Efficient Infectious Cell Culture Systems of the Hepatitis C Virus (HCV) Prototype Strains HCV-1 and H77

Yi-Ping Li, Santseharay Ramirez, Lotte Mikkelsen, Jens Bukh

The first discovered and sequenced hepatitis C virus (HCV) genome and the first in vivo infectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell-culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77C in vivo infectious clones. We initially adapted a genome with the HCV-1 5' UTR-NS5A and the JFH1 NS5B-3' UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2 mutation S399F. Combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with seventeen mutations (HCV1cc) replicated efficiently in Huh7.5 cells, and produced supernatant infectivity titers of 10(4.0) focus-forming-units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 viruses, and the A970T/I1312V/C2419R/A2919T mutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R identified here from culture-adapted full-length TN-viruses and a common NS3-helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants initiated replication and culture-adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harbouring nineteen mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naïve Huh7.5 cells, reaching titers of 10(3.5) and 10(4.4) FFU/ml, respectively.