Reovirus σNS protein is required for nucleation of viral assembly complexes and formation of viral inclusions
Reovirus σNS protein is required for nucleation of viral assembly complexes and formation of viral inclusions
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DOI：
10.1128/jvi.75.3.1459-1475.2001
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发表时间：
2001-02-01
影响因子：
5.4
通讯作者：
Dermody, TS
中科院分区：
文献类型：
--
作者：
Becker, MM;Goral, MI;Dermody, TS
哺乳动物呼肠孤病毒的子代病毒粒子聚集在感染细胞的细胞质中的离散位置，称为病毒包涵体。对温度敏感(Ts)突变病毒的研究表明，非结构蛋白Sigma NS和核心蛋白MU2是合成双链(DS)RNA所必需的，双链RNA是发生在病毒组装部位的一个过程。我们使用共聚焦免疫荧光显微镜和ts突变呼肠孤病毒来确定Sigma NS和MU2在病毒包涵体形成中的作用。在被感染的细胞中，野生型呼肠孤病毒、Sigma NS和MU2共同定位于与病毒包涵体相对应的大的核周结构。在不允许的温度下感染Sigma NS-突变病毒tsE320的细胞中，Sigma NS弥漫分布在细胞质中，MU2存在于不像病毒包涵体的小的点状病灶中。在不允许的温度下感染MU2突变病毒tsH11.2的细胞中，MU2弥漫分布在细胞质和细胞核中。然而，Sigma NS定位于细胞质中包含其他病毒蛋白的离散结构，在形态上与感染呼肠孤病毒的细胞中看到的病毒包涵体无法区分。在一段时间内对感染呼肠孤病毒的细胞的检查表明，Sigma NS对病毒包涵体的定位先于MU2。这些发现表明，含有Sigma NS的病毒RNA-蛋白质复合体使包括MU2在内的其他病毒蛋白被招募到病毒复制位点以开始dsRNA合成。
Progeny virions of mammalian reoviruses are assembled in the cytoplasm of infected cells at discrete sites termed viral inclusions. Studies of temperature-sensitive (ts) mutant viruses indicate that nonstructural protein sigma NS and core protein mu2 are required for synthesis of double-stranded (ds) RNA, a process that occurs at sites of viral assembly. We used confocal immunofluorescence microscopy and ts mutant reoviruses to define the roles of sigma NS and mu2 in viral inclusion formation. In cells infected,vith wild-type (wt) reovirus, sigma NS and mu2 colocalize to large, perinuclear structures that correspond to viral inclusions. In cells infected at a nonpermissive temperature with sigma NS-mutant virus tsE320, sigma NS is distributed diffusely in the cytoplasm and mu2 is contained in small, punctate foci that do not resemble viral inclusions. In cells infected at a nonpermissive temperature with mu2-mutant virus tsH11.2, mu2 is distributed diffusely in the cytoplasm and the nucleus. However, sigma NS localizes to discrete structures in the cytoplasm that contain other viral proteins and are morphologically indistinguishable from viral inclusions seen in cells infected with wt reovirus. Examination of cells infected with wt reovirus over a time course demonstrates that sigma NS precedes mu2 in localization to viral inclusions. These findings suggest that viral RNA-protein complexes containing sigma NS nucleate sites of viral replication to which other viral proteins, including mu2, are recruited to commence dsRNA synthesis.