Claims
- 1. A nucleic acid molecule comprising a region selected from the group consisting of:
a) an altered HCV NS3 encoding region coding for one or more NS3 mutations, wherein at least one of said NS3 mutations, identified by reference to the amino acid sequence numbering of SEQ. ID. NO. 1, is selected from the group consisting of:
amino acid 1095 being Ala, amino acid 1202 being Gly, and amino acid 1347 being Thr; b) an altered HCV NS5A encoding region coding for one or more NS5A mutations, wherein at least one of said NS5A mutations, identified by reference to the amino acid sequence numbering of SEQ. ID. NO. 1, is selected from the group consisting of:
amino acid 2041 being Thr, a Lys insertion between residue 2039 and 2040. amino acid 2173 being Phe, amino acid 2197 being Phe, amino acid 2198 being Ser, amino acid 2199 being Thr, and amino acid 2204 being Arg; and c) an altered encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) region containing one or more EMCV IRES mutations, wherein at least one of said EMCV IRES mutations, identified by reference to the nucleotide number of SEQ. ID. NO. 3, is an insertion at nucleotide 1736 of adenine.
- 2. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises said NS5A encoding region.
- 3. The nucleic acid molecule of claim 2, wherein at least two of said NS5A adaptive mutations are present.
- 4. The nucleic acid molecule of claim 2, further comprising a region encoding for a HCV NS3 region, wherein said NS3 region may be the same or different than said altered NS3 region.
- 5. The nucleic acid molecule of claim 4, wherein said nucleic acid molecule is an HCV replicon comprising a HCV 5′ UTR-PC region, said NS3 encoding region, an HCV NS4A encoding region, an HCV NS4B encoding region, said NS5A encoding region, an HCV NS5B encoding region, and a HCV 3′ UTR.
- 6. The nucleic acid molecule of claim 5, wherein said HCV replicon further comprises a sequence encoding for a reporter protein.
- 7. The nucleic acid molecule of claim 5, wherein said HCV replicon further comprises a sequence encoding for a selection protein.
- 8. The nucleic acid molecule of claim 5, wherein said HCV replicon further comprises a HCV core encoding region, a HCV E1 encoding region, a HCV E2 encoding region, a HCV p7 encoding region, and a HCV NS2 encoding region.
- 9. A nucleic acid molecule comprising a region selected from the group consisting of:
a) an altered HCV NS3 encoding region containing one or more NS3 mutations, wherein at least one of said NS3 mutations, identified by reference to the nucleotide numbering of SEQ. ID. NO. 2, is selected from the group consisting of:
nucleotide 3625 being cytosine, nucleotide 3946 being guanine, nucleotide 4380 being adenine, b) an altered HCV NS5A encoding region containing one or more NS5A mutations, wherein at least one of said NS5A mutations, identified by reference to the nucleotide numbering of SEQ. ID. NO. 2, is selected from the group consisting of:
an insertion of 3 adenine residues between nucleotide 6458 and 6459, nucleotide 6463 being cytosine, nucleotide 6859 being thymine or uracil, nucleotide 6931 being thymine or uracil, nucleotide 6934 being cytosine, nucleotide 6936 being adenine, and nucleotide 6953 being adenine or guanine; and c) an altered encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) region containing one or more EMCV IRES mutations, wherein at least one of said EMCV IRES mutations, identified by reference to the nucleotide number of SEQ. ID. NO. 3, is an insertion at nucleotide 1736 of adenine.
- 10. The nucleic acid molecule of claim 9, wherein said molecule comprises said altered NS5A encoding region, and the nucleotide sequence of said altered NS5A region is provided for by bases 6258-7598 of SEQ. ID. NO. 2, or the RNA version thereof, modified with one or more of said NS5A modifications selected from the group consisting of:
an insertion of 3 adenine residues between nucleotide 6458 and 6459, nucleotide 6463 being cytosine, nucleotide 6859 being thymine or uracil, nucleotide 6931 being thymine or uracil, nucleotide 6934 being cytosine, nucleotide 6936 being adenine, and nucleotide 6953 being adenine or guanine.
- 11. The nucleic acid molecule of claim 10, wherein said molecule is an HCV replicon comprising a HCV 5′ UTR-PC region, a modified HCV NS3-NS5B region, and a HCV 3′ UTR, wherein said modified NS3-NS5B region comprises said altered NS5A region.
- 12. The nucleic acid molecule of claim 11, wherein said 5′ UTR-PC region is the RNA version of bases 1-377 of SEQ. ID. NO. 2 and said 3′ UTR is the RNA version of bases 9374-9605 of SEQ. ID. NO. 2.
- 13. The nucleic acid molecule of claim 10, wherein said molecule is an HCV replicon comprising a HCV 5′ UTR-PC region, a modified HCV NS3-NS5B region, and a HCV 3′ UTR, wherein
said 5′ UTR-PC region is the RNA version of bases 1-377 of SEQ. ID. NO. 2; said 3′ UTR is the RNA version of bases 9374-9605 of SEQ. ID. NO. 2; and, said modified NS3-NS5B region consists of the RNA version of bases 3420-9371 of SEQ. ID. NO. 2 modified with one or more modifications selected from the group consisting of:
nucleotide 4380 being adenine, nucleotide 3625 being cytosine, nucleotide 3946 being guanine, an insertion of 3 adenine residues between nucleotide 6458 and nucleotide 6459, nucleotide 6463 being cytosine, nucleotide 6859 being uracil, nucleotide 6931 being uracil, nucleotide 6934 being cytosine, nucleotide 6936 being adenine, and nucleotide 6953 being adenine or guanine.
- 14. The nucleic acid molecule of claim 13, wherein said replicon is a genomic replicon that further comprises the RNA version of nucleotides 378-3419 of SEQ. ID. NO. 2.
- 15. A nucleic acid molecule comprising the nucleic acid base sequence of bases 1-7989 of SEQ. ID. NO. 3, or the RNA version thereof, consisting of one or more different modifications selected from the group consisting of:
a) nucleotides 5335-5337 modified to code for arginine; b) nucleotides 5242-5244 modified to code for phenylalanine; c) nucleotides 5314-5316 modified to code for phenylalanine; d) nucleotides 5317-5319 modified to code for serine; e) nucleotides coding for lysine inserted after nucleotide 4843; f) nucleotides 2329-2331 modified to code for glycine, nucleotides 2764-2766 modified to code for threonine, nucleotides 5242-5244 modified to code for phenylalanine, and an extra adenosine inserted after nucleotide 1736; g) nucleotides 4846-4848 modified to code for threonine, and nucleotides 5242-5244 modified to modified to code for phenylalanine; h) nucleotides 4846-4848 modified to code for threonine, and nucleotides 5314-5316 modified to code for phenylalanine; i) nucleotides 4846-4848 modified to code for threonine, and nucleotides 5317-5319 modified to code for serine; j) nucleotides 2329-2331 modified to code for glycine, and nucleotides coding for lysine inserted after nucleotides 4843; k) nucleotides 5314-5316 modified to code for phenylalanine and nucleotides 5320-5322 modified to code for threonine; l) nucleotides 4846-4848 modified to code for threonine, nucleotides 5314-5316 modified to code for phenylalanine, and nucleotides 5320-5322 modified to code for threonine; m) nucleotides 4846-4848 modified to code for threonine, nucleotides 5314-5316 modified to code for phenylalanine, and an extra adenosine inserted after nucleotide 1736; and n) nucleotides 5314-5316 modified to code for phenylalanine, nucleotides 5320-5322 modified to code for threonine, and an extra adenosine inserted after nucleotide 1736; and o) nucleotides 5320-5322 modified to code for threonine.
- 16. The nucleic acid of claim 15, wherein said one or more different modifications is selected from the group consisting of:
a) C5337A; b) C5243T or U; c) C5315T or U; d) T or U5318C; e) AAA inserted after 4843; f) A2330G, G2764A, C5243T or U, and adenosine inserted 1736; g) A4847C and C5243T or U; h) A4847C and C5315T or U; i) A4847C and T or U5318C; j) A2330G and AAA inserted after 4843; k) C5315T or U and G5320A; l) A4847C, C5315T or U, and G5320A; m) A4847C, C5315T or U, and adenosinc inserted 1736; n) C5315T or U, G5320A and adenosine inserted 1736; and o) G5320A.
- 17. The nucleic acid of claim 16, wherein said nucleic acid is RNA and comprises said nucleic acid base sequence.
- 18. The nucleic acid of claim 17, wherein said nucleic acid is RNA and consists of said nucleic acid base sequence.
- 19. An expression vector comprising a nucleotide sequence coding for the nucleic acid molecule of any one of claims 1-18, wherein said nucleotide sequence is transcriptionally coupled to an exogenous promoter.
- 20. A recombinant cell human hepatoma cell, wherein said cell comprises the nucleic acid of any one of claims 5-8 and 11-18.
- 21. The recombinant cell of claim 20, wherein said hepatoma cell is an Huh-7 cell.
- 22. The recombinant cell of claim 20, wherein said cell is derived from a Huh-7 cell.
- 23. A recombinant cell made by a process comprising the step of introducing into a human hepatoma cell the nucleic acid of any one of claims 5-8 and 11-18.
- 24. A method of making an HCV replicon enhanced cell comprising the steps of:
a) introducing and maintaining a HCV replicon in a cell; and b) curing said cell of said HCV replicon to produce said replicon enhanced cell.
- 25. The method of claim 24, wherein said cell is a human hepatoma cell.
- 26. The method of claim 24, wherein said cell is a Huh-7 cell or is derived from a Huh-7 cell.
- 27. The method of claim 26, further comprising the step of confirming the ability of said replicon enhanced cell to maintain an HCV replicon.
- 28. A method of making an HCV replicon enhanced cell containing a functional HCV replicon comprising the steps of:
a) introducing and maintaining a first HCV replicon in a cell; b) curing said cell of said first replicon to produce a cured cell; and c) introducing and maintaining a second HCV replicon into said cured cell, wherein said second HCV replicon may be the same or different than said first HCV replicon.
- 29. The method of claim 28, wherein said cell is a human hepatoma cell.
- 30. The method of claim 29, wherein said human hepatoma cell is a Huh-7 cell.
- 31. The method of claim 30, wherein said human hepatoma cell is derived from a Huh-7 cell.
- 32. An HCV replicon enhanced cell made by the method of any one of claims 24-27.
- 33. An HCV replicon enhanced cell containing a HCV replicon made by the method of any one of claims 28-31.
- 34. A method of measuring the ability of a compound to affect HCV activity comprising the steps of:
a) providing said compound to the HCV replicon enhanced cell of claim 33; and b) measuring the ability of said compound to effect one or more replicon activities as a measure of the effect on HCV activity.
- 35. The method of claim 34, wherein said compound is a ribozyme.
- 36. The method of claim 34, wherein said compound in an antisense nucleic acid.
- 37. The method of claim 34, wherein compound is an organic compound.
- 38. The method of claim 34, wherein said step (b) measures HCV protein production.
- 39. The method of claim 33, wherein said step (b) measures production of RNA transcripts.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Ser. No. 60/263,479, filed Jan. 23, 2001, hereby incorporated by reference herein.
PCT Information
Filing Document |
Filing Date |
Country |
Kind |
PCT/EP02/00526 |
1/16/2002 |
WO |
|