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
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丙型肝炎病毒基因型 3a 分离株的全长细胞培养感染性克隆和亚基因组复制子的开发
DOI：
10.1099/jgv.0.001704
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发表时间：
2021-01-01
影响因子：
3.8
通讯作者：
Li, Yi-Ping
中科院分区：
文献类型：
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作者：
Chen, Mingxiao;Xu, Yi;Li, Yi-Ping
丙型肝炎病毒(HCV3型)广泛分布，感染3型肝炎病毒的患者在直接作用抗病毒(DAA)治疗中治愈率较低，与其他基因型患者相比，发生肝脏脂肪变性的风险更高。因此，对3型丙型肝炎病毒的病毒学和致病机制的研究日益重要。在这里，我们为3a型分离株CH3a开发了一个全长感染性克隆和一个亚基因组复制子。从感染的血清中，我们构建了CH3a的全长克隆，然而，它在Huh7.5.1细胞中不能存活。接下来，我们系统地改编了几个基因间型重组体，其中包含来自CH3a的Core-NS2和5‘UTR-NS5A，以及来自复制能力强的基因2 a克隆JFH1的其他序列。鉴定了适应性突变，其中几种组合促进了CH3a-JFH1重组体的复制，但它们未能适应全长CH3a和含有CH3a NS5B的重组体。因此，我们试图通过从感染型3a克隆DBN3acc中使用5‘UTR-NS5A构建一个基因内重组体来分别适应CH3a NS5B-3’UTR，从中鉴定出NS5B中的L3004P/M和3‘UTRPolyU/UC道下游的11个核苷酸(Delta 11nt)的缺失，并证明该重组能有效地提高病毒产量。最后，我们将携带所选突变的功能性5‘UTR-NS5A和NS5B-3’UTR序列结合在一起，产生具有26或27个替换(CH3acc)的全长CH3a，两者都显示了有效的复制和病毒在感染细胞中的传播，释放了10(4.2)f.f.u的丙型肝炎病毒。Ml(-1)。针对NS3/4A、NS5A和NS5B的DAA对CH3acc的抑制作用呈剂量依赖关系。所选择的突变允许开发亚基因组复制子CH3a-SGRep，其中L3004P、L3004M和Delta 11nt被证明与单循环病毒生产试验一起，促进了病毒的组装、释放和RNA复制。CH3acc克隆和CH3a-SGRep复制子为研究丙型肝炎病毒3型提供了新的工具。
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 (Delta 11nt) 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(4.2) 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 Delta 11nt 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.