Magyar G, Chung H K, Dobos P
Department of Biology, Konkuk University, Seoul, Korea.
Virology. 1998 May 25;245(1):142-50. doi: 10.1006/viro.1998.9137.
The RNA polymerase VP1 of IPNV (a bisegmented dsRNA containing virus) is present in the virion both as a free polypeptide and as a genome-linked protein (VPg). Virion VP1 primes viral RNA synthesis in vitro (P. Dobos, 1995, Virology 208, 19-25), and here we present data which suggest that protein-primed RNA synthesis may also take place in infectious pancreatic necrosis virus (IPNV)-infected cells. Anti-VP1 serum immunoprecipitated several polypeptides larger than the 94-kDa VP1 of IPNV from [35S]methionine-labeled infected cell lysates. During denaturing, SDS-polyacrylamide gel electrophoresis these polypeptides formed a characteristic "ladder" which was resistant to alkaline phosphatase but sensitive to RNases, indicating that it consisted of VP1 polypeptides with oligoribonucleotides of various lengths attached to them. Probing the ladder with 5' and 3' end-specific, as well as plus-, or minus-strand-specific oligonucleotides revealed that they represent VP1 linked to 5' terminal sequences of genome segment A- and B-specific plus strands. Pulse-chase experiments in combination with two-dimensional polyacrylamide gel electrophoresis revealed that labeled VP1 could be chased to replicative intermediate, to ssRNA, to dsRNA, and eventually to virion VPg-dsRNA and that VP1 could be released from all these structures by RNase treatment. We suggest that these results are most compatible with the model where a VP1-pN structure acts as a primer for viral RNA synthesis in vivo, a mechanism that has been shown to occur in vitro.
传染性胰腺坏死病毒（IPNV，一种含双链RNA的双节段病毒）的RNA聚合酶VP1以游离多肽和基因组连接蛋白（VPg）的形式存在于病毒粒子中。病毒粒子VP1在体外可引发病毒RNA合成（P. 多博斯，1995年，《病毒学》208卷，第19 - 25页），在此我们提供的数据表明，蛋白质引发的RNA合成也可能发生在感染传染性胰腺坏死病毒（IPNV）的细胞中。抗VP1血清从[35S]甲硫氨酸标记的感染细胞裂解物中免疫沉淀出几种比IPNV的94 kDa VP1更大的多肽。在变性SDS - 聚丙烯酰胺凝胶电泳过程中，这些多肽形成了一个特征性的“梯状条带”，该条带对碱性磷酸酶有抗性，但对核糖核酸酶敏感，表明它由附着有不同长度寡核糖核苷酸的VP1多肽组成。用5'和3'末端特异性以及正链或负链特异性寡核苷酸探测该梯状条带，发现它们代表与基因组A节段和B节段特异性正链5'末端序列相连的VP1。脉冲追踪实验结合二维聚丙烯酰胺凝胶电泳表明，标记的VP1可以追踪到复制中间体→单链RNA→双链RNA，最终到病毒粒子VPg - 双链RNA，并且VP1可以通过核糖核酸酶处理从所有这些结构中释放出来。我们认为，这些结果与以下模型最为相符：VP1 - pN结构在体内作为病毒RNA合成的引物，这一机制已在体外得到证实。
