Khromykh A A, Meka H, Guyatt K J, Westaway E G
Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, University of Queensland, Herston Rd., Herston, Brisbane, QLD 4029, Australia.
J Virol. 2001 Jul;75(14):6719-28. doi: 10.1128/JVI.75.14.6719-6728.2001.
A possible role in RNA replication for interactions between conserved complementary (cyclization) sequences in the 5'- and 3'-terminal regions of Flavivirus RNA was previously suggested but never tested in vivo. Using the M-fold program for RNA secondary-structure predictions, we examined for the first time the base-pairing interactions between the covalently linked 5' genomic region (first ~160 nucleotides) and the 3' untranslated region (last ~115 nucleotides) for a range of mosquito-borne Flavivirus species. Base-pairing occurred as predicted for the previously proposed conserved cyclization sequences. In order to obtain experimental evidence of the predicted interactions, the putative cyclization sequences (5' or 3') in the replicon RNA of the mosquito-borne Kunjin virus were mutated either separately, to destroy base-pairing, or simultaneously, to restore the complementarity. None of the RNAs with separate mutations in only the 5' or only the 3' cyclization sequences was able to replicate after transfection into BHK cells, while replicon RNA with simultaneous compensatory mutations in both cyclization sequences was replication competent. This was detected by immunofluorescence for expression of the major nonstructural protein NS3 and by Northern blot analysis for amplification and accumulation of replicon RNA. We then used the M-fold program to analyze RNA secondary structure of the covalently linked 5'- and 3'-terminal regions of three tick-borne virus species and identified a previously undescribed additional pair of conserved complementary sequences in locations similar to those of the mosquito-borne species. They base-paired with DeltaG values of approximately -20 kcal, equivalent or greater in stability than those calculated for the originally proposed cyclization sequences. The results show that the base-pairing between 5' and 3' complementary sequences, rather than the nucleotide sequence per se, is essential for the replication of mosquito-borne Kunjin virus RNA and that more than one pair of cyclization sequences might be involved in the replication of the tick-borne Flavivirus species.
此前曾有人提出黄病毒RNA 5'和3'末端区域保守互补（环化）序列之间的相互作用在RNA复制中可能发挥作用，但从未在体内进行过测试。我们使用M-fold程序进行RNA二级结构预测，首次检测了一系列蚊媒黄病毒物种中5'基因组区域（最初的约160个核苷酸）和3'非翻译区（最后的约115个核苷酸）之间的碱基配对相互作用。碱基配对如先前提出的保守环化序列所预测的那样发生。为了获得预测相互作用的实验证据，对蚊媒库宁病毒复制子RNA中的假定环化序列（5'或3'）进行单独突变以破坏碱基配对，或同时突变以恢复互补性。仅在5'或仅在3'环化序列中具有单独突变的RNA在转染到BHK细胞后均无法复制，而在两个环化序列中同时具有补偿性突变的复制子RNA具有复制能力。这通过免疫荧光检测主要非结构蛋白NS3的表达以及通过Northern印迹分析检测复制子RNA的扩增和积累来得以证实。然后，我们使用M-fold程序分析了三种蜱传病毒物种5'和3'末端共价连接区域的RNA二级结构，并在与蚊媒病毒物种相似的位置鉴定出一对先前未描述的额外保守互补序列。它们的碱基配对自由能值约为 -20千卡，稳定性与最初提出的环化序列计算值相当或更高。结果表明，5'和3'互补序列之间的碱基配对而非核苷酸序列本身对于蚊媒库宁病毒RNA的复制至关重要，并且可能有不止一对环化序列参与蜱传黄病毒物种的复制。
