Wang Jibin, Fang Shouguo, Xiao Han, Chen Bo, Tam James P, Liu Ding Xiang
School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
PLoS One. 2009;4(3):e4908. doi: 10.1371/journal.pone.0004908. Epub 2009 Mar 16.
Coronavirus M protein is an essential component of virion and plays pivotal roles in virion assembly, budding and maturation. The M protein is integrated into the viral envelope with three transmembrane domains flanked by a short amino-terminal ectodomain and a large carboxy-terminal endodomain. In this study, we showed co-purification of the M protein from coronavirus infectious bronchitis virus (IBV) with actin. To understand the cellular factors that may be involved in virion assembly, budding and maturation processes, IBV M was used as the bait in a yeast two-hybrid screen, resulting in the identification of beta-actin as a potentially interacting partner. This interaction was subsequently confirmed by coimmunoprecipitation and immunofluorescence microscopy in mammalian cells, and mutation of amino acids A159 and K160 on the M protein abolished the interaction. Introduction of the A159-K160 mutation into an infectious IBV clone system blocks the infectivity of the clone, although viral RNA replication and subgenomic mRNA transcription were actively detected. Disruption of actin filaments with cell-permeable agent cytochalasin D at early stages of the infection cycle led to the detection of viral protein synthesis in infected cells but not release of virus particles to the cultured media. However, the same treatment at late stages of the infection cycle did not affect the release of virus particles to the media, suggesting that disruption of the actin filaments might block virion assembly and budding, but not release of the virus particles. This study reveals an essential function of actin in the replication cycle of coronavirus.
冠状病毒M蛋白是病毒粒子的重要组成部分，在病毒粒子的组装、出芽和成熟过程中起关键作用。M蛋白通过三个跨膜结构域整合到病毒包膜中，其两侧分别是一个短的氨基末端胞外结构域和一个大的羧基末端胞内结构域。在本研究中，我们发现冠状病毒传染性支气管炎病毒（IBV）的M蛋白与肌动蛋白共纯化。为了了解可能参与病毒粒子组装、出芽和成熟过程的细胞因子，我们将IBV M用作酵母双杂交筛选的诱饵，结果鉴定出β-肌动蛋白是一个潜在的相互作用伴侣。随后，通过共免疫沉淀和哺乳动物细胞免疫荧光显微镜证实了这种相互作用，并且M蛋白上的氨基酸A159和K160突变消除了这种相互作用。将A159-K160突变引入传染性IBV克隆系统会阻断该克隆的感染性，尽管可以检测到病毒RNA复制和亚基因组mRNA转录。在感染周期早期用细胞可渗透剂细胞松弛素D破坏肌动蛋白丝，导致在感染细胞中检测到病毒蛋白合成，但病毒颗粒未释放到培养基中。然而，在感染周期后期进行相同处理并不影响病毒颗粒释放到培养基中，这表明肌动蛋白丝的破坏可能会阻断病毒粒子的组装和出芽，但不会影响病毒颗粒的释放。本研究揭示了肌动蛋白在冠状病毒复制周期中的重要功能。
