Dobos P
Department of Microbiology, College of Biological Science, University of Guelph, Ontario, Canada.
Virology. 1995 Apr 1;208(1):19-25. doi: 10.1006/viro.1995.1125.
The 94-kDa virion-associated RNA-dependent RNA polymerase (RdRp) is present in infectious pancreatic necrosis virus in two forms: (i) as a free polypeptide (VP1) and (ii) as a genome linked protein (VPg) (J. G. Calvert et al., 1991, J. Gen. Virol. 72, 2563-2567). VP1 was guanylylated in vitro by incubating purified virus in the presence of [alpha2P]GTP. During further incubation in an in vitro RNA polymerase reaction mixture (in the presence of unlabeled GTP), the radiolabeled VP1-pG complex was "chased" via nascent RNA strands and replicative intermediates to a VP1-dsRNA complex. Labeled VP1-pG was recovered from the intermediate as well as from the final reaction products by digestion with RNase A and RNase V1, a dsRNA-specific nuclease. Analysis of the reaction products indicated that only the plus strands of the two genome segments were being synthesized in vitro which remained base-paired to their templates. The results suggest that in vitro transcription by the virion RdRp is primed by VP1 and then proceeds via an asymmetric, semiconservative, strand-displacement mechanism.
94千道尔顿的病毒粒子相关RNA依赖性RNA聚合酶（RdRp）以两种形式存在于传染性胰腺坏死病毒中：（i）作为游离多肽（VP1），（ii）作为基因组连接蛋白（VPg）（J.G.卡尔弗特等人，1991年，《普通病毒学杂志》72卷，2563 - 2567页）。通过在[α2P]GTP存在下孵育纯化的病毒，VP1在体外被鸟苷酸化。在体外RNA聚合酶反应混合物中进一步孵育期间（在未标记的GTP存在下），放射性标记的VP1 - pG复合物通过新生RNA链和复制中间体被“追踪”到VP1 - dsRNA复合物。通过用RNase A和dsRNA特异性核酸酶RNase V1消化，从中间体以及最终反应产物中回收标记的VP1 - pG。对反应产物的分析表明，体外仅合成了两个基因组片段的正链，它们与模板保持碱基配对。结果表明，病毒粒子RdRp的体外转录由VP1引发，然后通过不对称、半保留、链置换机制进行。
