Genetic analysis of the hepatitis C virus (HCV) genome from HCV-infected human T cells

We recently established a cell culture system for the replication of hepatitis C virus (HCV) by using a human T cell leukaemia virus type I-infected cell line, MT-2, and showed that the quasi-species of the hypervariable region 1 observed in the original inoculum became homogeneous in MT-2 cells 10 days after inoculation of HCV. In this study, we obtained HCV cDNA clones covering the whole viral genome by RT-nested PCR using RNA from HCV-infected cloned MT-2C cells, which support viral replication more efficiently, at 12 days after inoculation. A total of 41 distinct HCV cDNA clones covering almost the whole viral genome were also isolated from a cDNA library derived from the original inoculum. Molecular evolutional analyses comparing the sequences of the HCV clones obtained from both sources revealed that the HCV populations became homogeneous in more than half of the compared regions. This finding suggests that limited HCV populations are able to replicate in MT-2C cells. In addition, we isolated cDNA clones containing a 3' X-tail sequence, which was recently identified as a bona fide 3' terminus of the HCV genome, in the HCV-infected MT-2C cells and confirmed that the nucleotide sequence of the 3' X-tail was highly conserved, suggesting its implication in HCV replication. Finally, on the basis of the sequences of HCV cDNA clones obtained from HCV-infected MT-2C cells, we determined the entire nucleotide sequence of the HCV genome containing the 3' X-tail as a candidate for an infectious HCV molecular clone.