Development of hepatitis C virus genotype 3a cell culture system

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It has been proposed that the hepatitis C virus (HCV) NS4B protein triggers the membranous HCV replication compartment, but the underlying molecular mechanism is not fully understood. Here, we screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis and identified 202 host proteins. Subsequent screening of replicon cells with small interfering RNA identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. The interaction between PREB and NS4B was confirmed by immunoprecipitation, immunofluorescence, and proximity ligation assays. PREB colocalized with double-stranded RNA and the newly synthesized HCV RNA labeled with bromouridine triphosphate in HCV replicon cells. Furthermore, PREB shifted to detergent-resistant membranes (DRMs), where HCV replication complexes reside, in the presence of NS4B expression in Huh7 cells. However, a PREB mutant lacking the NS4B-binding region (PREBd3) could not colocalize with double-stranded RNA and did not shift to the DRM in the presence of NS4B. These results indicate that PREB locates at the HCV replication complex by interacting with NS4B. PREB silencing inhibited the formation of the membranous HCV replication compartment and increased the protease and nuclease sensitivity of HCV replicase proteins and RNA in DRMs, respectively. Collectively, these data indicate that PREB promotes HCV RNA replication by participating in the formation of the membranous replication compartment and by maintaining its proper structure by interacting with NS4B. Furthermore, PREB was induced by HCV infection in vitro and in vivo. Our findings provide new insights into HCV host cofactors. IMPORTANCEThe hepatitis C virus (HCV) protein NS4B can induce alteration of the endoplasmic reticulum and the formation of a membranous web structure, which provides a platform for the HCV replication complex. The molecular mechanism by which NS4B induces the membranous HCV replication compartment is not understood. We screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis, followed by screening with small interfering RNA. We identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. PREB is induced by HCV infection and recruited into the replication complex by interaction with NS4B. Recruited PREB promotes HCV RNA replication by participating in the formation of the membranous HCV replication compartment. To our knowledge, the effect of NS4B-binding protein on the formation of the membranous HCV replication compartment is newly described in this report. Our findings are expected to provide new insights into HCV host cofactors. H epatitis C virus (HCV) infects about 2% of the world's population and leads to several diseases, including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). HCV is the most common reason for patients requiring a liver transplant in developed countries and is thus a significant health burden worldwide (1). To date,...

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Prolactin Regulatory Element Binding Protein Is Involved in Hepatitis C Virus Replication by Interaction with NS4B

Kong

Fujimoto

Nakamura

et al. 2016

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“…Recent advances have made it possible to infect cells in culture with human-derived HCV isolates and observe genome replication, but new infectious particles still cannot be generated readily from clinical samples (6). The study of cell culture infectious HCV relies on a few relatively slow-growing infectious clones (7)(8)(9)(10)(11)(12)(13). These clones do not acquire growth-adaptive mutations rapidly during passaging despite the high error rate of the HCV polymerase, possibly because of the limited production of new infectious virions.…”

mentioning

confidence: 99%

A Library of Infectious Hepatitis C Viruses with Engineered Mutations in the E2 Gene Reveals Growth-Adaptive Mutations That Modulate Interactions with Scavenger Receptor Class B Type I

Zuiani

Chen

Schwarz

et al. 2016

While natural hepatitis C virus (HCV) infection results in highly diverse quasispecies of related viruses over time, mutations accumulate more slowly in tissue culture, in part because of the inefficiency of replication in cells. To create a highly diverse population of HCV particles in cell culture and identify novel growth-enhancing mutations, we engineered a library of infectious HCV with all codons represented at most positions in the ectodomain of the E2 gene. We identified many putative growth-adaptive mutations and selected nine highly represented E2 mutants for further study: Q412R, T416R, S449P, T563V, A579R, L619T, V626S, K632T, and L644I. We evaluated these mutants for changes in particle-to-infectious-unit ratio, sensitivity to neutralizing antibody or CD81 large extracellular loop (CD81-LEL) inhibition, entry factor usage, and buoyant density profiles. Q412R, T416R, S449P, T563V, and L619T were neutralized more efficiently by anti-E2 antibodies and T416R, T563V, and L619T by CD81-LEL. Remarkably, all nine variants showed reduced dependence on scavenger receptor class B type I (SR-BI) for infection. This shift from SR-BI usage did not correlate with a change in the buoyant density profiles of the variants, suggesting an altered E2-SR-BI interaction rather than changes in the virus-associated lipoprotein-E2 interaction. Our results demonstrate that residues influencing SR-BI usage are distributed across E2 and support the development of large-scale mutagenesis studies to identify viral variants with unique functional properties. IMPORTANCECharacterizing variant viruses can reveal new information about the life cycle of HCV and the roles played by different viral genes. However, it is difficult to recapitulate high levels of diversity in the laboratory because of limitations in the HCV culture system. To overcome this limitation, we engineered a library of mutations into the E2 gene in the context of an infectious clone of the virus. We used this library of viruses to identify nine mutations that enhance the growth rate of HCV. These growth-enhancing mutations reduced the dependence on a key entry receptor, SR-BI. By generating a highly diverse library of infectious HCV, we mapped regions of the E2 protein that influence a key virus-host interaction and provide proof of principle for the generation of large-scale mutant libraries for the study of pathogens with great sequence variability. H epatitis C virus (HCV) infection causes a major global health burden, as approximately 3% of the world's population is affected (1). Up to 30% of individuals with HCV will resolve infection spontaneously, whereas the majority develop chronic infections that can cause fibrosis, cirrhosis, and hepatocellular carcinoma (2). HCV is a member of the hepacivirus genus of the Flaviviridae family of RNA viruses and is a positive-sense, enveloped virus with a 9.6-kb genome. The genome comprises a single open reading frame encoding an approximately 3,000-amino-acid polyprotein flanked by 5=-and 3=-noncoding regions. HC...

“…Therefore, we used the long-term culture of cells transfected with J6CF/JFH-1 chimeric RNAs and obtained replication-competent cell culture-adapted viruses. This strategy of inducing adaptive mutations has been broadly used, and the established replication-competent HCV strains containing cell culture-adaptive mutations were as follows: TNcc (GT1a) (13), HCV-1cc (GT1a) (14), H77Ccc (GT1a) (14), a NC1 mutant (GT1b) (15), J6cc (GT2a) (16), a JFH-2 mutant (GT2a) (17), J8cc (GT2b) (16), DH8cc (GT2b) (18), DH10cc (GT2b) (18), and an S310 mutant (GT3a) (19).…”

Section: Discussionmentioning

confidence: 99%

“…To date, several HCV strains have been reported to be competent for infection and replication in cultured cells. However, all these clones, aside from JFH-1, require many adaptive mutations (13)(14)(15)(16)(17)(18)(19). The molecular clone J6CF was constructed from the consensus sequence of HCV isolated from a chimpanzee inoculated with an HCV-infected patient's plasma (20).…”

Section: Importancementioning

confidence: 99%

Amino Acid Mutations in the NS4A Region of Hepatitis C Virus Contribute to Viral Replication and Infectious Virus Production

Murayama

Sugiyama

Suzuki

et al. 2017

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Hepatitis C virus (HCV) strain JFH-1, which belongs to genotype 2a, replicates autonomously in cultured cells, whereas another genotype 2a strain, J6CF, does not. Previously, we found that replacement of the NS3 helicase and NS5B-to-3=X regions of J6CF with those of JFH-1 confers J6CF replication competence. In this study, we aimed to identify the minimum modifications within these genomic regions needed to establish replication-competent J6CF. We previously identified 4 mutations in the NS5B-to-3=X region that could be used instead of replacement of this region to confer J6CF replication competence. Here, we induced cell cultureadaptive mutations in J6CF by the long-term culture of J6CF/JFH-1 chimeras composed of JFH-1 NS5B-to-3=X or individual parts of this but not the NS3 helicase region. After 2 months of culture, efficient HCV replication and infectious virus production in chimeric RNA-transfected cells were observed, and several amino acid mutations in NS4A were identified in replicating HCV genomes. The introduction of NS4A mutations into the J6CF/JFH-1 chimeras enhanced viral replication and infectious virus production. Immunofluorescence microscopy demonstrated that some of these mutations altered the subcellular localization of the coexpressed NS3 protein and affected the interaction between NS3 and NS4A. Finally, introduction of the most effective NS4A mutation, A1680E, into J6CF contributed to its replication competence in cultured cells when introduced in conjunction with four previously identified adaptive mutations in the NS5B-to-3=X region. In conclusion, we identified an adaptive mutation in NS4A that confers J6CF replication competence when introduced in conjunction with 4 mutations in NS5B-to-3=X and established a replicationcompetent J6CF strain with minimum essential modifications in cultured cells. IMPORTANCEThe HCV cell culture system using the JFH-1 strain and HuH-7 cells can be used to assess the complete HCV life cycle in cultured cells. This cell culture system has been used to develop direct-acting antivirals against HCV, and the ability to use various HCV strains within this system is important for future studies. In this study, we aimed to establish a novel HCV cell culture system using another HCV genotype 2a strain, J6CF, which replicates in chimpanzees but not in cultured cells. We identified an effective cell culture-adaptive mutation in NS4A and established a replication-competent J6CF strain in cultured cells with minimum essential modifications. The described strategy can be used in establishing a novel HCV cell culture system, and the replication-competent J6CF clone composed of the minimum essential modifications needed for cell culture adaptation will be valuable as another representative of genotype 2a strains.