Summary auto-generated
Researchers studied hepatitis C virus (HCV) genetic variation in cultured human T cells (MT-2C cells) to understand viral replication and select for infectious clones. They compared HCV sequences from two sources: the original viral inoculum (serum 1B-1) containing genotype 1b virus with 23 distinct HVR1 species, and HCV-infected MT-2C cells harvested 12 days after inoculation. Using RT-nested PCR and cDNA cloning, they obtained sequences covering the entire HCV genome. Molecular evolutionary analysis revealed that HCV populations became homogeneous in more than half of the analyzed genome regions during cell culture, with limited viral populations able to replicate efficiently in MT-2C cells. One HVR1 species (II-1) became predominant despite representing only 13% of the original inoculum. They also characterized the conserved 3′ X-tail sequence and found the poly(U) stretch upstream of it became shortened in cultured cells (29-32 nucleotides versus 47-71 in serum). Based on consensus sequences from these clones, they determined the complete HCV genome sequence (HCV-JS), which may serve as a candidate infectious molecular clone for studying HCV replication mechanisms.
Key findings
- HCV populations converged to homogeneous sequences in cultured MT-2C cells despite complex quasi-species in the original inoculum, with one HVR1 species (II-1) becoming predominant at 50% frequency versus 13% in serum
- Sequence homogenization occurred in at least 10 of 17 analyzed genomic regions throughout the HCV genome during 12-day culture period
- The conserved 3′ X-tail sequence remained highly conserved in cultured cells, and the poly(U) stretch upstream shortened to 29-32 nucleotides from 47-71 nucleotides in serum, suggesting importance for viral replication
- Complete HCV genome sequence (HCV-JS) was determined and is proposed as a candidate for an infectious molecular clone for understanding HCV replication mechanisms
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Abstract
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.