Reverse Genetics System for a Human Group A Rotavirus
Reverse Genetics System for a Human Group A Rotavirus
复制标题
DOI：
10.1128/jvi.00963-19
复制
发表时间：
2020-01-01
影响因子：
5.4
通讯作者：
Kobayashi, Takeshi
中科院分区：
文献类型：
--
作者：
Kawagishi, Takahiro;Nurdin, Jeffery A.;Kobayashi, Takeshi
A组轮状病毒（RV）是全球婴幼儿急性胃肠炎的主要原因。最近，我们建立了一个完全基于质粒的猴RV株SA 11的反向遗传学系统。尽管该系统足够稳健以产生包括人RV基因区段的重组RV，并且使得能够更好地理解动物和人RV毒株之间的生物学差异，但是需要用于人RV毒株的完整反向遗传学系统。在这里，我们建立了一个基于质粒的反向遗传学系统的G4 P [8]人RV株Odelia。该技术用于在人和猿猴RV毒株之间产生一组单克隆抗体病毒，其所有11个基因片段证明人和猿猴RV毒株之间完全相容。此外，我们产生的重组病毒缺乏的病毒非结构蛋白NSP 1的C-末端区域，并使用它来定义在病毒复制过程中的NSP 1和它的靶蛋白β-transducin重复包含蛋白（β-TrCP）之间的相互作用的生物学功能。虽然缺乏C-末端13个氨基酸的NSP 1截短突变体显示出较低的β-TrCP降解活性，但其复制效率与野生型病毒一样。相比之下，缺失NSP 1的C-末端166个氨基酸的截短突变体复制较差，表明NSP 1的C-末端区域在病毒复制中起关键作用。这里报道的系统将允许产生工程重组病毒窝藏所需的突变，增加我们对人类RV的分子生物学的理解，并促进新的治疗和疫苗的开发。重要反向遗传学，一种用于产生病毒的克隆cDNA的方法，增加了我们对病毒生物学的理解。世界范围内的研究导致了一个完全基于质粒的反向遗传学系统的猴RV实验室菌株的发展。尽管该技术允许产生基因修饰的重组RV，但动物和人RV之间的生物学差异意味着仍然需要用于人RV毒株的反向遗传学系统。在这里，我们描述了一个反向遗传学系统的高产人类RV菌株Odelia，复制效率高，适合在体外分子研究。猴和人RV株之间的单重突变病毒和由拯救系统产生的NSP 1突变病毒使得能够研究病毒基因片段的生物学功能。这种人RV反向遗传学系统将促进RV生物学的研究和疫苗和载体的开发。
Group A rotavirus (RV) is a major cause of acute gastroenteritis in infants and young children worldwide. Recently, we established an entirely plasmid-based reverse genetics system for simian RV strain SA11. Although that system was robust enough to generate reassortant RVs, including human RV gene segments, and enabled better understanding of the biological differences between animal and human RV strains, a complete reverse genetics system for human RV strains is desirable. Here, we established a plasmid-based reverse genetics system for G4P[8] human RV strain Odelia. This technology was used to generate a panel of monoreassortant viruses between human and simian RV strains for all of the 11 gene segments demonstrating full compatibility between human and simian RV strains. Furthermore, we generated recombinant viruses lacking the C-terminal region of the viral nonstructural protein NSP1 and used it to define the biological function of the interaction between NSP1 and its target protein beta-transducin repeat-containing protein (beta-TrCP) during viral replication. While the NSP1 truncation mutant lacking the C-terminal 13 amino acids displayed lower beta-TrCP degradation activity, it replicated as efficiently as the wildtype virus. In contrast, the truncation mutant lacking the C-terminal 166 amino acids of NSP1 replicated poorly, suggesting that the C-terminal region of NSP1 plays critical roles in viral replication. The system reported here will allow generation of engineered recombinant virus harboring desired mutations, increase our understanding of the molecular biology of human RV, and facilitate development of novel therapeutics and vaccines.IMPORTANCE Reverse genetics, an approach used to generate viruses from cloned cDNA, has increased our understanding of virus biology. Worldwide research led to the development of an entirely plasmid-based reverse genetics system for the simian RV laboratory strain. Although the technique allows generation of gene-modified recombinant RVs, biological differences between animal and human RVs mean that reverse genetics systems for human RV strains are still needed. Here, we describe a reverse genetics system for the high-yield human RV strain Odelia, which replicates efficiently and is suitable for in vitro molecular studies. Monoreassortant viruses between simian and human RV strains and NSP1 mutant viruses generated by the rescue system enabled study of the biological functions of viral gene segments. This human RV reverse genetics system will facilitate study of RV biology and development of vaccines and vectors.