Komoto Satoshi, Kanai Yuta, Fukuda Saori, Kugita Masanori, Kawagishi Takahiro, Ito Naoto, Sugiyama Makoto, Matsuura Yoshiharu, Kobayashi Takeshi, Taniguchi Koki
Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi, Japan
Department of Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
J Virol. 2017 Oct 13;91(21). doi: 10.1128/JVI.00695-17. Print 2017 Nov 1.
The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two nonstructural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid-only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6-deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture, although the NSP6 protein seemed to promote efficient virus growth. Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmid-only-based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can hardly be addressed by conventional approaches.
对于多种病毒，已有利用重叠开放阅读框（ORF）从单个mRNA合成一种以上独特蛋白质的相关描述。轮状病毒基因组的第11节段编码两种非结构蛋白，即NSP5和NSP6。NSP6的开放阅读框存在于绝大多数轮状病毒株中，因此预计NSP6蛋白在病毒复制中具有一定功能。然而，目前尚无其在病毒复制周期中的功能或需求的直接证据。在此，利用最近建立的仅基于质粒的反向遗传学系统，该系统可从克隆的cDNA中完全拯救重组轮状病毒，我们构建了NSP6缺陷型病毒，以直接探讨其在病毒复制周期中的重要性。可构建出有活力的重组NSP6缺陷型病毒。NSP6缺陷型病毒的单步生长曲线和噬斑形成证实，尽管NSP6蛋白似乎能促进病毒高效生长，但NSP6表达对细胞培养中RVA复制的意义有限。轮状病毒是全球幼儿严重腹泻最重要的病原体之一。轮状病毒基因组由11个双链RNA节段组成，编码六种结构蛋白（VP1至VP4、VP6和VP7）和六种非结构蛋白（NSP1至NSP6）。尽管已赋予这12种病毒蛋白各自特定的功能，但NSP6在病毒复制周期中的作用仍不清楚。在本研究中，我们利用最近开发的仅基于质粒的反向遗传学系统证明，NSP6蛋白对细胞培养中的病毒复制并非必需。这种反向遗传学方法将成功应用于解答有关轮状病毒蛋白在复制和致病性方面作用的重大问题，而这些问题用传统方法几乎无法解决。
