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Background: Hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis C, end-stage liver diseases, and hepatocellular carcinoma. The development of infectious HCV cell culture systems primarily relied on the replication enhancement effect of adaptive mutations. Although the mode of action may vary, those adaptive mutations could direct the study of virus-host interactions required for efficient virus infection. We previously identified a substitution D559G in NS5B (RNA dependent RNA polymerase) critical for the replication of HCV genomes. In this study, we set out to study whether D559G-NS5B specifically interacted with some host factors crucial for HCV infection.Methods: Through mass spectrometry analysis of immunoprecipitation mixture of ectopically expressed wild-type and D559G-mutated NS5B, we identified candidate factors showing potential interactions with NS5B and D559G-NS5B. The requirement of selected host factor in HCV infection in vitro was demonstrated by gene knockout, overexpression, virus infection, and co-immunoprecipitation approaches.Results: From the results of immunoprecipitation and mass spectrometry analysis, we selected protein phosphatase 2 regulatory subunit B’delta (PPP2R5D) for further characterization. Co-immunoprecipitation confirmed that both wild-type and D559G NS5B proteins interacted with PPP2R5D, but the interaction between D559G-NS5B and PPP2R5D was more efficient. Silencing of PPP2R5D decreased HCV infection, and knockout of PPP2R5D nearly eliminated HCV infection in Huh7.5 cells. Transient and stably overexpression of PPP2R5D in PPP2R5D-knockout cells restored HCV infection to a level close to that seen for wild-type Huh7.5 cells. Conclusions: PPP2R5D is required for HCV infection in cultured hepatoma cells, and PPP2R5D may function through binding to HCV NS5B. The underlying mechanism of PPP2R5D in the complete HCV life cycle requires further investigation.

Currently, no vaccine to prevent hepatitis C virus (HCV) infection is available. A major challenge in developing an HCV vaccine is the high diversity of HCV sequences. The purpose of immunization with viral glycoproteins is to induce a potent and long-lasting cellular and humoral immune response. However, this strategy only achieves limited protection, and antigen selection plays a crucial role in vaccine design. In this study, we investigated the humoral immune responses induced by intraperitoneal injection of keyhole limpet hemocyanin conjugated with 4 highly conserved peptides, including amino acids [aa]317-325 from E1 and aa418-429, aa502-518, and aa685-693 from E2, or 3 peptides from hypervariable region 1 (HVR1) of E2, including the N terminus of HVR1 (N-HVR1, aa384-396), C terminus of HVR1 (C-HVR1, aa397-410), and HVR1 in BALB/c mice. The neutralizing activity against HCV genotypes 1-6 was assessed using the cell culture HCV (HCVcc) system. The results showed that the 4 conserved peptides efficiently induced antibodies with potent neutralizing activity against 3 or 4 genotypes. Antibodies induced by aa685-693 conferred potent protection (>50%) against genotypes 2, 4, and 5. Peptide N-HVR1 elicited antibodies with the most potent neutralization activities against 3 HCV genotypes: TNcc(1a), S52(3a), and ED43(4a). These findings suggested that peptides within HCV glycoproteins could serve as potent immunogens for vaccine design and development.

Extra‑hepatic manifestations are frequently observed in hepatitis C virus (HCV)‑infected patients; however the underlying mechanisms remain largely unknown. In the present study, the human glioblastoma SF268 cell line (the precise origin of the cell type is not clear) was infected with HCV using HCV‑positive serum, and viral replication was assessed by immunofluorescence, reverse transcription‑polymerase chain reaction (PCR), quantitative PCR and western blotting following infection. HCV core protein and HCV RNA were detected in HCV‑positive serum‑infected SF268 cells at day 4 post‑infection, while no infection was observed in cells exposed to HCV‑negative serum. The mean HCV RNA levels at day 4 post‑infection were up to 5.00 IU/ml log10; however, HCV RNA and immunostaining for core protein were negative when cultured to day 6 or longer. The data suggest that human glioblastoma SF268 cells were transiently infected with HCV. AKT serine/threonine kinase phosphorylation was also detected in HCV‑infected SF268 cells at day 4 post‑infection. To the best of our knowledge, this is the first demonstration that a human glioblastoma cell line can be infected with serum‑derived HCV. The results provide evidence that HCV infection can occur in cells of the central nervous system. Neurological disorder‑associated phosphoinositide 3‑kinase‑AKT signaling pathway was activated in parallel with HCV infection, suggesting that SF268 may serve as an in vitro model for investigating HCV‑nervous system cell interactions.

Genotype 1b strain Con1 represents an important reference in the study of hepatitis C virus (HCV). Here, we aimed to develop an advanced infectious Con1 recombinant. We found that previously identified mutations A1226G/F1464L/A1672S/Q1773H permitted culture adaption of Con1 Core-NS5A (C-5A) recombinant containing 5'UTR and NS5B-3'UTR from JFH1 (genotype 2a), thus acquired additional mutations L725H/F886L/D2415G. C-5A containing all seven mutations (C-5A_7m) replicated efficiently in Huh7.5 and Huh7.5.1 cells and had an increased infectivity in SEC14L2-expressing Huh7.5.1 cells. Incorporation of Con1 NS5B was deleterious to C-5A_7m, however Con1 5'UTR was permissive but attenuated the virus. Nucleotides G1, A4, and G35 primarily accounted for the viral attenuation without affecting RNA translation. C-5A_7m was inhibited dose-dependently by simeprevir and daclatasvir, and substitutions at A4, A29, A34, and G35 conferred resistance to miR-122 antagonism. The novel Con1 5'UTR-NS5A recombinant, adaptive mutations, and critical nucleotides described here will facilitate future studies of HCV culture systems and virus-host interaction.