Replication of hepatitis C virus subgenomes in nonhepatic epithelial and mouse hepatoma cells

Abstract

The hepatitis C virus (HCV) pandemic affects the health of more than 170 million people and is the major indication for orthotopic liver transplantations. Although the human liver is the primary site for HCV replication, it is not known whether extrahepatic tissues are also infected by the virus and whether nonprimate cells are permissive for RNA replication. Because HCV exists as a quasispecies, it is conceivable that a viral population may include variants that can replicate in different cell types and in other species. We have tested this hypothesis and found that subgenomic HCV RNAs can replicate in mouse hepatoma and nonhepatic human epithelial cells. Replicons isolated from these cell lines carry new mutations that could be involved in the control of tropism of the virus. Our results demonstrated that translation and RNA-directed RNA replication of HCV do not depend on hepatocyte or primate-specific factors. Moreover, our results could open the path for the development of animal models for HCV infection.

FIG. 1.

Physical map of HCV. The open reading frame is flanked by the IRES and the 3′ untranslated region containing a polypyrimidine tract (poly U-UC), respectively. Subgenomes contain the neomycin phosphotransferase gene (neo) in lieu of the structural genes (C, core; gpE1 and gpE2, envelope proteins; p7) and the NS protein 2 (NS2 protease). Translation of the remaining NS proteins (NS3, protease; NS4A, cofactor for protease; NS4B; NS5A; NS5B, polymerase) is regulated by the IRES derived from EMCV. Proteins of unknown function for viral replication are marked with white rectangles. The cleavage sites for the three proteases required for processing of the polyprotein are indicated with circles.

FIG. 2.

Replication of HCV subgenomic replicons in HeLa cells. (A) Detection of HCV viral RNA. Total RNA (5 μg) was isolated from HeLa cell lines that were established from G418-resistant cell colonies and analyzed by Northern blot analysis. Blots were hybridized with radiolabeled RNA probes corresponding to the HCV NS5 region to detect viral RNA (vRNA) and the ΔE1 region of HPV present in HeLa cells. In vitro-transcribed HCV RNA (1 ng plus 5 μg of total RNA from Huh7 cells, lane 7) served as a marker (M) and control for the hybridization reaction, and 28S rRNA served as a control for the amount of RNA present in each sample analyzed. GS4.1 is a Huh7-derived cell line expressing HCV subgenomic replicons. RNA in SL1 cells was analyzed from cells harvested at the indicated passage (p). SL3 to SL7 were analyzed at passage 3. (B) Immunohistochemical analysis of HCV replication in HeLa cells. Expression of NS5A in GS4.1 and SL1 cells (passage 26) was detected with a monoclonal antibody bound to fluorescein isothiocyanate-conjugated antibody. Parental Huh7 and HeLa cells served as controls.

FIG. 3.

Sequence analysis of HCV subgenomes in HeLa and mouse hepatoma Hepa1-6 cells. (Left) Physical map of HCV subgenomic RNA including the positions of the first amino acid NS3 and the last residue of the polyprotein. The IRES for the translation of the NS genes is indicated (EMCV-IRES). (Right) Mutations causing amino acid changes identified in cDNAs isolated from subgenomic replicons present in GS4.1 (passage 21) and the indicated HeLa (SL1, passage 26; SL2, passage 5) and Hepa1-6 (MH1 [passage 12], MH2 [passage 4], and MH4 [passage 4]) cell lines are depicted with horizontal bars. Four independent clones were sequenced from each PCR fragment that was amplified from cDNAs obtained from total RNA purified from the indicated cell lines. Mutations present in more than one cell line are indicated with their amino acid positions. Mutations that occurred in 50% of the clones analyzed are indicated with an asterisk. Mutations that occurred in only one of four clones analyzed are not included in the figure. A deletion identified in cDNA clones obtained from SL1 cells spanning amino acids 2371 to 2413 is indicated (Δ).

FIG. 4.

Replication of HCV subgenomes in mouse hepatoma cells. (A) Detection of HCV viral RNA. RNA in Hepa1-6 cell lines (MH1 to MH5) that were established from G418-resistant cell colonies was analyzed as described in the legend to Fig. 2 except that a radiolabeled probe specific to the mouse albumin cDNA was used in lieu of the probe against HPV. MH4 and MH5 were analyzed at passage 4. (B) Immunohistochemical analysis of HCV replication in MH1 cells (passage 3). Expression of NS5A was detected as described in the legend to Fig. 2. Hepa1-6 cells served as a negative control.