Increased ubiquitination and other covariant phenotypes attributed to a strain- and temperature-dependent defect of reovirus core protein μ2
Increased ubiquitination and other covariant phenotypes attributed to a strain- and temperature-dependent defect of reovirus core protein μ2
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DOI：
10.1128/jvi.78.19.10291-10302.2004
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发表时间：
2004-10-01
影响因子：
5.4
通讯作者：
Nibert, ML
中科院分区：
文献类型：
--
作者：
Miller, CL;Parker, JSL;Nibert, ML
呼肠孤病毒的复制和组装被认为发生在细胞质包涵体内，我们称之为病毒工厂。这些结构形态上的毒株依赖性差异反映了某些病毒株的 mu2 核心蛋白（形成丝状工厂）比其他病毒株（形成球状工厂）更有效的微管关联。在本报告中，我们确定并表征了工厂的另一个菌株依赖性属性，即它们与缀合泛素 (cUb) 共定位的程度。在 16 个实验室菌株和现场分离株中，cUb 工厂共染色的程度与工厂形态平行，其中球状菌株迄今为止表现出更高的水平。在重配病毒中，cUb 的工厂共染色主要映射到编码 mu2 的 M1 基因组片段，尽管编码 lambda3 和 lambda2 的 L1 和 L2 基因组片段的贡献也很明显。免疫沉淀显示，被球状菌株感染的细胞含有较高水平的泛素化 mu2 (Ub-mu2)。在 M1 转染的细胞中，cUb 通常与来自球状菌株的 mu2 形成的聚集体共定位，但不与来自丝状菌株的 mu2 包被的微管共定位，并且免疫沉淀显示来自球状菌株的 mu2 显示出更高水平的 Ub-mu2。 mu2 残基 208 处的等位基因变化决定了这些差异。诺考达唑处理感染丝状菌株的细胞产生球状工厂，但仍显示出低水平的 cUb 共染色，表明较高水平的共染色并不是微管关联减少的直接结果。此外，当细胞在低温下生长时，球状菌株的工厂或其在转染细胞中表达的 mu2 蛋白被进一步证明获得微管关联并失去与 cUb 的共定位。根据这些发现的总和，我们提出来自球状菌株的 mu2 更容易发生温度依赖性错误折叠，因此表现出聚集增加、Ub-mu2 水平增加以及与微管的关联减少。由于很少有病毒株形成通常与泛素化蛋白相关的工厂，因此我们得出结论，呼肠孤病毒工厂通常与细胞聚集体不同。
Reovirus replication and assembly are thought to occur within cytoplasmic inclusion bodies, which we call viral factories. A strain-dependent difference in the morphology of these structures reflects more effective microtubule association by the mu2 core proteins of some viral strains, which form filamentous factories, than by those of others, which form globular factories. For this report, we identified and characterized another strain-dependent attribute of the factories, namely, the extent to which they colocalized with conjugated ubiquitin (cUb). Among 16 laboratory strains and field isolates, the extent of factory costaining for cUb paralleled factory morphology, with globular strains exhibiting higher levels by far. In reassortant viruses, factory costaining for cUb mapped primarily to the mu2-encoding M1 genome segment, although contributions by the lambda3- and lambda2-encoding L1 and L2 genome segments were also evident. Immunoprecipitations revealed that cells infected with globular strains contained higher levels of ubiquitinated mu2 (Ub-mu2). In M1-transfected cells, cUb commonly colocalized with aggregates formed by mu2 from globular strains but not with microtubules coated by mu2 from filamentous strains, and immunoprecipitations revealed that mu2 from globular strains displayed higher levels of Ub-mu2. Allelic changes at mu2 residue 208 determined these differences. Nocodazole treatment of cells infected with filamentous strains resulted in globular factories that still showed low levels of costaining for cUb, indicating that higher levels of costaining were not a direct result of decreased microtubule association. The factories of globular strains, or their mu2 proteins expressed in transfected cells, were furthermore shown to gain microtubule association and to lose colocalization with cUb when cells were grown at reduced temperature. From the sum of these findings, we propose that mu2 from globular strains is more prone to temperature-dependent misfolding and as a result displays increased aggregation, increased levels of Ub-mu2, and decreased association with microtubules. Because so few of the viral strains formed factories that were regularly associated with ubiquitinated proteins, we conclude that reovirus factories are generally distinct from cellular aggresomes.