Random Insertion Mutagenesis of Sindbis Virus Nonstructural Protein 2 and Selection of Variants Incapable of Downregulating Cellular Transcription
Random Insertion Mutagenesis of Sindbis Virus Nonstructural Protein 2 and Selection of Variants Incapable of Downregulating Cellular Transcription
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DOI：
10.1128/jvi.00850-09
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发表时间：
2009-09-15
影响因子：
5.4
通讯作者：
Frolova, Elena I.
中科院分区：
文献类型：
--
作者：
Frolov, Ilya;Garmashova, Natalia;Frolova, Elena I.
辛德毕斯病毒非结构蛋白2（SINV nsP2）是病毒致病和下调病毒诱导的细胞反应的重要决定因素。这种蛋白质有效地抑制细胞信使和核糖体RNA的转录，因此能够抑制其产物参与抗病毒反应发展的基因的活化。甲病毒nsP2具有许多预测的功能结构域，其中一些通过晶体结构证实。我们目前的研究表明，没有一个假定的或已知的结构域单独或其组合能够发挥转录抑制作用。通过使用随机的转座子介导的诱变，我们产生了具有短肽插入的SINV nsP2变体的文库，并选择了那些失去抑制细胞转录和引起细胞病变效应的能力的变体。在三个不同功能的nsP2结构域中发现了废除蛋白质核功能的插入。一些突变的蛋白变体保留了病毒基因组复制所需的酶功能。这些病毒能够在干扰素（IFN）信号传导缺陷的细胞中有效、生产性复制，但被减毒并且不能在具有完整I型IFN应答的细胞中传播。这些结果揭示了SINV nsP2结构及其结构域相互作用的新信息。
Sindbis virus nonstructural protein 2 (SINV nsP2) is an important determinant of virus pathogenesis and downregulation of virus-induced cell response. This protein efficiently inhibits transcription of cellular messenger and ribosomal RNAs and, thus, is capable of inhibiting the activation of genes whose products are involved in development of the antiviral response. Alphavirus nsP2 has a number of predicted functional domains, some of which were confirmed by crystal structure. Our current study demonstrated that none of the putative or known structural domains alone or their combinations was capable of functioning in transcription inhibition. By using random, transposon-mediated mutagenesis, we generated a library of SINV nsP2 variants having short peptide insertions and selected those that lost the ability to inhibit cellular transcription and cause a cytopathic effect. Insertions abrogating the nuclear functions of the protein were found in the three different functional nsP2 domains. Some of the mutated protein variants retained the enzymatic functions required for replication of the viral genome. Such viruses were capable of efficient, productive replication in cells defective in interferon (IFN) signaling but were attenuated and incapable of spreading in cells with an intact type I IFN response. These results revealed new information about the structure of SINV nsP2 and interaction of its domains.