Khromykh Alexander A, Kondratieva Natasha, Sgro Jean-Yves, Palmenberg Ann, Westaway Edwin G
Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Clinical Medical Virology Centre, University of Queensland, Brisbane, Queensland, Australia.
J Virol. 2003 Oct;77(19):10623-9. doi: 10.1128/jvi.77.19.10623-10629.2003.
Point mutations that resulted in a substitution of the conserved 3'-penultimate cytidine in genomic RNA or the RNA negative strand of the self-amplifying replicon of the Flavivirus Kunjin virus completely blocked in vivo replication. Similarly, substitutions of the conserved 3'-terminal uridine in the RNA negative or positive strand completely blocked replication or caused much-reduced replication, respectively. The same preference for cytidine in the 3'-terminal dinucleotide was noted in reports of the in vitro activity of the RNA-dependent RNA polymerase (RdRp) for the other genera of Flaviviridae that also employ a double-stranded RNA (dsRNA) template to initiate asymmetric semiconservative RNA positive-strand synthesis. The Kunjin virus replicon results were interpreted in the context of a proposed model for initiation of RNA synthesis based on the solved crystal structure of the RdRp of phi6 bacteriophage, which also replicates efficiently using a dsRNA template with conserved 3'-penultimate cytidines and a 3'-terminal pyrimidine. A previously untested substitution of the conserved pentanucleotide at the top of the 3'-terminal stem-loop of all Flavivirus species also blocked detectable in vivo replication of the Kunjin virus replicon RNA.
导致黄病毒科库京病毒基因组RNA或自我扩增复制子的RNA负链中保守的3'-倒数第二个胞苷被取代的点突变完全阻断了体内复制。同样，RNA负链或正链中保守的3'-末端尿苷的取代分别完全阻断了复制或导致复制大幅减少。在黄病毒科其他属的RNA依赖性RNA聚合酶（RdRp）的体外活性报告中也注意到对3'-末端二核苷酸中胞苷的相同偏好，这些属也使用双链RNA（dsRNA）模板启动不对称半保留RNA正链合成。库京病毒复制子的结果是在基于phi6噬菌体RdRp的解析晶体结构提出的RNA合成起始模型的背景下进行解释的，phi6噬菌体也使用具有保守的3'-倒数第二个胞苷和3'-末端嘧啶的dsRNA模板高效复制。所有黄病毒属物种3'-末端茎环顶部保守五核苷酸的先前未经测试的取代也阻断了库京病毒复制子RNA在体内的可检测复制。
