDsec secondary structure alignment algorithm and are also generally consistent with both Chou-Fasman and Robson-Garnier predictions. An important feature of the E2 model is the predominance of beta sheet structures in the amino terminal two-thirds of the molecule and alpha helical structures in the carboxyl terminal third of the molecule. Dicysteine linkages were predicted on the basis of comparison to the envelope glycoproteins of retroviruses. In retroviral envelope proteins adjacent cysteines that are separated by greater than 15 amino acids are typically covalently bonded to each other. Clusters of four cysteines that are each separated by fewer than 15 amino acids are typically covalently bonded to a nonadjacient cysteine in the cluster. The first two long predicted alpha helices in the carboxyl terminal third of E2 form a predicted stem structure analogous to the stem region of other flavivirus envelope proteins (Allison et al., 1999). The depicted transmembrane domain structure was predicted by the TMPRED algorithm.
[0070]HCV E2 inhibitory peptides map to regions on the HCV E2 model that correspond to an amino terminal region, a cysteine cluster, the stem and transmembrane domain (shaded regions in FIG. 3). These domains of HCV E2 can be involved in hepatitis C virus receptor binding, E2 structural rearrangements or protein-protein interactions, or other pre-fusion steps. HCV E2 inhibitory peptides can be used to interfere with these early steps in HCV infection as depicted in FIG. 4.
[0071]The present invention is not to be to be construed as limited in scope by the specific embodiments described herein. Rather, the specific embodiments described are only illustrative. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the claims. Various publications are cited herein, the disclosures of each of which is incorporated by reference in its entirety. Citation or identification of any reference in any section of this application shall not be construed as an admission that such reference is available as prior art to the present invention
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Sequence CWU
1
141126PRTArtificial sequenceSynthetic peptide 1Leu Val Gly Leu Leu Thr Pro
Gly Ala Lys Gln Asn Ile Gln Leu Ile1 5 10
15Asn Thr Asn Gly Ser Trp His Ile Asn Ser 20
25217PRTArtificial sequenceSynthetic peptide 2Cys Asn Glu
Ser Leu Asn Thr Gly Trp Leu Ala Gly Leu Phe Tyr Gln1 5
10 15His334PRTArtificial sequenceSynthetic
peptide 3Tyr Ser Trp Gly Ala Asn Asp Thr Asp Val Phe Val Leu Asn Asn Thr1
5 10 15Arg Pro Pro Leu
Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr 20
25 30Gly Phe427PRTArtificial sequenceSynthetic
peptide 4Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Ile Asn Tyr Thr1
5 10 15Ile Phe Lys Val
Arg Met Tyr Val Gly Gly Val 20
25535PRTArtificial sequenceSynthetic peptide 5Ala Leu Ser Thr Gly Leu Ile
His Leu His Gln Asn Ile Val Asp Val1 5 10
15Gln Tyr Leu Tyr Gly Val Gly Ser Ser Ile Ala Ser Trp
Ala Ile Lys 20 25 30Trp Glu
Tyr 35628PRTArtificial sequenceSynthetic peptide 6Val Val Leu Leu
Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ser Cys1 5
10 15Leu Trp Met Met Leu Leu Ile Ser Gln Ala
Glu Ala 20 25726PRTArtificial
sequenceSynthetic peptide 7Phe Thr Ser Leu Phe Ser Ser Gly Ala Ser Gln
Lys Ile Gln Leu Val1 5 10
15Asn Thr Asn Gly Ser Trp His Ile Asn Arg 20
25826PRTArtificial sequenceSynthetic peptide 8Leu Ala Gly Leu Phe Thr Ser
Gly Ala Lys Gln Asn Ile Gln Leu Ile1 5 10
15Asn Thr Asn Gly Ser Trp His Ile Asn Arg 20
25926PRTArtificial sequenceSynthetic peptide 9Phe Thr Ser
Phe Phe Thr Arg Gly Pro Ser Gln Asp Leu Gln Leu Val1 5
10 15Asn Ser Asn Gly Ser Trp His Ile Asn
Ser 20 251026PRTArtificial sequenceSynthetic
peptide 10Leu Ala Asn Leu Phe Ser Ser Gly Ser Lys Gln Asn Leu Gln Leu
Ile1 5 10 15Asn Ser Asn
Gly Ser Trp His Ile Asn Arg 20
251126PRTArtificial sequenceSynthetic peptide 11Leu Thr Ser Phe Phe Asn
Pro Gly Pro Gln Arg Gln Leu Gln Phe Val1 5
10 15Asn Thr Asn Gly Ser Trp His Ile Asn Ser
20 251226PRTArtificial sequenceSynthetic peptide 12Phe
Ala Ser Leu Leu Thr Pro Gly Ala Lys Gln Asn Ile Gln Leu Ile1
5 10 15Asn Thr Asn Gly Ser Trp His
Ile Asn Arg 20 251317PRTArtificial
sequenceSynthetic peptide 13Cys Asn Asp Ser Leu His Thr Gly Phe Leu Ala
Ala Leu Phe Tyr Thr1 5 10
15His1417PRTArtificial sequenceSynthetic peptide 14Cys Asn Asp Ser Leu
Asn Thr Gly Phe Ile Ala Ser Leu Phe Tyr Thr1 5
10 15Tyr1517PRTArtificial sequenceSynthetic peptide
15Cys Asn Asp Ser Leu Asn Thr Gly Phe Ile Ala Gly Leu Phe Tyr Tyr1
5 10 15His1617PRTArtificial
sequenceSynthetic peptide 16Cys Asn Asp Ser Leu Asn Thr Gly Phe Leu Ala
Ser Leu Phe Tyr Thr1 5 10
15His1717PRTArtificial sequenceSynthetic peptide 17Cys Asn Asp Ser Leu
Gln Thr Gly Phe Ile Ala Gly Leu Met Tyr Ala1 5
10 15His1817PRTArtificial sequenceSynthetic peptide
18Cys Asn Asp Ser Leu Gln Thr Gly Phe Leu Ala Ser Leu Phe Tyr Thr1
5 10 15His1934PRTArtificial
sequenceSynthetic peptide 19Tyr Ser Trp Gly Glu Asn Glu Thr Asp Val Met
Leu Leu Asn Asn Thr1 5 10
15Arg Pro Pro Gln Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr
20 25 30Gly Phe2034PRTArtificial
sequenceSynthetic peptide 20Tyr Thr Trp Gly Glu Asn Glu Thr Asp Val Phe
Ile Leu Asn Ser Thr1 5 10
15Arg Pro Pro Gly Gly Ser Trp Phe Gly Cys Thr Trp Met Asn Ser Thr
20 25 30Gly Phe2134PRTArtificial
sequenceSynthetic peptide 21Tyr Arg Phe Gly Val Asn Glu Ser Asp Val Phe
Leu Leu Thr Ser Leu1 5 10
15Arg Pro Pro Gln Gly Arg Trp Phe Gly Cys Val Trp Met Asn Ser Thr
20 25 30Gly Phe2234PRTArtificial
sequenceSynthetic peptide 22Tyr Thr Trp Gly Glu Asn Glu Thr Asp Val Phe
Leu Leu Asn Ser Thr1 5 10
15Arg Pro Pro His Gly Ala Trp Phe Gly Cys Val Trp Met Asn Ser Thr
20 25 30Gly Phe2334PRTArtificial
sequenceSynthetic peptide 23Tyr Asn Trp Gly Ser Asn Glu Thr Asp Ile Leu
Leu Leu Asn Asn Ile1 5 10
15Arg Pro Pro Ala Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr
20 25 30Gly Phe2434PRTArtificial
sequenceSynthetic peptide 24Tyr Thr Trp Gly Glu Asn Glu Thr Asp Val Phe
Met Leu Glu Ser Leu1 5 10
15Arg Pro Pro Thr Gly Gly Trp Phe Gly Cys Thr Trp Met Asn Ser Thr
20 25 30Gly Phe2527PRTArtificial
sequenceSynthetic peptide 25Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys
Thr Leu Asn Phe Ser1 5 10
15Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val 20
252627PRTArtificial sequenceSynthetic peptide 26Asp Tyr Pro Tyr Arg
Leu Trp His Tyr Pro Cys Thr Ile Asn Tyr Thr1 5
10 15Ile Phe Lys Ile Arg Met Tyr Val Gly Gly Val
20 252727PRTArtificial sequenceSynthetic peptide
27Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn Phe Ser1
5 10 15Ile Phe Lys Val Arg Met
Phe Val Gly Gly His 20 252827PRTArtificial
sequenceSynthetic peptide 28Asp Tyr Pro Tyr Arg Leu Trp His Phe Pro Cys
Thr Ala Asn Phe Ser1 5 10
15Val Phe Asn Ile Arg Thr Phe Val Gly Gly Ile 20
252927PRTArtificial sequenceSynthetic peptide 29His Tyr Pro Tyr Arg
Leu Trp His Tyr Pro Cys Thr Val Asn Tyr Thr1 5
10 15Ile Phe Lys Val Arg Met Phe Ile Gly Gly Leu
20 253027PRTArtificial sequenceSynthetic peptide
30Asp Tyr Ala Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn Phe Thr1
5 10 15Leu His Lys Val Arg Met
Phe Val Gly Gly Thr 20 253135PRTArtificial
sequenceSynthetic peptide 31Ala Leu Ser Thr Gly Leu Ile His Leu His Gln
Asn Ile Val Asp Val1 5 10
15Gln Tyr Leu Tyr Gly Val Gly Ser Ala Phe Val Ser Phe Ala Ile Lys
20 25 30Trp Glu Tyr
353235PRTArtificial sequenceSynthetic peptide 32Ala Leu Ser Thr Gly Leu
Leu His Leu His Gln Asn Ile Val Asp Val1 5
10 15Gln Tyr Met Tyr Gly Leu Ser Pro Ala Leu Thr Lys
Tyr Ile Val Arg 20 25 30Trp
Glu Trp 353335PRTArtificial sequenceSynthetic peptide 33Arg Leu
Ser Thr Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val1 5
10 15Gln Tyr Leu Tyr Gly Val Gly Ser
Ala Val Val Gly Trp Ala Leu Lys 20 25
30Trp Glu Phe 353435PRTArtificial sequenceSynthetic
peptide 34Ala Leu Ser Thr Gly Leu Ile His Leu His Gln Asn Ile Val Asp
Val1 5 10 15Gln Tyr Leu
Tyr Gly Val Gly Ser Ala Val Val Ser Trp Ala Leu Lys 20
25 30Trp Glu Tyr 353535PRTArtificial
sequenceSynthetic peptide 35Ala Leu Ser Thr Gly Leu Ile His Leu His Gln
Asn Ile Val Asp Thr1 5 10
15Gln Tyr Leu Tyr Gly Leu Ser Ser Ser Ile Val Ser Trp Ala Val Lys
20 25 30Trp Glu Tyr
353635PRTArtificial sequenceSynthetic peptide 36Ala Leu Ser Thr Gly Leu
Ile His Leu His Gln Asn Ile Val Asp Val1 5
10 15Gln Tyr Leu Tyr Gly Val Ser Thr Asn Val Thr Ser
Trp Val Val Lys 20 25 30Trp
Glu Tyr 353728PRTArtificial sequenceSynthetic peptide 37Ile Leu
Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ala Cys1 5
10 15Leu Trp Met Met Leu Leu Ile Ala
Gln Ala Glu Ala 20 253828PRTArtificial
sequenceSynthetic peptide 38Val Val Leu Leu Phe Leu Leu Leu Ala Asp Ala
Arg Val Cys Ala Cys1 5 10
15Leu Trp Met Leu Ile Leu Leu Gly Gln Ala Glu Ala 20
253928PRTArtificial sequenceSynthetic peptide 39Val Val Leu Val
Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Val Ala1 5
10 15Leu Trp Met Met Leu Leu Ile Ser Gln Ala
Glu Ala 20 254028PRTArtificial
sequenceSynthetic peptide 40Val Val Leu Ala Phe Leu Leu Leu Ala Asp Ala
Arg Val Ser Ala Tyr1 5 10
15Leu Trp Met Met Phe Met Val Ser Gln Val Glu Ala 20
254128PRTArtificial sequenceSynthetic peptide 41Ile Met Leu Val
Phe Leu Leu Leu Ala Asp Ala Arg Ile Cys Thr Cys1 5
10 15Leu Leu Ile Leu Leu Leu Ile Cys Gln Ala
Glu Ala 20 254228PRTArtificial
sequenceSynthetic peptide 42Ile Val Leu Met Phe Leu Val Leu Ala Asp Ala
Arg Ile Cys Thr Cys1 5 10
15Leu Trp Leu Met Leu Leu Ile Ser Thr Val Glu Ala 20
254318PRTArtificial sequenceSynthetic peptide 43Ala Gly Val Asp
Ala Glu Thr His Val Thr Gly Gly Ser Ala Gly Arg1 5
10 15Thr Thr4418PRTArtificial sequenceSynthetic
peptide 44His Val Thr Gly Gly Ser Ala Gly Arg Thr Thr Ala Gly Leu Val
Gly1 5 10 15Leu
Leu4518PRTArtificial sequenceSynthetic peptide 45Gly Arg Thr Thr Ala Gly
Leu Val Gly Leu Leu Thr Pro Gly Ala Lys1 5
10 15Gln Asn4618PRTArtificial sequenceSynthetic peptide
46Val Gly Leu Leu Thr Pro Gly Ala Lys Gln Asn Ile Gln Leu Ile Asn1
5 10 15Thr Asn4718PRTArtificial
sequenceSynthetic peptide 47Ala Lys Gln Asn Ile Gln Leu Ile Asn Thr Asn
Gly Ser Trp His Ile1 5 10
15Asn Ser4818PRTArtificial sequenceSynthetic peptide 48Ile Asn Thr Asn
Gly Ser Trp His Ile Asn Ser Thr Ala Leu Asn Cys1 5
10 15Asn Glu4918PRTArtificial sequenceSynthetic
peptide 49His Ile Asn Ser Thr Ala Leu Asn Cys Asn Glu Ser Leu Asn Thr
Gly1 5 10 15Trp
Leu5018PRTArtificial sequenceSynthetic peptide 50Asn Cys Asn Glu Ser Leu
Asn Thr Gly Trp Leu Ala Gly Leu Phe Tyr1 5
10 15Gln His5118PRTArtificial sequenceSynthetic peptide
51Phe Tyr Gln His Lys Phe Asn Ser Ser Gly Cys Pro Glu Arg Leu Ala1
5 10 15Ser Cys5218PRTArtificial
sequenceSynthetic peptide 52Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys
Arg Arg Leu Thr Asp1 5 10
15Phe Ala5318PRTArtificial sequenceSynthetic peptide 53Leu Ala Ser Cys
Arg Arg Leu Thr Asp Phe Ala Gln Gly Trp Gly Pro1 5
10 15Ile Ser5418PRTArtificial sequenceSynthetic
peptide 54Thr Asp Phe Ala Gln Gly Trp Gly Pro Ile Ser Tyr Ala Asn Gly
Ser1 5 10 15Gly
Leu5518PRTArtificial sequenceSynthetic peptide 55Gly Pro Ile Ser Tyr Ala
Asn Gly Ser Gly Leu Asp Glu Arg Pro Tyr1 5
10 15Cys Trp5618PRTArtificial sequenceSynthetic peptide
56Gly Ser Gly Leu Asp Glu Arg Pro Tyr Cys Trp His Tyr Pro Pro Arg1
5 10 15Pro Cys5718PRTArtificial
sequenceSynthetic peptide 57Pro Tyr Cys Trp His Tyr Pro Pro Arg Pro Cys
Gly Ile Val Pro Ala1 5 10
15Lys Ser5818PRTArtificial sequenceSynthetic peptide 58Pro Arg Pro Cys
Gly Ile Val Pro Ala Lys Ser Val Cys Gly Pro Val1 5
10 15Tyr Cys5918PRTArtificial sequenceSynthetic
peptide 59Pro Ala Lys Ser Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser
Pro1 5 10 15Val
Val6018PRTArtificial sequenceSynthetic peptide 60Pro Val Tyr Cys Phe Thr
Pro Ser Pro Val Val Val Gly Thr Thr Asp1 5
10 15Arg Ser6118PRTArtificial sequenceSynthetic peptide
61Ser Pro Val Val Val Gly Thr Thr Asp Arg Ser Gly Ala Pro Thr Tyr1
5 10 15Ser Trp6218PRTArtificial
sequenceSynthetic peptide 62Thr Tyr Ser Trp Gly Ala Asn Asp Thr Asp Val
Phe Val Leu Asn Asn1 5 10
15Thr Arg6318PRTArtificial sequenceSynthetic peptide 63Asp Thr Asp Val
Phe Val Leu Asn Asn Thr Arg Pro Pro Leu Gly Asn1 5
10 15Trp Phe6418PRTArtificial sequenceSynthetic
peptide 64Asn Asn Thr Arg Pro Pro Leu Gly Asn Trp Phe Gly Cys Thr Trp
Met1 5 10 15Asn
Ser6518PRTArtificial sequenceSynthetic peptide 65Gly Asn Trp Phe Gly Cys
Thr Trp Met Asn Ser Thr Gly Phe Thr Lys1 5
10 15Val Cys6618PRTArtificial sequenceSynthetic peptide
66Trp Met Asn Ser Thr Gly Phe Thr Lys Val Cys Gly Ala Pro Pro Cys1
5 10 15Val Ile6718PRTArtificial
sequenceSynthetic peptide 67Thr Lys Val Cys Gly Ala Pro Pro Cys Val Ile
Gly Gly Val Gly Asn1 5 10
15Asn Thr6818PRTArtificial sequenceSynthetic peptide 68Pro Cys Val Ile
Gly Gly Val Gly Asn Asn Thr Leu Leu Cys Pro Thr1 5
10 15Asp Cys6918PRTArtificial sequenceSynthetic
peptide 69Gly Asn Asn Thr Leu Leu Cys Pro Thr Asp Cys Phe Arg Lys His
Pro1 5 10 15Glu
Ala7018PRTArtificial sequenceSynthetic peptide 70Pro Thr Asp Cys Phe Arg
Lys His Pro Glu Ala Thr Tyr Ser Arg Cys1 5
10 15Gly Ser7118PRTArtificial sequenceSynthetic peptide
71His Pro Glu Ala Thr Tyr Ser Arg Cys Gly Ser Gly Pro Trp Ile Thr1
5 10 15Pro Arg7218PRTArtificial
sequenceSynthetic peptide 72Arg Cys Gly Ser Gly Pro Trp Ile Thr Pro Arg
Cys Met Val Asp Tyr1 5 10
15Pro Tyr7318PRTArtificial sequenceSynthetic peptide 73Ile Thr Pro Arg
Cys Met Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr1 5
10 15Pro Cys7418PRTArtificial sequenceSynthetic
peptide 74Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Ile Asn Tyr
Thr1 5 10 15Ile
Phe7518PRTArtificial sequenceSynthetic peptide 75His Tyr Pro Cys Thr Ile
Asn Tyr Thr Ile Phe Lys Val Arg Met Tyr1 5
10 15Val Gly7618PRTArtificial sequenceSynthetic peptide
76Tyr Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg1
5 10 15Leu Glu7718PRTArtificial
sequenceSynthetic peptide 77Met Tyr Val Gly Gly Val Glu His Arg Leu Glu
Ala Ala Cys Asn Trp1 5 10
15Thr Arg7818PRTArtificial sequenceSynthetic peptide 78His Arg Leu Glu
Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp1 5
10 15Leu Glu7918PRTArtificial sequenceSynthetic
peptide 79Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp Arg Asp Arg
Ser1 5 10 15Glu
Leu8018PRTArtificial sequenceSynthetic peptide 80Cys Asp Leu Glu Asp Arg
Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu1 5
10 15Ser Thr8118PRTArtificial sequenceSynthetic peptide
81Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Gln Trp Gln Val1
5 10 15Leu Pro8218PRTArtificial
sequenceSynthetic peptide 82Leu Leu Ser Thr Thr Gln Trp Gln Val Leu Pro
Cys Ser Phe Thr Thr1 5 10
15Leu Pro8318PRTArtificial sequenceSynthetic peptide 83Gln Val Leu Pro
Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr Gly1 5
10 15Leu Ile8418PRTArtificial sequenceSynthetic
peptide 84Thr Thr Leu Pro Ala Leu Ser Thr Gly Leu Ile His Leu His Gln
Asn1 5 10 15Ile
Val8518PRTArtificial sequenceSynthetic peptide 85Thr Gly Leu Ile His Leu
His Gln Asn Ile Val Asp Val Gln Tyr Leu1 5
10 15Tyr Gly8618PRTArtificial sequenceSynthetic peptide
86Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr Gly Val Gly Ser Ser Ile1
5 10 15Ala Ser8718PRTArtificial
sequenceSynthetic peptide 87Tyr Leu Tyr Gly Val Gly Ser Ser Ile Ala Ser
Trp Ala Ile Lys Trp1 5 10
15Glu Tyr8818PRTArtificial sequenceSynthetic peptide 88Ser Ile Ala Ser
Trp Ala Ile Lys Trp Glu Tyr Val Val Leu Leu Phe1 5
10 15Leu Leu8918PRTArtificial sequenceSynthetic
peptide 89Lys Trp Glu Tyr Val Val Leu Leu Phe Leu Leu Leu Ala Asp Ala
Arg1 5 10 15Val
Cys9018PRTArtificial sequenceSynthetic peptide 90Leu Phe Leu Leu Leu Ala
Asp Ala Arg Val Cys Ser Cys Leu Trp Met1 5
10 15Met Leu9118PRTArtificial sequenceSynthetic peptide
91Ala Arg Val Cys Ser Cys Leu Trp Met Met Leu Leu Ile Ser Gln Ala1
5 10 15Glu Ala9218PRTArtificial
sequenceSynthetic peptide 92Trp Met Met Leu Leu Ile Ser Gln Ala Glu Ala
Ala Leu Glu Gln Leu1 5 10
15Val Ile9318PRTArtificial sequenceSynthetic peptide 93Ala Gly Val Asp
Gly Glu Thr His Thr Thr Gly Arg Val Ala Gly His1 5
10 15Thr Thr9418PRTArtificial sequenceSynthetic
peptide 94His Thr Thr Gly Arg Val Ala Gly His Thr Thr Ser Gly Phe Thr
Ser1 5 10 15Leu
Phe9518PRTArtificial sequenceSynthetic peptide 95Gly His Thr Thr Ser Gly
Phe Thr Ser Leu Phe Ser Ser Gly Ala Ser1 5
10 15Gln Lys9618PRTArtificial sequenceSynthetic peptide
96Thr Ser Leu Phe Ser Ser Gly Ala Ser Gln Lys Ile Gln Leu Val Asn1
5 10 15Thr Asn9718PRTArtificial
sequenceSynthetic peptide 97Ala Ser Gln Lys Ile Gln Leu Val Asn Thr Asn
Gly Ser Trp His Ile1 5 10
15Asn Arg9818PRTArtificial sequenceSynthetic peptide 98His Ile Asn Arg
Thr Ala Leu Asn Cys Asn Asp Ser Leu Gln Thr Gly1 5
10 15Phe Phe9918PRTArtificial sequenceSynthetic
peptide 99Asn Cys Asn Asp Ser Leu Gln Thr Gly Phe Phe Ala Ala Leu Phe
Tyr1 5 10 15Ala
His10018PRTArtificial sequenceSynthetic peptide 100Thr Gly Phe Phe Ala
Ala Leu Phe Tyr Ala His Lys Phe Asn Ser Ser1 5
10 15Gly Cys10118PRTArtificial sequenceSynthetic
peptide 101Phe Tyr Ala His Lys Phe Asn Ser Ser Gly Cys Pro Glu Arg Met
Ala1 5 10 15Ser
Cys10218PRTArtificial sequenceSynthetic peptide 102Ser Ser Gly Cys Pro
Glu Arg Met Ala Ser Cys Arg Pro Ile Asp Trp1 5
10 15Phe Ala10318PRTArtificial sequenceSynthetic
peptide 103Met Ala Ser Cys Arg Pro Ile Asp Trp Phe Ala Gln Gly Trp Gly
Pro1 5 10 15Ile
Thr10418PRTArtificial sequenceSynthetic peptide 104Asp Trp Phe Ala Gln
Gly Trp Gly Pro Ile Thr Tyr Thr Lys Pro Asn1 5
10 15Ser Ser10518PRTArtificial sequenceSynthetic
peptide 105Pro Asn Ser Ser Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro
Arg1 5 10 15Pro
Cys10618PRTArtificial sequenceSynthetic peptide 106Pro Tyr Cys Trp His
Tyr Ala Pro Arg Pro Cys Gly Val Val Pro Ala1 5
10 15Ser Gln10718PRTArtificial sequenceSynthetic
peptide 107Pro Arg Pro Cys Gly Val Val Pro Ala Ser Gln Val Cys Gly Pro
Val1 5 10 15Tyr
Cys10818PRTArtificial sequenceSynthetic peptide 108Pro Ala Ser Gln Val
Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro1 5
10 15Val Val10918PRTArtificial sequenceSynthetic
peptide 109Pro Val Tyr Cys Phe Thr Pro Ser Pro Val Val Val Gly Thr Thr
Asp1 5 10 15Arg
Ser11018PRTArtificial sequenceSynthetic peptide 110Ser Pro Val Val Val
Gly Thr Thr Asp Arg Ser Gly Val Pro Thr Tyr1 5
10 15Ser Trp11118PRTArtificial sequenceSynthetic
peptide 111Thr Asp Arg Ser Gly Val Pro Thr Tyr Ser Trp Gly Glu Asn Glu
Thr1 5 10 15Asp
Val11218PRTArtificial sequenceSynthetic peptide 112Thr Tyr Ser Trp Gly
Glu Asn Glu Thr Asp Val Met Leu Leu Asn Asn1 5
10 15Thr Arg11318PRTArtificial sequenceSynthetic
peptide 113Glu Thr Asp Val Met Leu Leu Asn Asn Thr Arg Pro Pro Gln Gly
Asn1 5 10 15Trp
Phe11418PRTArtificial sequenceSynthetic peptide 114Asn Asn Thr Arg Pro
Pro Gln Gly Asn Trp Phe Gly Cys Thr Trp Met1 5
10 15Asn Ser11518PRTArtificial sequenceSynthetic
peptide 115Trp Met Asn Ser Thr Gly Phe Thr Lys Thr Cys Gly Gly Pro Pro
Cys1 5 10 15Asn
Ile11618PRTArtificial sequenceSynthetic peptide 116Thr Lys Thr Cys Gly
Gly Pro Pro Cys Asn Ile Gly Gly Val Gly Asn1 5
10 15Arg Thr11718PRTArtificial sequenceSynthetic
peptide 117Pro Cys Asn Ile Gly Gly Val Gly Asn Arg Thr Leu Ile Cys Pro
Thr1 5 10 15Asp
Cys11818PRTArtificial sequenceSynthetic peptide 118Gly Asn Arg Thr Leu
Ile Cys Pro Thr Asp Cys Phe Arg Lys His Pro1 5
10 15Glu Ala11918PRTArtificial sequenceSynthetic
peptide 119Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala Thr Tyr Thr Lys
Cys1 5 10 15Gly
Ser12018PRTArtificial sequenceSynthetic peptide 120His Pro Glu Ala Thr
Tyr Thr Lys Cys Gly Ser Gly Pro Trp Leu Thr1 5
10 15Pro Arg12118PRTArtificial sequenceSynthetic
peptide 121Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys Leu Val Asp
Tyr1 5 10 15Pro
Tyr12218PRTArtificial sequenceSynthetic peptide 122Leu Thr Pro Arg Cys
Leu Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr1 5
10 15Pro Cys12318PRTArtificial sequenceSynthetic
peptide 123Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Leu Asn Phe
Ser1 5 10 15Ile
Phe12418PRTArtificial sequenceSynthetic peptide 124His Tyr Pro Cys Thr
Leu Asn Phe Ser Ile Phe Lys Val Arg Met Tyr1 5
10 15Val Gly12518PRTArtificial sequenceSynthetic
peptide 125Met Tyr Val Gly Gly Val Glu His Arg Leu Asn Ala Ala Cys Asn
Trp1 5 10 15Thr
Arg12618PRTArtificial sequenceSynthetic peptide 126His Arg Leu Asn Ala
Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asn1 5
10 15Leu Glu12718PRTArtificial sequenceSynthetic
peptide 127Asn Trp Thr Arg Gly Glu Arg Cys Asn Leu Glu Asp Arg Asp Arg
Ser1 5 10 15Glu
Leu12818PRTArtificial sequenceSynthetic peptide 128Cys Asn Leu Glu Asp
Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu1 5
10 15Ser Thr12918PRTArtificial sequenceSynthetic
peptide 129Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu Trp Gln
Ile1 5 10 15Leu
Pro13018PRTArtificial sequenceSynthetic peptide 130Leu Leu Ser Thr Thr
Glu Trp Gln Ile Leu Pro Cys Ala Phe Thr Thr1 5
10 15Leu Pro13118PRTArtificial sequenceSynthetic
peptide 131Gln Ile Leu Pro Cys Ala Phe Thr Thr Leu Pro Ala Leu Ser Thr
Gly1 5 10 15Leu
Ile13218PRTArtificial sequenceSynthetic peptide 132Thr Thr Leu Pro Ala
Leu Ser Thr Gly Leu Ile His Leu His Gln Asn1 5
10 15Ile Val13318PRTArtificial sequenceSynthetic
peptide 133Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr Gly Val Gly Ser Ala
Phe1 5 10 15Val
Ser13418PRTArtificial sequenceSynthetic peptide 134Ala Phe Val Ser Phe
Ala Ile Lys Trp Glu Tyr Ile Leu Leu Leu Phe1 5
10 15Leu Leu13518PRTArtificial sequenceSynthetic
peptide 135Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ala Cys Leu Trp
Met1 5 10 15Met
Leu13618PRTArtificial sequenceSynthetic peptide 136Ala Arg Val Cys Ala
Cys Leu Trp Met Met Leu Leu Ile Ala Gln Ala1 5
10 15Glu Ala137360PRTHepatitis C Virus 137Glu Thr
His Val Thr Gly Gly Asn Ala Gly Arg Thr Thr Ala Gly Leu1 5
10 15Val Gly Leu Leu Thr Pro Gly Ala
Lys Gln Asn Ile Gln Leu Ile Asn 20 25
30Thr Asn Gly Ser Trp His Ile Asn Ser Thr Ala Leu Asn Cys Asn
Glu 35 40 45Ser Leu Asn Thr Gly
Trp Leu Ala Gly Leu Phe Tyr Gln His Lys Phe 50 55
60Asn Ser Ser Gly Cys Pro Glu Arg Leu Thr Ser Cys Arg Arg
Leu Thr65 70 75 80Asp
Phe Ala Gln Gly Trp Gly Pro Ile Ser Tyr Ala Asn Gly Ser Gly
85 90 95Leu Asp Glu Arg Pro Tyr Cys
Trp His Tyr Pro Pro Arg Pro Cys Gly 100 105
110Ile Val Pro Ala Lys Ser Val Cys Gly Pro Val Tyr Cys Phe
Thr Pro 115 120 125Ser Pro Val Val
Val Gly Thr Thr Asp Arg Ser Gly Ala Pro Thr Tyr 130
135 140Ser Trp Gly Ala Asn Asp Thr Asp Val Phe Val Leu
Asn Asn Thr Arg145 150 155
160Pro Pro Leu Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly
165 170 175Phe Thr Lys Val Cys
Gly Ala Pro Pro Cys Val Ile Gly Gly Val Gly 180
185 190Asn Asn Thr Leu Leu Cys Pro Thr Asp Cys Phe Arg
Lys His Pro Glu 195 200 205Ala Thr
Tyr Ser Arg Cys Gly Ser Gly Pro Trp Ile Thr Pro Arg Cys 210
215 220Met Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr
Pro Cys Thr Ile Asn225 230 235
240Tyr Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg
245 250 255Leu Glu Ala Ala
Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu 260
265 270Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu
Leu Ser Thr Thr Gln 275 280 285Trp
Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr 290
295 300Gly Leu Ile His Leu His Gln Asn Ile Val
Asp Val Gln Tyr Leu Tyr305 310 315
320Gly Val Gly Ser Ser Ile Ala Ser Trp Ala Ile Lys Trp Glu Tyr
Val 325 330 335Val Leu Leu
Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ser Cys Leu 340
345 350Trp Met Met Leu Leu Ile Ser Gln
355 360138360PRTHepatitis C Virus 138Ala Thr Tyr Thr Ser
Gly Gly Val Ala Gly Arg Thr Thr Ser Gly Phe1 5
10 15Thr Ser Leu Phe Ser Ser Gly Ala Ser Gln Lys
Ile Gln Leu Val Asn 20 25
30Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp
35 40 45Ser Leu His Thr Gly Phe Leu Ala
Ala Leu Phe Tyr Thr His Lys Phe 50 55
60Asn Ser Ser Gly Cys Pro Glu Arg Met Ala Ser Cys Arg Pro Ile Asp65
70 75 80Gly Phe Ala Gln Gly
Trp Gly Pro Ile Thr Tyr Thr Glu Pro Asn Ser 85
90 95Pro Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala
Pro Arg Pro Cys Gly 100 105
110Ile Val Pro Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro
115 120 125Ser Pro Val Val Val Gly Thr
Thr Asp Arg Ser Gly Val Pro Thr Tyr 130 135
140Ser Trp Gly Glu Asn Glu Thr Asp Val Met Leu Leu Asn Asn Thr
Arg145 150 155 160Pro Pro
Gln Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly
165 170 175Phe Thr Lys Thr Cys Gly Gly
Pro Pro Cys Asn Ile Gly Gly Val Gly 180 185
190Asn Arg Thr Leu Ile Cys Pro Thr Asp Cys Phe Arg Lys His
Pro Glu 195 200 205Ala Thr Tyr Thr
Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys 210
215 220Leu Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro
Cys Thr Leu Asn225 230 235
240Phe Ser Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg
245 250 255Leu Asn Ala Ala Cys
Asn Trp Thr Arg Gly Glu Arg Cys Asn Leu Glu 260
265 270Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu
Ser Thr Thr Glu 275 280 285Trp Gln
Ile Leu Pro Cys Ala Phe Thr Thr Leu Pro Ala Leu Ser Thr 290
295 300Gly Leu Ile His Leu His Gln Asn Ile Val Asp
Val Gln Tyr Leu Tyr305 310 315
320Gly Val Gly Ser Ala Phe Val Ser Phe Ala Ile Lys Trp Glu Tyr Ile
325 330 335Leu Leu Leu Phe
Leu Leu Leu Ala Asp Ala Arg Val Cys Ala Cys Leu 340
345 350Trp Met Met Leu Leu Ile Ala Gln 355
360139360PRTHepatitis C Virus 139Glu Thr His Val Thr Gly Gly
Ser Ala Gly Arg Thr Thr Ala Gly Leu1 5 10
15Val Gly Leu Leu Thr Pro Gly Ala Lys Gln Asn Ile Gln
Leu Ile Asn 20 25 30Thr Asn
Gly Ser Trp His Ile Asn Ser Thr Ala Leu Asn Cys Asn Glu 35
40 45Ser Leu Asn Thr Gly Trp Leu Ala Gly Leu
Phe Tyr Gln His Lys Phe 50 55 60Asn
Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Arg Leu Thr65
70 75 80Asp Phe Ala Gln Gly Trp
Gly Pro Ile Ser Tyr Ala Asn Gly Ser Gly 85
90 95Leu Asp Glu Arg Pro Tyr Cys Trp His Tyr Pro Pro
Arg Pro Cys Gly 100 105 110Ile
Val Pro Ala Lys Ser Val Cys Gly Pro Val Tyr Cys Phe Thr Pro 115
120 125Ser Pro Val Val Val Gly Thr Thr Asp
Arg Ser Gly Ala Pro Thr Tyr 130 135
140Ser Trp Gly Ala Asn Asp Thr Asp Val Phe Val Leu Asn Asn Thr Arg145
150 155 160Pro Pro Leu Gly
Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly 165
170 175Phe Thr Lys Val Cys Gly Ala Pro Pro Cys
Val Ile Gly Gly Val Gly 180 185
190Asn Asn Thr Leu Leu Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu
195 200 205Ala Thr Tyr Ser Arg Cys Gly
Ser Gly Pro Trp Ile Thr Pro Arg Cys 210 215
220Met Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Ile
Asn225 230 235 240Tyr Thr
Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg
245 250 255Leu Glu Ala Ala Cys Asn Trp
Thr Arg Gly Glu Arg Cys Asp Leu Glu 260 265
270Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr
Thr Gln 275 280 285Trp Gln Val Leu
Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr 290
295 300Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val
Gln Tyr Leu Tyr305 310 315
320Gly Val Gly Ser Ser Ile Ala Ser Trp Ala Ile Lys Trp Glu Tyr Val
325 330 335Val Leu Leu Phe Leu
Leu Leu Ala Asp Ala Arg Val Cys Ser Cys Leu 340
345 350Trp Met Met Leu Leu Ile Ser Gln 355
360140360PRTHepatitis C Virus 140Glu Thr His Thr Thr Gly Arg Val
Ala Gly His Thr Thr Ser Gly Phe1 5 10
15Thr Ser Leu Phe Ser Ser Gly Ala Ser Gln Lys Ile Gln Leu
Val Asn 20 25 30Thr Asn Gly
Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp 35
40 45Ser Leu Gln Thr Gly Phe Phe Ala Ala Leu Phe
Tyr Ala His Lys Phe 50 55 60Asn Ser
Ser Gly Cys Pro Glu Arg Met Ala Ser Cys Arg Pro Ile Asp65
70 75 80Trp Phe Ala Gln Gly Trp Gly
Pro Ile Thr Tyr Thr Lys Pro Asn Ser 85 90
95Ser Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg
Pro Cys Gly 100 105 110Val Val
Pro Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro 115
120 125Ser Pro Val Val Val Gly Thr Thr Asp Arg
Ser Gly Val Pro Thr Tyr 130 135 140Ser
Trp Gly Glu Asn Glu Thr Asp Val Met Leu Leu Asn Asn Thr Arg145
150 155 160Pro Pro Gln Gly Asn Trp
Phe Gly Cys Thr Trp Met Asn Ser Thr Gly 165
170 175Phe Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile
Gly Gly Val Gly 180 185 190Asn
Arg Thr Leu Ile Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu 195
200 205Ala Thr Tyr Thr Lys Cys Gly Ser Gly
Pro Trp Leu Thr Pro Arg Cys 210 215
220Leu Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Leu Asn225
230 235 240Phe Ser Ile Phe
Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg 245
250 255Leu Asn Ala Ala Cys Asn Trp Thr Arg Gly
Glu Arg Cys Asn Leu Glu 260 265
270Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu
275 280 285Trp Gln Ile Leu Pro Cys Ala
Phe Thr Thr Leu Pro Ala Leu Ser Thr 290 295
300Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu
Tyr305 310 315 320Gly Val
Gly Ser Ala Phe Val Ser Phe Ala Ile Lys Trp Glu Tyr Ile
325 330 335Leu Leu Leu Phe Leu Leu Leu
Ala Asp Ala Arg Val Cys Ala Cys Leu 340 345
350Trp Met Met Leu Leu Ile Ala Gln 355
360141358PRTHepatitis C Virus 141Glu Thr His Val Thr Gly Gly Asn Ala Gly
Arg Thr Thr Ala Gly Leu1 5 10
15Val Gly Leu Leu Thr Pro Gly Ala Lys Gln Asn Ile Gln Leu Ile Asn
20 25 30Thr Asn Gly Ser Trp His
Ile Asn Ser Thr Ala Leu Asn Cys Asn Glu 35 40
45Ser Leu Asn Thr Gly Trp Leu Ala Gly Leu Phe Tyr Gln His
Lys Phe 50 55 60Asn Ser Ser Gly Cys
Pro Glu Arg Leu Thr Ser Cys Arg Arg Leu Thr65 70
75 80Asp Phe Ala Gln Gly Trp Gly Pro Ile Ser
Tyr Ala Asn Gly Ser Gly 85 90
95Leu Asp Glu Arg Pro Tyr Cys Trp His Tyr Pro Pro Arg Pro Cys Gly
100 105 110Ile Val Pro Ala Lys
Ser Val Cys Pro Val Tyr Cys Phe Thr Pro Ser 115
120 125Pro Val Val Val Gly Thr Thr Asp Arg Ser Gly Ala
Pro Thr Tyr Ser 130 135 140Trp Gly Ala
Asn Asp Thr Asp Val Phe Val Leu Asn Asn Thr Arg Pro145
150 155 160Pro Leu Gly Asn Trp Phe Gly
Cys Thr Trp Met Asn Ser Thr Gly Phe 165
170 175Thr Lys Val Cys Gly Ala Pro Pro Cys Val Ile Gly
Gly Val Gly Asn 180 185 190Asn
Thr Leu Leu Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala 195
200 205Thr Tyr Ser Arg Cys Gly Ser Gly Pro
Trp Ile Pro Met Val Asp Tyr 210 215
220Pro Tyr Arg Leu Trp Cys Thr His Tyr Pro Cys Thr Ile Asn Tyr Thr225
230 235 240Ile Phe Lys Val
Arg Met Tyr Val Gly Gly Val Glu His Arg Leu Glu 245
250 255Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg
Cys Asp Leu Glu Asp Arg 260 265
270Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Gln Val Gln
275 280 285Val Leu Pro Cys Ser Phe Thr
Thr Leu Pro Ala Leu Ser Thr Gly Leu 290 295
300Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr Gly
Val305 310 315 320Gly Ser
Ser Ile Ala Ser Trp Ala Ile Lys Trp Glu Tyr Val Val Leu
325 330 335Leu Phe Leu Leu Leu Ala Asp
Ala Arg Val Cys Ser Cys Leu Trp Met 340 345
350Met Leu Leu Ile Ser Gln 355
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