1887

Abstract

Hepatitis C virus (HCV) genotype 3 is widely distributed, and genotype 3-infected patients achieve a lower cure rate in direct-acting antiviral (DAA) therapy and are associated with a higher risk of hepatic steatosis than patients with other genotypes. Thus, the study of the virology and pathogenesis of genotype 3 HCV is increasingly relevant. Here, we developed a full-length infectious clone and a subgenomic replicon for the genotype 3a isolate, CH3a. From an infected serum, we constructed a full-length CH3a clone, however, it was nonviable in Huh7.5.1 cells. Next, we systematically adapted several intergenotypic recombinants containing Core-NS2 and 5′UTR-NS5A from CH3a, and other sequences from a replication-competent genotype 2 a clone JFH1. Adaptive mutations were identified, of which several combinations facilitated the replication of CH3a-JFH1 recombinants; however, they failed to adapt to the full-length CH3a and the recombinants containing CH3a NS5B. Thus, we attempted to separately adapt CH3a NS5B-3′UTR by constructing an intragenotypic recombinant using 5′UTR-NS5A from an infectious genotype 3a clone, DBN3acc, from which L3004P/M in NS5B and a deletion of 11 nucleotides (Δ11nt) downstream of the polyU/UC tract of the 3′UTR were identified and demonstrated to efficiently improve virus production. Finally, we combined functional 5′UTR-NS5A and NS5B-3′UTR sequences that carried the selected mutations to generate full-length CH3a with 26 or 27 substitutions (CH3acc), and both revealed efficient replication and virus spread in transfected and infected cells, releasing HCV of 10 f.f.u. ml. CH3acc was inhibited by DAAs targeting NS3/4A, NS5A and NS5B in a dose-dependent manner. The selected mutations permitted the development of subgenomic replicon CH3a-SGRep, by which L3004P, L3004M and Δ11nt were proven, together with a single-cycle virus production assay, to facilitate virus assembly, release, and RNA replication. CH3acc clones and CH3a-SGRep replicon provide new tools for the study of HCV genotype 3.

Funding
This study was supported by the:
  • Entrepreneurial Talent Team Award of Guangdong Province (Award 2016ZT06S252)
    • Principle Award Recipient: Yi-PingLi
  • National Basic Research Program of China (973 Program) (Award 2015CB554301)
    • Principle Award Recipient: Yi-PingLi
  • National Natural Science Foundation of China (Award 81901557)
    • Principle Award Recipient: PingYin
  • National Natural Science Foundation of China (Award 81971938)
    • Principle Award Recipient: Yi-PingLi
  • The Innovation Research Team for Basic and Clinical Studies on Chronic Liver Diseases of 2018 High-Level Health Teams of Zhuhai
    • Principle Award Recipient: Yi-PingLi
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/content/journal/jgv/10.1099/jgv.0.001704
2021-12-24
2022-01-28
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