Claims
- 1. A HCV polynucleotide molecule comprising a 5′-non translated region (NTR) wherein guanine at position 1 is substituted for adenine.
- 2. A HCV self-replicating polynucleotide comprising:
(a) a 5′-NTR consisting of ACCAGC (SEQ ID NO. 8); (b) a HCV polyprotein region coding for a HCV polyprotein; and (c) a 3′-NTR region.
- 3. The HCV polynucleotide according to claim 2, wherein said polyprotein comprises one or more amino acid substitution selected from the group consisting of: R(1135)K, S(1148)G, S(1560)G, K(1691)R, L(1701)F, L(1984)V, T(1993)A, G(2042)C, G(2042)R, S(2404)P, L(2155)P, P(2166)L and M(2992)T.
- 4. The HCV polynucleotide encoding a polyprotein comprising one or more of the amino acid substitutions as defined in claim 3, and further comprising the amino acid substitution E(1202)G.
- 5. The HCV polynucleotide according to claim 3, wherein said substitution is a G2042C or a G2042R mutation.
- 6. The HCV polynucleotide according to claim 3, wherein said substitution is selected from the group consisting of: K(1691)R, and G(2042)C.
- 7. The HCV polynucleotide according to claim 3, wherein said substitution is selected from the group consisting of: R(1135)K, S(1560)G, K(1691)R, T(1993)A, G(2042)C, and P(2166)L.
- 8. The HCV polynucleotide according to claim 3, wherein said substitution is selected from the group consisting of: R(1135)K, S(1560)G, K(1691)R, T(1993)A, G(2042)C, L(2155)P, and P(2166)L.
- 9. The HCV polynucleotide according to claim 3, wherein said substitution is selected from the group consisting of: E(1202)G, I(1984)V, G(2042)C, and M(2992)T.
- 10. The HCV polynucleotide according to claim 3, wherein said substitution is selected from the group consisting of: S(1148)G, E(1202)G, L(1701)F, G(2042)R, and S(2404)P.
- 11. The HCV polynucleotide according to claim 2, wherein said polynucleotide is an RNA molecule encoded by the DNA molecule selected from the group consisting of: SEQ ID Nos: 2, 4, 5, 6, 7, 24, and 25.
- 12. The HCV polynucleotide according to claim 2, wherein said polynucleotide is a DNA molecule selected from the group consisting of: SEQ ID Nos: 2, 4, 5, 6, 7, 24, and 25.
- 13. An expression vector comprising a DNA form of the polynucleotide according to claim 2 operably linked to a promoter.
- 14. A host cell transfected with the self-replicating polynucleotide molecule according to claim 2.
- 15. The host cell according to claim 14, wherein the host cell is a eukaryotic cell line.
- 16. The host cell according to claim 15, wherein said eukaryotic cell line is a hepatic cell line.
- 17. The host cell according to claim 16, wherein said hepatic cell line is Huh-7.
- 18. A method for determining RNA replication comprising the steps of:
(a) incubating the host cell according to claim 14 under conditions suitable for RNA replication; (b) isolating the total cellular RNA from the cells; and (c) analyzing the RNA so as to measure the amount of HCV RNA replicated.
- 19. The assay according to claim 18, wherein the analysis of RNA levels in step (c) is carried out by amplifying the RNA by real-time reverse transcriptase-polymerase chain reaction(RT-PCR) analysis using HCV specific primers so as to measure the amount of HCV RNA replicated.
- 20. The assay according to claim 18, wherein said polynucleotide encodes a reporter gene, and the analysis of RNA levels in step (c) is carried out by assessing the level of reporter expressed.
- 21. A method for testing a compound for inhibiting HCV replication, including the steps of:
(a) carrying out step a) according to claim 18, in the presence or absence of the compound; (b) isolating the total cellular RNA from the cells; and (c) analyzing the RNA so as to measure the amount of HCV RNA replicated. (d) comparing the levels of HCV RNA in the cells in the absence and presence of the inhibitor, wherein reduced RNA levels is indicative of the ability of the compound to inhibit replication.
- 22. The method according to claim 21, wherein said cell line is incubated with the test compound for about 3-4 days at a temperature of about 37° C.
- 23. A HCV polynucleotide molecule comprising:
(a) a 5′-NTR region; (b) a HCV polyprotein region coding for an HCV polyprotein comprising one or more amino acid substitutions selected from the group consisting of: R(1135)K, S(1148)G, S(1560)G, K(1691)R, L(1701)F, I(1984)V, T(1993)A, G(2042)C, G(2042)R, S(2404)P, L(2155)P, P(2166)L, and M(2992)T, and (c) a 3′-NTR region.
- 24. The HCV self-replicating polynucleotide encoding a polyprotein comprising any one of the amino acid substitutions as defined in claim 24, further comprising the amino acid substitution E(1202)G.
- 25. The polynucleotide according to claim 24, wherein said substitution is a G2042C or a G2042R mutation.
- 26. The HCV polynucleotide according to claim 24, wherein said substitution is selected from the group consisting of: K(1691)R, and G(2042)C.
- 27. The HCV polynucleotide according to claim 24, wherein said substitution is selected from the group consisting of: R(1135)K, S(1560)G, K(1691)R, T(1993)A, G(2042)C, and P(2166)L.
- 28. The HCV polynucleotide according to claim 24, wherein said substitution is selected from the group consisting of: R(1135)K, S(1560)G, K(1691)R, T(1993)A, G(2042)C, L(2155)P, and P(2166)L.
- 29. The HCV polynucleotide according to claim 24, wherein said substitution is selected from the group consisting of: E(1202)G, I(1984)V, G(2042)C, and M(2992)T.
- 30. The HCV polynucleotide according to claim 24, wherein said substitution is selected from the group consisting of: S(1148)G, E(1202)G, L(1701)F, G(2042)R, and S(2404)P.
- 31. The HCV polynucleotide according to claim 24, wherein said molecule is a RNA molecule encoded by the DNA molecule selected from the group consisting of:
,SEQ ID NOs: 2, 4, 5, 6, 7, 24, and 25.
- 32. The HCV polynucleotide according to claim 24, wherein said molecule is a DNA molecule selected from the group consisting of: SEQ ID Nos: 2, 4, 5, 6, 7, 24, and 25.
- 33. An expression vector comprising a DNA form of the polynucleotide according to claim 24 operably linked to a promoter.
- 34. A host cell transfected with the self-replicating polynucleotide according to claim 24.
- 35. A host cell according to claim 34, wherein the host cell is a eukaryotic cell line.
- 36. A host cell according to claim 35, wherein said eukaryotic cell line is a hepatic cell line.
- 37. A host cell according to claim 36, wherein said hepatic cell line is Huh-7.
- 38. A RNA replication assay comprising the steps of:
incubating the host cell according to claim 34 under conditions suitable for RNA replication; isolating the total cellular RNA from the cells; and analyzing the RNA so as to measure the amount of HCV RNA replicated.
- 39. The assay according to claim 38, wherein the analysis of RNA levels in step c) is carried out by amplifying the RNA by real-time RT-PCR analysis using HCV specific primers so as to measure the amount of HCV RNA replicated.
- 40. The assay according to claim 38, wherein said polynucleotide encodes for a reporter gene, and the analysis of RNA levels in step c) is carried out by assessing the level of reporter expressed.
- 41. A method for testing a compound for inhibiting HCV replication, including the steps of:
a) carrying step a) according to claim 38, in the presence or absence of the compound; b) isolating the total cellular RNA from the cells; and c) analyzing the RNA so as to measure the amount of HCV RNA replicated. d) comparing the levels of HCV RNA in cells in the absence and presence of the inhibitor, wherein reduced RNA levels is indicative of the ability of the compound to inhibit replication.
- 42. The method according to claim 41, wherein said cell line is incubated with the test compound for about 3-4 days at a temperature of about 37° C.
RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional Application Serial No. 60/257,857 filed on Dec. 22, 2000.
Provisional Applications (1)
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Number |
Date |
Country |
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60257857 |
Dec 2000 |
US |