HCV is known as a species- and tissue-specific virus. This report now shows that replication of HCV can occur in cells derived from tissues other than liver, indicating that cellular factors required for RNA replication are expressed in cell types other than hepatocytes. One interpretation of this result is that the apparent tropism of HCV for hepatocytes is determined primarily at the level of virus entry or assembly or, alternatively, that HCV can infect many other tissues but has escaped detection due to very low amounts of RNA replication or accumulation. Extrahepatic tissues could serve as reservoirs for HCV that, as with human immunodeficiency virus, could provide a source of viruses that are refractory to antiviral therapy and, importantly, can be responsible for infection of liver grafts following orthotopic liver transplantation (5
). Such a scenario would have profound implications for antiviral therapy. For example, the targeting of drugs to secondary sites of viral replication and the analysis of drug metabolism in cells other than hepatocytes would become important factors for the development of successful antiviral therapies. Although proof for HCV replication in cells of nonhepatic origin is still lacking, there is ample evidence for the presence of viral RNA in lymphocytes and other tissues (3
). The results of this study will encourage further investigations that might provide convincing evidence for HCV replication in extrahepatic tissues.
It is conceivable that HCV quasispecies in hepatocytes and other tissues exhibit differences in their composition due to the selection of variants with cell-type-specific adaptations. As shown in this report, replication of subgenomes in HeLa cells led to the accumulation of clusters of mutations in the NS3, NS4B, and NS5A regions including a deletion in NS5A (Fig. ). Mutations and deletions in NS5A have been found previously in genomes that replicated in Huh7 cells, which could suggest that expression of the natural form of this protein in cell culture somehow interferes with RNA replication (1
). However, mutations in the amino terminus of NS4B have previously not been observed. Notably, in both SL1 and SL2 cells, the mutations changed two or one glutamine residues, respectively, to one of the two basic amino acids arginine and histidine. Moreover, the mutation V1749A was present in all five cell lines examined (Table and Fig. ). Thus far, our results showed that these mutations appeared to be required for replication in HeLa cells, because only replicons pZS2 and pZS25 carrying these mutations yielded colonies after transfection with in vitro-transcribed RNA (Tables and ). However, due to the low efficiency in colony formation obtained with in vitro-transcribed RNA, our results did not yet provide definitive proof for such a conclusion (see below). The amino terminus of NS4B is predicted to reside on the cytoplasmic side of endoplasmic reticulum membranes and may interact with other host or viral proteins required for RNA replication (8
). As an integral endoplasmic reticulum membrane protein, NS4B might provide a scaffold for the assembly of replication complexes and act as a regulator for RNA replication. More importantly, a recent study revealed that NS4B can induce particular membrane structures, called membranous webs, proposed to be the site for HCV replication (6
). Interestingly, genetic analyses with an HCV-related pestivirus identified the amino-terminal region of NS4B as a determinant for cytotoxicity caused by high levels of virus replication (17
). Although the exact mechanism by which NS4B exerts this activity is still unknown, it might interact with cell-type-specific factors and cause the selection of variants with adaptive mutations as shown in this study.
For reasons that we do not yet understand, we could not yet obtain subgenomes that replicated with high efficiency, transiently or permanently, in HeLa or mouse cells (Tables and ). Although it is conceivable that we missed a critical mutation, because it was for some reason underrepresented in our cDNA clones, it is puzzling that it did not arise following the transfection of HeLa cells with RNA. Based on the experience with Huh7 cells, we would have expected that such an event would have occurred and eventually led to the identification of the critical adaptive mutation(s). Nevertheless, our results indicated that the mutations in NS4B and NS5A in replicons pZS2 and pZS25 were sufficient to establish replication in a small number of HeLa cells, because, based on our sequence analysis of cDNA clones, cell lines obtained with these subgenomes did not contain any additional mutations (results not shown). Moreover, transfection of HeLa and mouse cells with heterogeneous populations of subgenomes that should have represented the populations of amplified RNA in cells, with one exception, did not yield more colonies than in vitro-transcribed RNA (Table ). Hence, based on our results we were speculating that in vitro-transcribed RNA exhibited some toxicity in HeLa or mouse cells. However, this is an unlikely scenario, because we found that in vitro-transcribed RNA did not alter the colony formation efficiency in HeLa cells when added to total RNA isolated from HCV-containing cell lines, such as GS4.1 and SL1 (results not shown). Similarly, addition of small amounts of in vitro-transcribed RNA to total RNA from normal Huh7 or HeLa cells did not yield any G418-resistant colonies. These results also indicated that cellular mRNAs did not influence the colony formation efficiency. In addition, we did not observe an increase in colony formation efficiency when we used cured HeLa cells that were obtained through the treatment of subgenome-expressing cells with an HCV polymerase inhibitor. It is conceivable that establishment of HCV replicons in HeLa cells requires certain adaptive mutations that are not required for the maintenance of replicons during the expansion of G418-resistant colonies. Because the amount of viral RNA in total cellular RNA is too low to permit detection of transient replication, we have not been able to directly test this hypothesis. Finally, our results could indicate that amplified viral RNA isolated from cells exhibits physical differences from RNA that is transcribed in vitro, such as methylation of certain residues or other, so far unrecognized modifications at the termini of viral RNA that are required for initiation of replication in HeLa cells.
In summary, we have shown in this report that HCV RNA replication is not restricted to the human hepatoma cell line Huh7 but instead can occur in HeLa cells and hepatoma cells derived from mice. These findings suggest that it may be possible to develop a mouse model for HCV infection. Establishment of such a model will depend on the isolation of HCV variants that can infect mouse hepatocytes or transgenic mice that express the still elusive HCV receptor.