Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells

Abstract

Dicistronic, selectable subgenomic replicons derived from the Con1 strain of hepatitis C virus (HCV) are capable of autonomous replication in cultured Huh7 cells (Lohmann et al., Science 285:110-113, 1999). However, adaptive mutations in the NS3, NS5A, and/or NS5B proteins are required for efficient replication of these RNAs and increase by orders of magnitude the numbers of G418-resistant colonies selected following transfection of Huh7 cells. Here, we demonstrate that a subgenomic replicon (NNeo/3-5B) derived from an infectious molecular clone of a second genotype 1b virus, HCV-N (Beard et al., Hepatology 30:316-324, 1999) is also capable of efficient replication in Huh7 cells. G418-resistant cells selected following transfection with NNeo/3-5B RNA contained abundant NS5A antigen and HCV RNA detectable by Northern analysis. Replicon RNA in one of three clonally isolated cell lines contained no mutations in the NS3-NS5B polyprotein, confirming that adaptive mutations are not required for efficient replication in these cells. However, the deletion of a unique 4-amino-acid insertion that is present within the interferon sensitivity-determining region (ISDR) of the NS5A protein in wild-type HCV-N drastically decreased the number of G418-resistant colonies obtained following transfection of Huh7 cells. This effect could be reversed by inclusion of a previously described Con1 cell culture-adaptive mutation (S2005-->I), confirming that this natural insertion has a controlling role in determining the replication capacity of wild-type HCV-N RNA in Huh7 cells. Additional selectable, dicistronic RNAs encoding NS2-NS5B, E1-NS5B, or the full-length HCV polyprotein were also capable of replication and gave rise to G418-resistant cell clones following transfection of Huh7 cells. We conclude that RNA derived from this documented infectious molecular clone has a unique capacity for replication in Huh7 cells in the absence of additional cell culture-adaptive mutations.

FIG. 1.

(A) Organization of the subgenomic HCV RNA replicons employed in these studies. Open reading frames are depicted as boxes, and untranslated segments of the dicistronic RNAs are depicted as solid lines. The sequence of BNeo/3-5B (shaded box) is identical to that of I₃₇₇NS3-3′/wt, described previously by Lohmann et al. (16). NNeo/3-5B contains mostly HCV-N-derived sequence (open boxes). The amino acid sequence of NS3 in NNeo/3-5B differs from that of HCV-N at only 2 amino acid residues, as described in the text, while the 5′- and 3′UTR sequences are identical. “ΔC” indicates the N-terminal segment of the HCV core protein that is expressed as a fusion with Neo in these replicons. (B) Locations of the S2205I and R2889G BNeo/3-5B-adaptive mutations that were introduced into the replicons shown in panel A.

FIG. 2.

Mean number of G418-resistant cell colonies isolated per 10-cm-diameter cell culture dish 4 weeks following transfection of Huh7 cells with various amounts of the subgenomic NNeo/3-5B replicon RNA. Error bars indicate the range of values observed in duplicate experiments.

FIG. 3.

(Top panel) Northern analysis of HCV-specific RNA present in G418-resistant cell lines selected following transfection of Huh7 cells with the indicated replicon RNA. Lanes 1 and 2, synthetic RNA transcribed from pNNeo/3-5B (10⁸ and 10⁷ genome equivalents spiked into normal cellular RNA), respectively; lane 3, normal Huh7 cells; lanes 4 and 5, two independent, clonally isolated cell lines (cell lines 1 and 2) selected following transfection with NNeo/3-5B; lanes 6 to 9, other clonally isolated cell lines. (Lower panel) Northern analysis of β-actin mRNAs in the samples loaded into the gel shown in the top panel.

FIG. 4.

Indirect immunofluorescence detection of NS5A antigen in normal Huh7 cells (A) and clonally isolated cell lines selected following transfection of Huh7 cells with NNeo/3-5B (cell line 1) (B), NNeo/3-5B(SI) (C), and NNeo/3-5B(RG) (D).

FIG. 5.

Amino acid substitutions predicted to be present in the HCV nonstructural proteins expressed by replicons present in three clonally isolated, G418-resistant cell lines selected following transfection of Huh7 cells with NNeo/3-5B RNA. There were no mutations identified within the open reading frame of the replicon RNA present in cell line 1.

FIG. 6.

Mean number of G418-resistant cell colonies selected per 10-cm-diameter culture dish following transfection of Huh7 cells with the indicated replicon RNAs. A total of 1 μg of replicon RNA was transfected per culture dish. Error bars indicate the range of values obtained in replicate experiments.

FIG. 7.

G418-resistant cell colonies present 3 weeks after transfection with NNeo/3-5B and NNeo/3-5BΔi5A RNAs (20 μg of RNA per 10-cm-diameter culture dish) (top panel) or NNeo/3-5B(SI) and NNeo/3-5B(SI)Δi5A (1 μg of RNA per 10-cm-diameter culture dish) (bottom panel). Colonies were visualized by staining cells with crystal violet. wt, wild type.

FIG. 8.

Organization of selectable dicistronic RNAs containing HCV-N sequence encoding NS2, the envelope proteins E1 and E2, and/or the core protein within the 3′ cistron. UTR, nontranslated region.

FIG. 9.

Northern analysis of (lanes 1 to 2) synthetic T7 transcripts representing the dicistronic subgenomic NNeo/3-5B (solid arrowhead) and full-length NNeo/C-5B (open arrowhead) RNAs spiked into total cellular RNA extracted from normal Huh7 cells; (lanes 3 to 6) total cellular RNA isolated from G418-resistant cell lines, including three subcloned cell lines selected following transfection with NNeo/C-5B RNA (lanes 3 to 5) and a clonal cell line selected following transfection with NNeo/3-5B RNA (lane 6), and total cellular RNA from normal Huh7 cells (lane 7). The blot was hybridized to a β-actin probe as an internal control.

FIG. 10.

Indirect immunofluorescence for detection of E2 protein (A and B) or core protein (C and D) in normal Huh7 cells (A and C) or G418-resistant cells selected following transfection with NNeo/E1-5B (B) or NNeo/C-5B.

FIG. 11.

Alignment of the amino acid sequences of the NS5A proteins encoded by NNeo/3-5B and BNeo/3-5B. The ISDR is shaded, with the 4-amino-acid -Ser-Ser-Tyr-Asn- insertion in NNeo/3-5B shown in boldface type and enclosed in a box. Arrows indicate the location of single-base substitutions and insertions and the large 47-amino-acid deletion that has been shown previously to enhance the replication capacity of BNeo/3-5B (4, 14, 15). The asterisk indicates the S2005I mutation.