Identification of Functional Domains in Reovirus Replication Proteins μNS and μ2
Identification of Functional Domains in Reovirus Replication Proteins μNS and μ2
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DOI：
10.1128/jvi.01495-08
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发表时间：
2009-04-01
影响因子：
5.4
通讯作者：
Dermody, Terence S.
中科院分区：
文献类型：
--
作者：
Kobayashi, Takeshi;Ooms, Laura S.;Dermody, Terence S.
哺乳动物呼肠孤病毒是无包膜颗粒，含有 10 个双链 RNA (dsRNA) 基因片段的基因组。呼肠孤病毒复制发生在病毒包涵体内，病毒包涵体是由病毒非结构蛋白和结构蛋白形成的特殊非膜细胞质细胞器。尽管这些结构是呼肠孤病毒生命周期中几个主要事件的位点，包括 dsRNA 合成、基因片段分类和基因组衣壳化，但包涵体内病毒体形态发生的生化机制尚未阐明，因为包涵体解剖和功能组织仍有很多未知之处。为了更好地了解内含物如何支持病毒复制，我们使用 RNA 干扰 (RNAi) 和反向遗传学来定义两种内含物相关蛋白 mu NS 和 mu 2 的功能域，它们是内含物发育和病毒复制所必需的相互作用伙伴。去除与 mu 2 或另一种 mu NS 结合蛋白 mu NS 关联所需的 mu NS N 端序列，阻止了 mu NS 支持病毒复制而不影响包涵体形成的能力，表明 mu NS-mu 2 和 mu NS-sigma NS 相互作用对于包涵体功能是必需的，但不是建立所必需的。相比之下，在 mu NS C 末端区域引入变化，包括形成假定的寡聚化结构域的序列，可以阻止包涵体形成以及病毒复制。 mu 2 的突变分析揭示了病毒复制对完整核苷酸/RNA 三磷酸酶结构域和符合核定位基序的碱性氨基酸残基 N 端簇的关键依赖性。 mu 2 中的另一个结构域控制病毒包涵体与微管结合的能力，从而调节包涵体形态（球状或丝状）。然而，包涵体形态改变的病毒变体表现出相同的复制效率。这些研究揭示了包涵蛋白 mu NS 和 mu 2 的模块化功能组织，定义了这些蛋白质中特定氨基酸序列和基序对病毒复制的重要性，并证明了基于互补 RNAi 的反向遗传方法在呼肠孤病毒复制蛋白研究中的实用性。
Mammalian reoviruses are nonenveloped particles containing a genome of 10 double-stranded RNA (dsRNA) gene segments. Reovirus replication occurs within viral inclusions, which are specialized nonmembranous cytoplasmic organelles formed by viral nonstructural and structural proteins. Although these structures serve as sites for several major events in the reovirus life cycle, including dsRNA synthesis, gene segment assortment, and genome encapsidation, biochemical mechanisms of virion morphogenesis within inclusions have not been elucidated because much remains unknown about inclusion anatomy and functional organization. To better understand how inclusions support viral replication, we have used RNA interference (RNAi) and reverse genetics to define functional domains in two inclusion-associated proteins, mu NS and mu 2, which are interacting partners essential for inclusion development and viral replication. Removal of mu NS N-terminal sequences required for association with mu 2 or another mu NS-binding protein, mu NS, prevented the capacity of mu NS to support viral replication without affecting inclusion formation, indicating that mu NS-mu 2 and mu NS-sigma NS interactions are necessary for inclusion function but not establishment. In contrast, introduction of changes into the mu NS C-terminal region, including sequences that form a putative oligomerization domain, precluded inclusion formation as well as viral replication. Mutational analysis of mu 2 revealed a critical dependence of viral replication on an intact nucleotide/RNA triphosphatase domain and an N-terminal cluster of basic amino acid residues conforming to a nuclear localization motif. Another domain in mu 2 governs the capacity of viral inclusions to affiliate with microtubules and thereby modulates inclusion morphology, either globular or filamentous. However, viral variants altered in inclusion morphology displayed equivalent replication efficiency. These studies reveal a modular functional organization of inclusion proteins mu NS and mu 2, define the importance of specific amino acid sequences and motifs in these proteins for viral replication, and demonstrate the utility of complementary RNAi-based and reverse genetic approaches for studies of reovirus replication proteins.