The Respiratory Syncytial Virus M2-1 Protein Forms Tetramers and Interacts with RNA and P in a Competitive Manner
The Respiratory Syncytial Virus M2-1 Protein Forms Tetramers and Interacts with RNA and P in a Competitive Manner
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DOI：
10.1128/jvi.00335-09
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发表时间：
2009-07-01
影响因子：
5.4
通讯作者：
Eleouet, Jean-Francois
中科院分区：
文献类型：
--
作者：
Tran, Thi-Lan;Castagne, Nathalie;Eleouet, Jean-Francois
呼吸道合胞病毒（RSV）M2-1蛋白是病毒RNA聚合酶复合物的重要辅因子，并作为转录持续合成能力和抗终止因子发挥作用。M2-1以磷酸化或非磷酸化形式存在于感染细胞中，是一种RNA结合蛋白，也与病毒聚合酶复合物的一些其他组分相互作用。它包含一个CCCH基序，一个假定的锌结合结构域，是必不可少的M2-1功能，在N端。为了深入了解其结构组织，M2-1在大肠杆菌中作为重组蛋白产生，并通过使用谷胱甘肽S-转移酶（GST）标签纯化至>95%的均一性。GST-M2-1融合蛋白与细菌RNA共纯化，其可以通过高盐洗涤去除。圆二色谱分析表明，M2-1主要是α-螺旋。化学交联、动态光散射、沉降速度和电子显微镜分析得出结论，M2-1在微摩尔浓度下形成89 kDa的5.4S四聚体，直径与7.6 nm相似。通过使用一系列缺失突变体，M2-1的寡聚化结构域被定位到由氨基酸残基32至63组成的推定的α-螺旋。当在RSV微型基因组复制子系统中使用荧光素酶基因作为报告基因进行测试时，与野生型M2-1相比，缺乏该区域的M2-1缺失突变体显示出RNA转录的显著降低，表明M2-1寡聚化对于蛋白质的活性是必需的。我们还表明，该区域包括氨基酸残基59至178结合P和RNA的竞争性方式是独立的磷酸化状态的M2-1。
The respiratory syncytial virus (RSV) M2-1 protein is an essential cofactor of the viral RNA polymerase complex and functions as a transcriptional processivity and antitermination factor. M2-1, which exists in a phosphorylated or unphosphorylated form in infected cells, is an RNA-binding protein that also interacts with some of the other components of the viral polymerase complex. It contains a CCCH motif, a putative zinc-binding domain that is essential for M2-1 function, at the N terminus. To gain insight into its structural organization, M2-1 was produced as a recombinant protein in Escherichia coli and purified to >95% homogeneity by using a glutathione S-transferase (GST) tag. The GST-M2-1 fusion proteins were copurified with bacterial RNA, which could be eliminated by a high-salt wash. Circular dichroism analysis showed that M2-1 is largely alpha-helical. Chemical cross-linking, dynamic light scattering, sedimentation velocity, and electron microscopy analyses led to the conclusion that M2-1 forms a 5.4S tetramer of 89 kDa and similar to 7.6 nm in diameter at micromolar concentrations. By using a series of deletion mutants, the oligomerization domain of M2-1 was mapped to a putative alpha-helix consisting of amino acid residues 32 to 63. When tested in an RSV minigenome replicon system using a luciferase gene as a reporter, an M2-1 deletion mutant lacking this region showed a significant reduction in RNA transcription compared to wild-type M2-1, indicating that M2-1 oligomerization is essential for the activity of the protein. We also show that the region encompassing amino acid residues 59 to 178 binds to P and RNA in a competitive manner that is independent of the phosphorylation status of M2-1.