1. Academic Validation
  2. Development of full-length cell-culture infectious clone and subgenomic replicon for a genotype 3a isolate of hepatitis C virus

Development of full-length cell-culture infectious clone and subgenomic replicon for a genotype 3a isolate of hepatitis C virus

  • J Gen Virol. 2021 Dec;102(12). doi: 10.1099/jgv.0.001704.
Mingxiao Chen 1 Yi Xu 1 Ni Li 2 Ping Yin 3 Qing Zhou 2 Shengjun Feng 2 Tiantian Wu 2 Lai Wei 4 Haihe Wang 5 Yongshui Fu 6 Yi-Ping Li 2 7
Affiliations

Affiliations

  • 1 Joint Program in Pathology, Department of Internal Medicine, Guangzhou Women and Children's Medical Center, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510623, PR China.
  • 2 Institute of Human Virology, Zhongshan School of Medicine, and Key Laboratory of Tropical Disease Control of Ministry of Education, Sun Yat-sen University, Guangzhou 510080, PR China.
  • 3 Department of Clinical Laboratory, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, PR China.
  • 4 Beijing Tsinghua Changgung Hospital, Tsinghua University, Beijing 102218, PR China.
  • 5 Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, PR China.
  • 6 Guangzhou Blood Center, Guangzhou 510095, PR China.
  • 7 Department of Infectious Diseases, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai 519000, PR China.
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 104.2 f.f.u. ml-1. 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.

Keywords

RNA replication; genotype; hepatitis C virus; infectious clone; mutations; replicon.

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Products
  • Cat. No.
    Product Name
    Description
    Target
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  • HY-15005
    99.99%, HCV 抑制剂
    HCV