Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, Pennsylvania, USA.
J Virol. 2012 Apr;86(7):3474-85. doi: 10.1128/JVI.06021-11. Epub 2012 Jan 18.
Ubiquitin is important for the budding of many retroviruses and other enveloped viruses, but the precise role of ubiquitin in virus budding remains unclear. Here, we characterized the ubiquitination of the matrix (M) protein of a paramyxovirus, parainfluenza virus 5 (PIV5). The PIV5 M protein (but not the PIV5 nucleocapsid protein) was found to be targeted for monoubiquitination in transfected mammalian cells. Major sites of ubiquitin attachment identified by mass spectrometry analysis were lysine residues at amino acid positions 79/80, 130, and 247. The cumulative mutation of lysine residues 79, 80, and 130 to arginines led to an altered pattern of M protein ubiquitination and impaired viruslike particle (VLP) production. However, the cumulative mutation of lysine residues 79, 80, 130, and 247 to arginines restored M protein ubiquitination and VLP production, suggesting that ubiquitin is attached to alternative sites on the M protein when the primary ones have been removed. Additional lysine residues were targeted for mutagenesis based on the UbiPred algorithm. An M protein with seven lysine residues changed to arginines exhibited altered ubiquitination and poor VLP production. A recombinant virus encoding an M protein with seven lysines mutated was generated, and this virus exhibited a 6-fold-reduced maximum titer, with the defect being attributed mainly to the budding of noninfectious particles. The recombinant virus was assembly deficient, as judged by the redistribution of viral M and hemagglutinin-neuraminidase proteins in infected cells. Similar assembly defects were observed for the wild-type (wt) virus after treatment with a proteasome inhibitor. Collectively, these findings suggest that the monoubiquitination of the PIV5 M protein is important for proper virus assembly and for the budding of infectious particles.
泛素对于许多逆转录病毒和其他包膜病毒的出芽都很重要，但泛素在病毒出芽中的精确作用仍不清楚。在这里，我们对副黏病毒、副流感病毒 5（PIV5）的基质（M）蛋白的泛素化进行了表征。在转染的哺乳动物细胞中，发现 PIV5 M 蛋白（而不是 PIV5 核衣壳蛋白）被靶向单泛素化。通过质谱分析鉴定的泛素附着的主要位点是赖氨酸残基 79/80、130 和 247 位。赖氨酸残基 79、80 和 130 突变为精氨酸导致 M 蛋白泛素化模式改变和病毒样颗粒（VLP）产生受损。然而，赖氨酸残基 79、80、130 和 247 突变为精氨酸则恢复了 M 蛋白泛素化和 VLP 产生，表明当主要位点被去除时，泛素被附着在 M 蛋白的替代位点上。根据 UbiPred 算法，对额外的赖氨酸残基进行了诱变。M 蛋白的七个赖氨酸突变为精氨酸后，其泛素化发生改变，VLP 产生不良。生成了编码 M 蛋白七个赖氨酸突变的重组病毒，该病毒的最大滴度降低了 6 倍，缺陷主要归因于非感染性颗粒的出芽。重组病毒装配缺陷，如感染细胞中病毒 M 和血凝素神经氨酸酶蛋白的重新分布所示。在用蛋白酶体抑制剂处理野生型（wt）病毒后，也观察到类似的装配缺陷。总的来说，这些发现表明 PIV5 M 蛋白的单泛素化对于适当的病毒装配和感染性颗粒的出芽很重要。
