Hajjou M, Hill K R, Subramaniam S V, Hu J Y, Raju R
Department of Microbiology, School of Medicine, Meharry Medical College, Nashville, Tennessee 37208, USA.
J Virol. 1996 Aug;70(8):5153-64. doi: 10.1128/JVI.70.8.5153-5164.1996.
The mechanism of RNA-RNA recombination at the 3' nontranslated region (3'NTR) of the Sindbis virus (SIN) genome was studied by using nonreplicative RNA precursors. The 11.7-kb SIN genome was transcribed in vitro as two nonoverlapping RNA fragments. RNA-1 contained the entire 11.4-kb protein coding sequence of SIN and also carried an additional 1.8-kb nonviral sequence at its 3' end. RNA-2 carried the remaining 0.26 or 0.3 kb of the SIN genome containing the 3'NTR. Transfection of these two fragments into BHK cells resulted in vivo RNA-RNA recombination and release of infectious SIN recombinants. Eighteen plaque-purified recombinant viruses were sequenced to precisely map the RNA-RNA crossover sites at the 3'NTR. Sixteen of the 18 recombinants were found to be genetically heterogeneous at the 3'NTR. Two major clustered sites within the 3'NTR of RNA-2 were found to be fused to multiple locations on the nonviral sequence of RNA-1, resulting in insertions of 10 to 1,085 nucleotides at the 3'NTR. Sequence analysis of crossover sites suggested only limited homology and heteroduplex-forming capability between substrate RNAs. Analysis of additional 23 recombinant viruses generated by mutagenized donor and acceptor templates supports the occurrence of recombination hot spots on donor templates. Introduction of a 17-nucleotide rudimentary replicase recognition signal in the acceptor template alone did not induce the polymerase to reinitiate at the 17-nucleotide signal. Interestingly, deletion of a 24-nucleotide hot spot locus on the donor template abolished crossover events at one of the two sites and allowed the polymerase to reinitiate at the 17-nucleotide replicase recognition signal inserted at the acceptor template. The possible roles of RNA-protein and RNA-RNA interactions in the differential regulation of apparent pausing, template selection, and reinitiation are discussed.
利用非复制性RNA前体研究了辛德毕斯病毒（SIN）基因组3'非翻译区（3'NTR）的RNA-RNA重组机制。11.7 kb的SIN基因组在体外转录为两个不重叠的RNA片段。RNA-1包含SIN完整的11.4 kb蛋白质编码序列，并且在其3'端还携带一个额外的1.8 kb非病毒序列。RNA-2携带SIN基因组剩余的0.26或0.3 kb，包含3'NTR。将这两个片段转染到BHK细胞中导致体内RNA-RNA重组并释放出有感染性的SIN重组体。对18个噬斑纯化的重组病毒进行测序，以精确绘制3'NTR处的RNA-RNA交叉位点。18个重组体中的16个在3'NTR处基因异质。发现RNA-2的3'NTR内的两个主要聚类位点与RNA-1的非病毒序列上的多个位置融合，导致在3'NTR处插入10至1085个核苷酸。交叉位点的序列分析表明底物RNA之间只有有限的同源性和异源双链体形成能力。对由诱变的供体和受体模板产生的另外23个重组病毒的分析支持了供体模板上重组热点的存在。仅在受体模板中引入17个核苷酸的基本复制酶识别信号不会诱导聚合酶在17个核苷酸信号处重新起始。有趣的是，删除供体模板上一个24个核苷酸的热点位点消除了两个位点之一的交叉事件，并允许聚合酶在受体模板中插入的17个核苷酸复制酶识别信号处重新起始。讨论了RNA-蛋白质和RNA-RNA相互作用在明显停顿、模板选择和重新起始的差异调节中的可能作用。
