Regulation of alphavirus 26S mRNA transcription by replicase component nsP2.
Regulation of alphavirus 26S mRNA transcription by replicase component nsP2.
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DOI：
10.1099/0022-1317-79-2-309
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发表时间：
1998-02
期刊：
影响因子：
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通讯作者：
J. Suopanki;D. Sawicki;S. Sawicki;L. Kääriäinen
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文献类型：
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作者：
J. Suopanki;D. Sawicki;S. Sawicki;L. Kääriäinen
塞姆利基森林病毒（SFV）突变体ts4在合成亚基因组26S mRNA方面存在可逆的温度敏感缺陷。通过将病毒非结构蛋白nsP2编码序列从ts4转移到感染性SFV cDNA克隆（SFoto）中，鉴定出病毒非结构蛋白nsP2是26S合成的调控因子。测序鉴定出致病突变为C4038U，预测nsP2的氨基酸变化为M781T。从U到C的反向突变恢复了野生型表型。与同样表现出对温度敏感的Sindbis病毒nsP2突变体ts15、ts17、ts18、ts24和ts133相比，ts4和SFots4在温度变化后更快地降低了26S RNA的合成水平。在这些条件下，ts4和SFots4也表现出RFII+RFIII完全转化为RFI，并重新激活负链合成。转移到39℃后，ts4 nsP2从由15000 g （P15）膜沉积组成的粗RNA聚合酶制备中释放出来，剩余的未释放的nsP2能够与nsP1和nsP3以几乎等摩尔的比例交联。这支持了nsP2直接或间接结合26S RNA启动子的假设，并且它也是合成42S RNA +链的病毒复制酶的重要组成部分。在ts4突变体中，只有前者的活性对温度敏感。
Semliki Forest virus (SFV) mutant ts4 has a reversible temperature-sensitive defect in the synthesis the subgenomic 26S mRNA. The viral nonstructural protein nsP2 was identified as a regulator of 26S synthesis by transferring nsP2 coding sequences from ts4 into the infectious SFV cDNA clone (SFoto) to create SFots4. Sequencing identified the causal mutation as C4038U, predicting the amino acid change M781T in nsP2. A revertant was isolated in which a back mutation of U to C restored the wild-type phenotype. Compared to Sindbis virus nsP2 mutants ts15, ts17, ts18, ts24 and ts133, which also exhibit temperature-sensitive 26S RNA synthesis, ts4 and SFots4 reduced 26S RNA synthesis faster and to lower levels after temperature shift. Under these conditions, ts4 and SFots4 also displayed complete conversion of RFII+RFIII into RFI and reactivated minus-strand synthesis. After shift to 39 degrees C, ts4 nsP2 was released from a crude RNA polymerase preparation consisting of membranes sedimenting at 15,000 g (P15) and the remaining, unreleased nsP2 was capable of being cross-linked in almost equimolar ratio with nsP1 and nsP3. This supports the hypothesis that nsP2 binds directly or undirectly to the promoter for 26S RNA and that it is also an essential component of the viral replicase synthesizing 42S RNA plus strands. Only the former activity is temperature-sensitive in ts4 mutant.