The NS3 helicase and NS5B-to-3'X regions are important for efficient hepatitis C virus strain JFH-1 replication in Huh7 cells

Abstract

The JFH-1 strain of hepatitis C virus (HCV) is a genotype 2a strain that can replicate autonomously in Huh7 cells. The J6 strain is also a genotype 2a strain, but its full genomic RNA does not replicate in Huh7 cells. However, chimeric J6/JFH-1 RNA that has J6 structural-protein-coding regions and JFH-1 nonstructural-protein-coding regions can replicate autonomously and produce infectious HCV particles. In order to determine the mechanisms underlying JFH-1 RNA replication, we constructed various J6/JFH-1 chimeras and tested their RNA replication and virus particle production abilities in Huh7 cells. Via subgenomic-RNA-replication assays, we found that both the JFH-1 NS5B-to-3'X (N5BX) and the NS3 helicase (N3H) regions are important for the replication of the J6CF replicon. We applied these results to full-length genomic RNA replication and analyzed replication using Northern blotting. We found that a chimeric J6 clone with JFH-1 N3H and N5BX could replicate autonomously but that a chimeric J6 clone with only JFH-1 N5BX had no replication ability. Finally, we tested the virus production abilities of these clones and found that a chimeric J6 clone with JFH-1 N3H and N5BX could produce infectious HCV particles. In conclusion, the JFH-1 NS3 helicase and NS5B-to-3'X regions are important for efficient replication and virus particle formation of HCV genotype 2a strains.

FIG. 1.

G418-resistant colony formation of JFH-1, J6CF, JCH-1, and JCH-4. Subgenomic RNAs were synthesized in vitro, using pSGR-JFH1, pSGR-J6CF, pSGR-JCH1, and pSGR-JCH4 as templates. Transcribed subgenomic RNAs were electroporated into Huh7 cells, and cells were cultured with G418 for 3 weeks before staining with crystal violet as described in Materials and Methods. JFH-1 subgenomic RNA (0.1 μg) and 10 μg of J6CF, JCH-1, and JCH-4 subgenomic RNAs were transfected into Huh7 cells. Experiments were performed in triplicate, and representative staining examples are shown.

FIG. 2.

Luciferase activities of chimeric replicons with a JFH-1 backbone. (A) Structures of chimeric subgenomic replicons with a JFH-1 backbone. The restriction enzyme recognition sites used for the construction of plasmids are indicated. P, PmeI; C, ClaI; E, EcoT22I; M, MunI; B, BsrGI; S, StuI; X, XbaI; wt, wild type. (B) Subgenomic RNAs were synthesized in vitro from wild-type or chimeric replicon constructs. Transcribed subgenomic RNAs (5 μg) were electroporated into Huh7 cells, and cells were harvested serially at 4, 24, and 48 h after transfection. The harvested cells were lysed, and then luciferase activities in the cell lysates were measured. The assays were performed three times independently and the results expressed as luciferase activities (RLU). Each value was corrected for transfection efficiency as determined by measuring the luciferase activity 4 h after transfection. Data are presented as means and standard deviations for luciferase activity at 24 h (white bars) and 48 h (gray bars) after transfection.

FIG. 3.

Luciferase activities of chimeric replicons with a J6CF backbone. (A) Structures of chimeric subgenomic replicons with a J6CF backbone. The restriction enzyme recognition sites used for the construction of plasmids are indicated. C, ClaI; E, EcoT22I; B, BsrGI; S, StuI; X, XbaI; wt, wild type. (B) Wild-type or chimeric subgenomic RNAs were transfected into Huh7 cells, and the luciferase activities of the transfected cells were examined as described in the legend to Fig. 2B. Assays were performed three times independently, and data are presented as means and standard deviations for luciferase activity (RLU) at 24 h (white bars) and 48 h (gray bars) after transfection.

FIG. 4.

Restoration of genotype 2a and genotype 1 replicon replication by the insertion of JFH-1 sequences. Two genotype 2a replicons, JCH-1 and JCH-4, a genotype 1a replicon, H77c, and a genotype 1b replicon, Con-1, were used in this assay. Three kinds of chimeric replicons, N3H-JFH-1, N5BX-JFH1, and N3H+N5BX-JFH-1, were prepared for all four HCV replicons. Wild-type (wt) or chimeric subgenomic RNAs were transfected into Huh7 cells and the luciferase activities of the transfected cells examined as described in the legend to Fig. 2B. The assays were performed three times independently, and data are presented as means and standard deviations for luciferase activity (RLU) at 24 h (white bars) and 48 h (gray bars) after transfection.

FIG. 5.

Analysis of transient replication of genomic chimeric HCV RNA. (A) Structures of full-length chimeric HCV RNAs. Each chimeric full-length construct was prepared by the insertion of the restricted fragments as indicated. The restriction enzyme recognition sites used for the plasmid constructions are indicated. C, ClaI; E, EcoT22I; B, BsrGI; S, StuI; X, XbaI; wt, wild type. (B) Northern blot analysis of total RNA prepared from cells transfected with transcribed genomic HCV RNA. Numbers of synthetic JFH-1 RNA (control RNA), RNA isolated from naïve cells (Huh7), and hours after transfection (4, 10, 24, 48, and 72) are indicated. Arrowheads indicate full-length HCV RNA (HCV RNA) and 28S rRNA (28S). A representative autoradiogram (6-h exposure) of three independent experiments is presented. (C) HCV core protein secretion from the RNA-transfected cells. Transcribed wild-type or chimeric full-length HCV RNAs (10 μg) were transfected into Huh7 cells. Culture medium was harvested at 4, 10, 24, 48, and 72 h after transfection. The amounts of core proteins in the harvested culture medium were measured using an HCV core enzyme-linked immunosorbent assay. The assays were performed five times independently, and data are presented as means and standard deviations.