Aponte C, Poncet D, Cohen J
Laboratoire de Virologie et Immunologie Moleculaires, Institut National de la Recherche Agronomique, Jouy en Josas, France.
J Virol. 1996 Feb;70(2):985-91. doi: 10.1128/JVI.70.2.985-991.1996.
Replication of the rotavirus genome involves two steps: (i) transcription and extrusion of transcripts and (ii) minus-strand RNA synthesis in viral complexes containing plus-strand RNA. In this study, we showed evidence for the importance of the viral nonstructural protein of rotavirus, NSP2, in the replication of viral RNAs. RNA-binding properties of NSP2 were tested by UV cross-linking in vivo (in rotavirus-infected MA104 cells and recombinant baculovirus-expressing NSP2-infected Sf9 cells). In rotavirus-infected cells, NSP2 is bound to the 11 double-stranded RNA genomic segments of rotavirus. Quantitative analysis (using hydrolysis by RNase A) is consistent with NSP2 being directly bound to partially replicated viral RNA. Using various monoclonal antibodies and specific antisera against the structural (VP1, VP2, and VP6) and nonstructural (NSP1, NSP2, NSP3, and NSP5) proteins, we developed a solid-phase assay for the viral replicase. In this test, we recovered a viral RNA-protein complex with replicase activity only with a monoclonal antibody directed against NSP2. Our results indicated that these viral complexes contain the structural proteins VP1, VP2, and VP6 and the nonstructural protein NSP2. Our results show that NSP2 is closely associated in vivo with the viral replicase.
（i）转录物的转录和挤出，以及（ii）在含有正链RNA的病毒复合物中合成负链RNA。在本研究中，我们证明了轮状病毒非结构蛋白NSP2在病毒RNA复制中的重要性。通过体内紫外线交联（在轮状病毒感染的MA104细胞和表达重组杆状病毒的NSP2感染的Sf9细胞中）测试NSP2的RNA结合特性。在轮状病毒感染的细胞中，NSP2与轮状病毒的11个双链RNA基因组片段结合。定量分析（使用核糖核酸酶A水解）与NSP2直接结合部分复制的病毒RNA一致。使用针对结构蛋白（VP1、VP2和VP6）和非结构蛋白（NSP1、NSP2、NSP3和NSP5）的各种单克隆抗体和特异性抗血清，我们开发了一种针对病毒复制酶的固相测定法。在该测试中，我们仅用针对NSP2的单克隆抗体回收了具有复制酶活性的病毒RNA-蛋白质复合物。我们的结果表明，这些病毒复合物包含结构蛋白VP1、VP2和VP6以及非结构蛋白NSP2。我们的结果表明，NSP2在体内与病毒复制酶密切相关。
