In vitro synthesis of Sindbis virus genomic and subgenomic RNAs: influence of nsP4 mutations and nucleoside triphosphate concentrations.
In vitro synthesis of Sindbis virus genomic and subgenomic RNAs: influence of nsP4 mutations and nucleoside triphosphate concentrations.
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辛德比斯病毒基因组和亚基因组 RNA 的体外合成：nsP4 突变和三磷酸核苷浓度的影响。
DOI：
10.1128/jvi.01561-09
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发表时间：
2010
影响因子：
5.4
通讯作者：
Stollar,Victor
中科院分区：
文献类型：
--
作者：
Li,Mei-Ling;Wang,Hongtao;Stollar,Victor
在辛德毕斯病毒感染的细胞中产生两种正链mRNA，基因组（G）RNA和亚基因组（SG）RNA。在感染野生型（wt）辛德比斯病毒的蚊子细胞中，后者的合成量超过前者;然而，在感染SVpzf或SVcpc的细胞中，G RNA的合成量多于SG RNA。利用体外系统研究了SVpzf和SVcpc突变对SG和G RNA合成的影响。我们的研究结果表明，在标准反应条件下，SG/G RNA的比例在体外反映了SG与G RNA的比例在感染的蚊子细胞。我们进一步观察到，在体外观察到的RNA模式不仅受到SVpzf和SVcpc突变的影响，而且还受到反应混合物中三磷酸核苷浓度的影响，并且将这些突变引入nsP 4和启动子/模板改变了所产生的SG和G RNA的相对量，这可能是通过选择启动子来实现的。我们的结论是，相对于SVpzf和SVcpc突变，它主要是SG启动子中的核苷酸变化，而不是nsP 4中的氨基酸变化，决定了SG/G RNA比的结果。此外，观察到SVpzf突变在最低浓度的UTP/CTP下增强SG RNA的体外合成，并且单个SVcpc突变在所测试的最低浓度的CTP下增强G RNA的合成。我们还鉴定了nsP 4中的三个Arg残基，R545，R546和R547，它们是合成G RNA而不是SG RNA所需的。
Two positive-strand mRNAs are made in Sindbis virus-infected cells, the genomic (G) RNA and the subgenomic (SG) RNA. In mosquito cells infected with wild-type (wt) Sindbis virus, the latter is made in excess over the former; however, in cells infected with SVpzf or SVcpc more G RNA is made than SG RNA. Use was made ofin vitrosystems to investigate the effects of the SVpzf and SVcpc mutations on the synthesis of SG and G RNAs. Our findings indicate that under standard reaction conditions, the SG/G RNA ratioin vitroreflects the ratio of SG to G RNA made in infected mosquito cells. We observed further that the RNA patterns seenin vitroare affected not only by the SVpzf and SVcpc mutations but also by the nucleoside triphosphate concentrations in the reaction mixtures and that introduction of these mutations into nsP4 and the promoter/template change the relative amounts of SG and G RNAs that are made, likely through the choice of promoter. We conclude that with respect to the SVpzf and SVcpc mutations, it is mainly the nucleotide changes in the SG promoter, not the amino acid changes in nsP4, that determine the SG/G RNA ratio that results. Further, it was observed that the SVpzf mutations enhance thein vitrosynthesis of SG RNA at the lowest concentrations of UTP/CTP and that the single SVcpc mutation enhances the synthesis of G RNA at the lowest concentrations of CTP tested. We also identified three Arg residues in nsP4, R545, R546, and R547, that are needed for the synthesis of G RNA but not SG RNA.