A cell-free system for the synthesis of Sindbis virus subgenomic RNA: importance of the concentration of the initiating NTP.
A cell-free system for the synthesis of Sindbis virus subgenomic RNA: importance of the concentration of the initiating NTP.
复制标题
用于合成辛德比斯病毒亚基因组 RNA 的无细胞系统：起始 NTP 浓度的重要性。
DOI：
10.1016/j.virol.2005.07.007
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发表时间：
2005
期刊：
影响因子：
3.7
通讯作者：
Stollar,Victor
中科院分区：
文献类型：
--
作者：
Li,Mei-Ling;Lin,Yen-Huei;Stollar,Victor
我们在这里描述了一个体外系统模板依赖的起始和合成Sindbis病毒（SV）亚基因组（SG） RNA转录物。该系统的关键组成部分是(1)与SV基因组nt 7441 - nt 7772区域对应的负链启动子模板（相对于SG RNA转录起始位点nt 7598 - 157 - +175），以及(2)来自表达SV非结构蛋白P123和nsP4的重组痘苗病毒感染细胞的p15片段（P123的nsP2编码区含有突变，导致P123比正常加工更快）。我们的数据表明，SG RNA转录物具有预期的大小，正极性，并且在预期的位点启动。将+1 nt从A改变为G、U或C导致SG RNA转录物的合成减少。然而，在每种情况下，增加初始NTP的浓度将转录本的合成恢复到野生型水平。这是体外合成依赖于外源启动子模板的甲病毒SG RNA转录物的首次演示。因此，它将为研究SG RNA的合成是如何被调节的提供新的方法。
We describe here an in vitro system for template-dependent initiation and synthesis of a Sindbis virus (SV) subgenomic (SG) RNA transcript. The critical components of this system were (1) a minus-strand promoter-template corresponding to the region of the SV genome from nt 7441 to nt 7772 (−157 to +175 relative to the SG RNA transcription initiation site at nt 7598), and (2) a p15 fraction from cells infected with recombinant vaccinia viruses expressing the SV nonstructural proteins, P123 and nsP4 (the nsP2 coding region in P123 contained a mutation which results in more rapid than normal processing of P123). Our data indicate that the SG RNA transcript is of the expected size, of positive polarity, and is initiated at the expected site. Changing the +1 nt from A to G, U, or C resulted in decreased synthesis of the SG RNA transcript. However, in each case, increasing the concentration of the initiating NTP restored synthesis of the transcript to the wild-type level. This is the first demonstration of an in vitro synthesis of an alphavirus SG RNA transcript which is dependent on the addition of an exogenous promoter-template. As such, it will make possible new approaches for learning how the synthesis of SG RNA is regulated.