Patent ID: 7235394

Claim:
A method of selecting for an HCV sequence with an adaptive mutation that permits higher levels of HCV replication in a cell line, said method comprising: (a) transfecting a cell line in culture with a polynucleotide that comprises a non-naturally occurring HCV sequence capable of replication, said sequence comprising from 5′ to 3′ on the positive-sense nucleic acid: (i) an HCV 5′ non-translated region (5′ NTR) sequence, wherein said HCV 5′NTR sequence comprises at the 5′ terminus at least one of NCCAGCC, GCCAGCC; GGCCAGCC; UGCCAGCC; AGCCAGCC; AAGCCAGCC; GAGCCAGCC; GUGCCAGCC; and GCGCCAGCC; (ii) an HCV non-structural protein coding region consensus sequence comprising an NS3 serine proteinase/helicase and an NS5B RNA-dependent RNA polymerase encoding region; (iii) a selectable marker under the control of the HCV replication machinery; and (iv) an HCV 3′ non-translated region (3′ NTR) that comprises a poly (u/c) tract of variable length and an HCV extreme 3′ terminal conserved sequence of about 98 nucleotides; wherein said HCV 5′ NTR, said HCV non-structural protein coding region, said selectable marker and said 3′ NTR are operably associated; (b) culturing said cell line in vitro; (c) selecting for cell populations displaying the phenotype conferred by said selectable marker; and (d) isolating an adaptive HCV sequence from said cell populations of (c), thereby selecting for an HCV sequence with an adaptive mutation that permits higher levels of HCV replication in a cell line.