Characterization of rotavirus NSP2/NSP5 interactions and the dynamics of viroplasm formation
Characterization of rotavirus NSP2/NSP5 interactions and the dynamics of viroplasm formation
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DOI：
10.1099/vir.0.19611-0
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发表时间：
2004-03-01
影响因子：
3.8
通讯作者：
Burrone, OR
中科院分区：
文献类型：
--
作者：
Eichwald, C;Rodriguez, JF;Burrone, OR
病毒颗粒是在轮状病毒感染细胞的细胞质中形成的离散结构，并构成病毒的复制机制。非结构蛋白NSP 2和NSP 5与其他病毒蛋白（包括聚合酶VP 1、VP 3和主要内核蛋白VP 2）一起定位于病毒载体中。NSP 2和NSP 5相互作用，激活NSP 5过度磷酸化和病毒质样结构（VLSs）的形成。我们已经使用NSP 2和NSP 5融合到增强型绿色荧光蛋白（EGFP），以研究这两种蛋白质在病毒感染的细胞中的定位，以及病毒质形成的动力学。随着感染后时间的推移，病毒颗粒的数量先是增加，然后减少，而每一个的面积增加，表明发生融合。NSP 2和一系列NSP 5突变体之间的相互作用进行了研究，使用两种不同的测定，酵母双杂交系统和体内结合/免疫沉淀测定。这两种方法给出了相当的结果，表明NSP 5的N-末端区域（33 aa）以及C-末端部分（aa 131-198）是与NSP 2结合所必需的。当分别融合到EGFP的N和C末端时，这两个区域能够赋予定位在病毒质中并与NSP 2形成VLS的能力。
Viroplasms are discrete structures formed in the cytoplasm of rotavirus-infected cells and constitute the replication machinery of the virus. The non-structural proteins NSP2 and NSP5 localize in viroplasms together with other viral proteins, including the polymerase VP1, VP3 and the main inner-core protein, VP2. NSP2 and NSP5 interact with each other, activating NSP5 hyperphosphorylation and the formation of viroplasm-like structures (VLSs). We have used NSP2 and NSP5 fused to the enhanced green fluorescent protein (EGFP) to investigate the localization of both proteins within viroplasms in virus-infected cells, as well as the dynamics of viroplasm formation. The number of viroplasms was shown first to increase and then to decrease with time post-infection, while the area of each one increased, suggesting the occurrence of fusions. The interaction between NSP2 and a series of NSP5 mutants was investigated using two different assays, a yeast two-hybrid system and an in vivo binding/immunoprecipitation assay. Both methods gave comparable results, indicating that the N-terminal region (33 aa) as well as the C-terminal part (aa 131-198) of NSP5 are required for binding to NSP2. When fused to the N and C terminus of EGFP, respectively, these two regions were able to confer the ability to localize in the viroplasm and to form VLSs with NSP2.