Takeda N, Kuhn R J, Yang C F, Takegami T, Wimmer E
J Virol. 1986 Oct;60(1):43-53. doi: 10.1128/JVI.60.1.43-53.1986.
An in vitro poliovirus RNA-synthesizing system derived from a crude membrane fraction of infected HeLa cells was used to analyze the mechanism of initiation of poliovirus plus-strand RNA synthesis. This system contains an activity that synthesizes the nucleotidyl proteins VPg-pU and VPg-pUpU. These molecules represent the 5'-terminal structure of nascent RNA molecules and of virion RNA. The membranous replication complex is also capable of synthesizing nucleotidyl proteins containing nine or more of the poliovirus 5'-proximal nucleotides as assayed by the formation of the RNase T1-resistant oligonucleotide VPg-pUUAAAACAGp or by fingerprint analysis of the in vitro-synthesized RNA. Incubation of preformed VPg-pUpU with unlabeled nucleoside triphosphates resulted in the formation of VPg-pUUAAAACAGp. This reaction, which appeared to be an elongation of VPg-pUpU, was stimulated by the addition of a soluble fraction (S-10) obtained from uninfected HeLa cells. Preformed VPg-pU could be chased into VPg-pUpU in the presence of UTP. Our data are consistent with a model that VPg-pU can function as a primer for poliovirus plus-strand RNA synthesis in the membranous replication complex and that the elongation reaction may be stimulated by a host cellular factor.
利用从感染的HeLa细胞粗膜部分获得的体外脊髓灰质炎病毒RNA合成系统，分析脊髓灰质炎病毒正链RNA合成起始机制。该系统含有一种能合成核苷酸蛋白VPg-pU和VPg-pUpU的活性。这些分子代表新生RNA分子和病毒粒子RNA的5'-末端结构。通过形成抗核糖核酸酶T1的寡核苷酸VPg-pUUAAAACAGp或对体外合成RNA进行指纹分析测定，膜复制复合物也能够合成含有九个或更多脊髓灰质炎病毒5'-近端核苷酸的核苷酸蛋白。将预先形成的VPg-pUpU与未标记的核苷三磷酸一起温育，导致形成VPg-pUUAAAACAGp。该反应似乎是VPg-pUpU的延伸，通过添加从未感染的HeLa细胞获得的可溶性部分（S-10）而受到刺激。在UTP存在下，预先形成的VPg-pU可以追踪到VPg-pUpU。我们的数据与一个模型一致，即VPg-pU可以作为膜复制复合物中脊髓灰质炎病毒正链RNA合成的引物，并且延伸反应可能受到宿主细胞因子的刺激。
