Arnold Michelle M, Murray Kenneth E, Nibert Max L
Department of Microbiology and Molecular Genetics, Harvard Medical School, Harvard University, Boston, MA 02115, USA.
Virology. 2008 Jun 5;375(2):412-23. doi: 10.1016/j.virol.2008.02.024. Epub 2008 Apr 18.
Genome replication of mammalian orthoreovirus (MRV) occurs in cytoplasmic inclusion bodies called viral factories. Nonstructural protein microNS, encoded by genome segment M3, is a major constituent of these structures. When expressed without other viral proteins, microNS forms cytoplasmic inclusions morphologically similar to factories, suggesting a role for microNS as the factory framework or matrix. In addition, most other MRV proteins, including all five core proteins (lambda1, lambda2, lambda3, micro2, and sigma2) and nonstructural protein sigmaNS, can associate with microNS in these structures. In the current study, small interfering RNA targeting M3 was transfected in association with MRV infection and shown to cause a substantial reduction in microNS expression as well as, among other effects, a reduction in infectious yields by as much as 4 log(10) values. By also transfecting in vitro-transcribed M3 plus-strand RNA containing silent mutations that render it resistant to the small interfering RNA, we were able to complement microNS expression and to rescue infectious yields by ~100-fold. We next used microNS mutants specifically defective at forming factory-matrix structures to show that this function of microNS is important for MRV growth; point mutations in a C-proximal, putative zinc-hook motif as well as small deletions at the extreme C terminus of microNS prevented rescue of viral growth while causing microNS to be diffusely distributed in cells. We furthermore confirmed that an N-terminally truncated form of microNS, designed to represent microNSC and still able to form factory-matrix structures, is unable to rescue MRV growth, localizing one or more other important functions to an N-terminal region of microNS known to be involved in both micro2 and sigmaNS association. Thus, factory-matrix formation is an important, though not a sufficient function of microNS during MRV infection; microNS is multifunctional in the course of viral growth.
哺乳动物正呼肠孤病毒（MRV）的基因组复制发生在称为病毒工厂的细胞质包涵体中。由基因组片段M3编码的非结构蛋白microNS是这些结构的主要成分。当microNS在没有其他病毒蛋白的情况下表达时，会形成形态上类似于病毒工厂的细胞质包涵体，这表明microNS作为病毒工厂框架或基质发挥作用。此外，大多数其他MRV蛋白，包括所有五种核心蛋白（λ1、λ2、λ3、micro2和σ2）和非结构蛋白σNS，都可以在这些结构中与microNS结合。在本研究中，与MRV感染相关地转染靶向M3的小干扰RNA，结果显示其导致microNS表达大幅降低，以及除其他影响外，感染性病毒产量降低多达4个对数（10）值。通过转染含有使其对小干扰RNA具有抗性的沉默突变的体外转录M3正链RNA，我们能够补充microNS表达并将感染性病毒产量挽救约100倍。接下来，我们使用在形成病毒工厂基质结构方面存在特异性缺陷的microNS突变体来表明microNS的这一功能对MRV生长很重要；microNS C近端假定锌钩基序中的点突变以及microNS极端C末端的小缺失阻止了病毒生长的挽救，同时导致microNS在细胞中呈弥漫性分布。我们进一步证实，设计用来代表microNSC且仍能形成病毒工厂基质结构的N末端截短形式的microNS无法挽救MRV生长，从而将一个或多个其他重要功能定位到已知参与micro2和σNS结合的microNS的N末端区域。因此，病毒工厂基质的形成是MRV感染期间microNS的一项重要功能，但并非充分功能；microNS在病毒生长过程中具有多种功能。
