Raju R, Subramaniam S V, Hajjou M
Department of Microbiology, School of Medicine, Meharry Medical College, Nashville, Tennessee 37208, USA.
J Virol. 1995 Dec;69(12):7391-401. doi: 10.1128/JVI.69.12.7391-7401.1995.
Genetically engineered RNA transcripts coding for various Sindbis virus (SIN) genes were used to study structure and sequence requirements of RNA recombination in BHK cells. Three different groups of RNA transcripts were made: (i) RNAs which retain the ability to replicate and which carry sequences coding for either viral polymerase or viral structural proteins; (ii) RNAs which lack the complete 3' end of the SIN genome and thus are incapable of replicating; and (iii) RNAs which lack the complete 5' end of the SIN genome and also are incapable of replicating. BHK cells were transfected with specific combinations of these precursor RNAs, and virus production and RNA synthetic abilities of the released virus were determined. We demonstrate in vivo generation of infectious SIN by fusion of (i) replicative RNAs to nonreplicative RNAs and (ii) two nonreplicative RNA precursors. Both homologous and nonhomologous types of recombinations were observed. In the homologous type of recombination, a 694-nucleotide overlap at the crossover region of the first pair of precursors resulted in the addition of an A residue converting the UAG stop codon of nonstructural protein P4 to a UAA stop codon. In the nonhomologous type of recombination, the crossover sites contained deletion of up to 76 nucleotides from one of the precursors and complete preservation of junction sequence from the other precursor. This is also the first report that a cytoplasmic RNA virus can be generated from biologically nonreplicative RNA precursors. These results have implications for initiation of viral RNA synthesis and recombination between RNA viral genomes in general. We favor template switching as a mechanism for the fusion events described here and suggest inclusion of polymerase scanning of diverse nonreplicative RNAs as an inherent feature of the copy choice model of RNA recombination. Very importantly, the facile nature of RNA recombination occurring between nonreplicative RNA precursors should speed up the production and analysis of targeted mutants of SIN and possibly other RNA viruses.
利用编码各种辛德毕斯病毒（SIN）基因的基因工程RNA转录本，研究了BHK细胞中RNA重组的结构和序列要求。制备了三组不同的RNA转录本：（i）保留复制能力且携带编码病毒聚合酶或病毒结构蛋白序列的RNA；（ii）缺乏SIN基因组完整3'端因而无法复制的RNA；（iii）缺乏SIN基因组完整5'端且同样无法复制的RNA。用这些前体RNA的特定组合转染BHK细胞，并测定释放病毒的病毒产生情况和RNA合成能力。我们证明了通过（i）复制性RNA与非复制性RNA融合以及（ii）两种非复制性RNA前体融合，在体内产生感染性SIN。观察到了同源和非同源类型的重组。在同源重组类型中，第一对前体交叉区域的694个核苷酸重叠导致添加了一个A残基，将非结构蛋白P4的UAG终止密码子转换为UAA终止密码子。在非同源重组类型中，交叉位点包含一个前体中多达76个核苷酸的缺失以及另一个前体连接序列的完全保留。这也是关于可从生物学上非复制性RNA前体产生细胞质RNA病毒的首次报道。这些结果总体上对病毒RNA合成的起始以及RNA病毒基因组之间的重组具有启示意义。我们倾向于模板转换作为此处描述的融合事件的机制，并建议将对多种非复制性RNA的聚合酶扫描纳入RNA重组拷贝选择模型的固有特征。非常重要的是，非复制性RNA前体之间发生的RNA重组的简便性质应会加速SIN及可能其他RNA病毒靶向突变体的产生和分析。
