4月30日，Plos Pathogens在线发表了上海巴斯德所丰田哲也研究组关于丙型肝炎病毒体外复制机制的最新研究成果。这项研究由丰田哲也研究组的研究人员与日本国立感染症研究室的Takaji Wakita研究室共同完成。
由于体外感染系统模型有限，丙型肝炎病毒（HCV）领域的科学研究进展受到限制。到目前为止，只有JFH1（2a）株病毒可以在体外培养的Huh7细胞中复制并产生具有感染性的病毒。为了找到JFH1复制的机制，研究人员首先比较了JFH1，HCR6（1b）和J6CF（2a）三株病毒RNA聚合酶的结构与生化特性等差异，并找到了能激活HCR6和J6CF聚合酶活性的JFH1特异性的氨基酸位点。在含有JFH1 N3H和3’ UTR区域时，在J6CF的RNA聚合酶中引入A450S，R517K和Y561F三个突变就可以使J6CF基因组在培养的细胞中复制并产生子代病毒粒子。研究人员在深入分析了其机制后，发现Y561F位于NS5B的编码区5BSL3.2（CRE）内，突变后，使得CRE和3’XSL2之间的配对区域延伸了。随后，经过分析比较了HCV病毒RNA基因组的3’UTR结构，发现JFH1的polyU/UC比J6CF的短，更有利于病毒在体外的高效复制。此外，位于JFH1 3’UTR可变区的9458G是形成RNA复制所需要的特定RNA结构所必不可少的。
在该研究中，研究人员分析了HCV基因组复制，子代病毒的活性和N5BX结构之间的关系。发现JFH1的NSH具有较高的聚合酶活性，增强了的RNA二级结构配对和最适合的3’UTR RNA结构是J6CF在体外细胞中复制所必不可少的，即HCV病毒在体外的高效复制需要较高的RNA聚合酶活性和特异性的RNA结构。
这项研究得到了科技部“973”和国家科技重大专项“艾滋病和病毒性肝炎等传染病防控”项目的支持。
原文出处：
PLoS Pathogens doi:10.1371/journal.ppat.1000885
RNA Polymerase Activity and Specific RNA Structure Are Required for Efficient HCV Replication in Cultured Cells
Asako Murayama1#, Leiyun Weng2#, Tomoko Date1, Daisuke Akazawa1,3, Xiao Tian2, Tetsuro Suzuki1, Takanobu Kato1, Yasuhito Tanaka4, Masashi Mizokami5, Takaji Wakita1*, Tetsuya Toyoda2*
1 Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan, 2 Unit of Viral Genome Regulation, Key Laboratory of Molecular Virology " Immunology, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, People's Republic of China, 3 Pharmaceutical Research Lab, Toray Industries, Inc., Kanagawa, Japan, 4 Department of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medical Sciences, Aichi, Japan, 5 Research Center for Hepatitis and Immunology, Kohnodai Hospital, International Medical Center of Japan, Chiba, Japan
We have previously reported that the NS3 helicase (N3H) and NS5B-to-3′X (N5BX) regions are important for the efficient replication of hepatitis C virus (HCV) strain JFH-1 and viral production in HuH-7 cells. In the current study, we investigated the relationships between HCV genome replication, virus production, and the structure of N5BX. We found that the Q377R, A450S, S455N, R517K, and Y561F mutations in the NS5B region resulted in up-regulation of J6CF NS5B polymerase activity in vitro. However, the activation effects of these mutations on viral RNA replication and virus production with JFH-1 N3H appeared to differ. In the presence of the N3H region and 3′ untranslated region (UTR) of JFH-1, A450S, R517K, and Y561F together were sufficient to confer HCV genome replication activity and virus production ability to J6CF in cultured cells. Y561F was also involved in the kissing-loop interaction between SL3.2 in the NS5B region and SL2 in the 3′X region. We next analyzed the 3′ structure of HCV genome RNA. The shorter polyU/UC tracts of JFH-1 resulted in more efficient RNA replication than J6CF. Furthermore, 9458G in the JFH-1 variable region (VR) was responsible for RNA replication activity because of its RNA structures. In conclusion, N3H, high polymerase activity, enhanced kissing-loop interactions, and optimal viral RNA structure in the 3′UTR were required for J6CF replication in cultured cells.
