Development of recombinant hepatitis C virus with NS5A from strains of genotypes 1 and 2
Development of recombinant hepatitis C virus with NS5A from strains of genotypes 1 and 2
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DOI：
10.1016/j.bbrc.2011.05.144
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发表时间：
2011-07-08
影响因子：
3.1
通讯作者：
Kato, Takanobu
中科院分区：
文献类型：
--
作者：
Okamoto, Yuka;Masaki, Takahiro;Kato, Takanobu
丙型肝炎病毒（HCV）的非结构蛋白5A（NS 5A）在病毒的生命周期中发挥着多种多样的作用，是目前公认的抗病毒治疗的新靶点。为了建立具有各种株的NS 5A的HCV细胞培养系统，通过用基因型1（H77：1a和Con 1; 1b）和2（J6 CF：2a和MA; 2b）的株的NS 5A替换株JFH-1的NS 5A来产生重组病毒。所有这些重组病毒都能够复制和感染性病毒生产。与JFH-1野生型相比，用基因型1菌株的NS 5A替换JFH-1 NS 5A导致相似或略微降低的病毒产生，而用基因型2菌株的NS 5A替换增强了病毒产生。用CD 81阴性细胞系进行的单循环病毒生产测定显示，通过用基因型2毒株替换引起的有效病毒生产依赖于增强的病毒组装，并且NS 5A的C-末端中的取代是导致这种表型的原因。脉冲追踪分析表明，这些取代在C-末端的NS 5A可能与加速切割动力学在NS 5A-NS 5 B网站。使用具有NS 5A取代的重组病毒的该细胞培养系统，然后检查NS 5A抑制剂的抗病毒效果。基因型1和2的菌株之间对抑制剂的敏感性存在300至1000倍的差异。该系统不仅有助于更好地理解NS 5A在HCV生命周期中的株特异性作用，而且还能够评估NS 5A抑制剂的基因型和株依赖性。(C)2011 Elsevier Inc. All rights reserved.
Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) plays multiple and diverse roles in the viral lifecycle, and is currently recognized as a novel target for anti-viral therapy. To establish an HCV cell culture system with NS5A of various strains, recombinant viruses were generated by replacing NS5A of strain JFH-1 with those of strains of genotypes 1 (H77; 1 a and Con1; 1b) and 2 (J6CF: 2a and MA; 2b). All these recombinant viruses were capable of replication and infectious virus production. The replacement of JFH-1 NS5A with those of genotype 1 strains resulted in similar or slightly reduced virus production, whereas replacement with those of genotype 2 strains enhanced virus production as compared with JFH-1 wild-type. A single cycle virus production assay with a CD81-negative cell line revealed that the efficient virus production elicited by replacement with genotype 2 strains depended on enhanced viral assembly, and that substitutions in the C-terminus of NS5A were responsible for this phenotype. Pulse-chase assays revealed that these substitutions in the C-terminus of NS5A were possibly associated with accelerated cleavage kinetics at the NS5A-NS5B site. Using this cell culture system with NS5A-substituted recombinant viruses, the anti-viral effects of an NS5A inhibitor were then examined. A 300- to 1000-fold difference in susceptibility to the inhibitor was found between strains of genotypes 1 and 2. This system will facilitate not only a better understanding of strain-specific roles of NS5A in the HCV lifecycle, but also enable the evaluation of genotype and strain dependency of NS5A inhibitors. (C) 2011 Elsevier Inc. All rights reserved.