RNA polymerase activity and specific RNA structure are required for efficient HCV replication in cultured cells

Abstract

We have previously reported that the NS3 helicase (N3H) and NS5B-to-3'X (N5BX) regions are important for the efficient replication of hepatitis C virus (HCV) strain JFH-1 and viral production in HuH-7 cells. In the current study, we investigated the relationships between HCV genome replication, virus production, and the structure of N5BX. We found that the Q377R, A450S, S455N, R517K, and Y561F mutations in the NS5B region resulted in up-regulation of J6CF NS5B polymerase activity in vitro. However, the activation effects of these mutations on viral RNA replication and virus production with JFH-1 N3H appeared to differ. In the presence of the N3H region and 3' untranslated region (UTR) of JFH-1, A450S, R517K, and Y561F together were sufficient to confer HCV genome replication activity and virus production ability to J6CF in cultured cells. Y561F was also involved in the kissing-loop interaction between SL3.2 in the NS5B region and SL2 in the 3'X region. We next analyzed the 3' structure of HCV genome RNA. The shorter polyU/UC tracts of JFH-1 resulted in more efficient RNA replication than J6CF. Furthermore, 9458G in the JFH-1 variable region (VR) was responsible for RNA replication activity because of its RNA structures. In conclusion, N3H, high polymerase activity, enhanced kissing-loop interactions, and optimal viral RNA structure in the 3'UTR were required for J6CF replication in cultured cells.

Figure 1. Relative HCV RNA polymerase activity of JFH-1 and J6CF wild-type and mutant RdRP.

HCV RdRP activity was measured using the purified HCV RdRP (Fig. S1A) and the average RdRP activity and the standard deviation (error bar) relative to that of JFH-1 RdRP wt were calculated from three independent experiments (Representative gel images are shown in Fig. S1B). The relative activity values are presented above the graph. SNF, A450S+S455N+Y561F; SKF, A450S+R517K+Y561F; NKF, S455N+R517K+Y561F.

Figure 2. Luciferase activity of J6CF backbone replicons containing substitutions in the NS5B region.

(A) Schematic structures of wt JFH-1 and J6CF constructs and the chimeric subgenomic replicons containing 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) Schematic diagram of the mutations introduced into J6/N3H+3′UTR-JFH1-Luc and J6/N3H+3′UTR-JFH1. (C) Replication activity of J6CF-based replicons. Subgenomic RNA was synthesized in vitro from wild-type or chimeric replicon constructs. Transcribed subgenomic RNA (5 µg) was then electroporated into HuH-7 cells and the cells harvested at 4, 24, and 48 h after transfection. The harvested cells were lysed, and the luciferase activity in the cell lysates was 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 the mean ± standard deviation for luciferase activity at 24 h (white bars) and 48 h (gray bars) after transfection. Asterisks indicate significant differences relative to the replication activity of J6/N3H+N5BX-JFH1 (p<0.05) at 48 h and the values represent the relative values against J6/N3H+N5BX-JFH1 at 48 h after transfection. SNF, A450S+S455N+Y561F; SKF, A450S+R517K+Y561F; SNKF, A450S+ S455N+R517K+Y561F.

Figure 3. Analysis of transient replication of genomic chimeric HCV RNA.

(A) The structure of full-length chimeric HCV RNA. Each chimeric full-length construct was prepared by the replacement of the indicated restricted fragments. 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) HCV core protein production in the culture medium from RNA-transfected cells. Transcribed wild-type or chimeric full-length HCV RNA (10 µg) was transfected into Huh-7.5.1 cells. Culture medium was harvested at 4, 24, 48, and 72 h after transfection. The amount of core protein in the harvested culture medium was measured using a HCV core chemiluminescence enzyme immunoassay (Lumipulse II HCV core assay). The assays were performed three times independently, and the data are presented as the mean ± standard deviation. Values in the right panel represent the relative core values against J6/N3H+N5BX-JFH1 at 72 h after transfection and infectious titers of the media from chimeric HCV RNA-transfected cells at 72h after transfection determined using Huh7.5.1 cells. SNF, A450S+S455N+Y561F; SKF, A450S+R517K+Y561F; SNKF, A450S+ S455N+R517K+Y561F.

Figure 4. Replication activity of J6CF-based replicons containing variants or substitutions.

(A) Comparison of the nucleotide sequence of 5BSL3.2 to 3′X of JFH-1 and J6CF. Boxes indicate nucleotide differences in VR and stop codon. Shaded boxes indicate non-synonymous variants in this region. 5BSL3.2, 5BSL3.3, Variable Region (VR), Poly U/UC tract, and 3′X tail are indicated by double-headed arrows in the figure. Stem-loop structures of VR (VRSL1 and VRSL2) are underlined. Asterisk; conserved nucleotides between JFH-1 and J6CF. (B, C, D) Replication activity of J6CF-based replicons. Five micrograms of in vitro synthesized RNA was electroporated into HuH-7 cells and the cells were harvested at 4, 24, and 48 h after transfection. The harvested cells were then lysed, and the luciferase activity in the cell lysates was measured. The assays were performed three times independently, and the results were expressed as luciferase activities (RLU). Data are presented as the mean ± standard deviation for luciferase activity at 24 h (white bars) and 48 h (gray bars) after transfection. (E) The predicted secondary structure of the VR. The RNA secondary structures of JFH-1, JFH-1 m3, J6CF, and J6CF m3 were predicated by Mfold. The stem-loop structure 1 (VRSL1) and 2 (VRSL2) are indicated. Nucleotide 9458 is circled and the mutated nucleotides are indicated by arrowheads. (F) Schematic structures of the 5BSL3.2 and X tail. The predicted stem loop structure of 5BSL3.2 and SL2 of 3′X of JFH-1 and J6CF strains are indicated. The sequences forming kissing interaction with 3′X SL2 are shaded. Codons encoding aa 561 and 571 are circled and the mutated sequences are indicated. The reported kissing-loop interactions are indicated by the connecting lines. The predicted interaction of the JFH-1 strain is indicated by the dotted connecting line.