Fujii Hikaru, Kato Akihisa, Mugitani Michio, Kashima Yukie, Oyama Masaaki, Kozuka-Hata Hiroko, Arii Jun, Kawaguchi Yasushi
Division of Molecular Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan Department of Infectious Disease Control, International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
Department of Infectious Disease Control, International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
J Virol. 2014 Sep;88(18):10624-34. doi: 10.1128/JVI.01634-14. Epub 2014 Jul 2.
The herpes simplex virus 1 (HSV-1) UL12 protein (pUL12) is a nuclease that is critical for viral replication in vitro and neurovirulence in vivo. In this study, mass spectrometric analysis of pUL12 and phosphate-affinity SDS-polyacrylamide gel electrophoresis analysis identified tyrosine at pUL12 residue 371 (Tyr-371) as a pUL12 phosphorylation site: Tyr-371 is conserved in pUL12 homologs in herpesviruses in all Herpesviridae subfamilies. Replacement of Tyr-371 with phenylalanine (Y371F) in pUL12 (i) abolished its exonuclease activity in HSV-1-infected Vero, HEL, and A549 cells, (ii) reduced viral replication, cell-cell spread, and pUL12 expression in infected cells in a cell type-dependent manner, (iii) led to aberrant subcellular localization of pUL12 in infected cells in a cell type-dependent manner, and (iv) reduced HSV-1 neurovirulence in mice. The effects of the pUL12 Y371F mutation in cell cultures and mice were similar to those of a nuclease-dead double mutation in pUL12, although the Y371F mutation reduced viral replication severalfold more than the nuclease-dead double mutation in a cell type- and multiplicity-of-infection-dependent manner. Replacement of Tyr-371 with glutamic acid, which mimics constitutive phosphorylation, restored the wild-type phenotype in cell cultures and mice. These results suggested that phosphorylation of pUL12 Tyr-371 was essential for pUL12 to express its nuclease activity in HSV-1-infected cells and that this phosphorylation promoted viral replication and cell-cell spread in cell cultures and neurovirulence in mice mainly by upregulating pUL12 nuclease activity and, in part, by regulating the subcellular localization and expression of pUL12 in HSV-1-infected cells.
Herpesviruses encode a considerable number of enzymes for their replication. Like cellular enzymes, the viral enzymes need to be properly regulated in infected cells. Although the functional aspects of herpesvirus enzymes have gradually been clarified, information on how most of these enzymes are regulated in infected cells is lacking. In the present study, we report that the enzymatic activity of the herpes simplex virus 1 alkaline nuclease pUL12 was regulated by phosphorylation of pUL12 Tyr-371 in infected cells and that this phosphorylation promoted viral replication and cell-cell spread in cell cultures and neurovirulence in mice, mainly by upregulating pUL12 nuclease activity. Interestingly, pUL12 and tyrosine at pUL12 residue 371 appeared to be conserved in all herpesviruses in the family Herpesviridae, raising the possibility that the herpesvirus pUL12 homologs may also be regulated by phosphorylation of the conserved tyrosine residue.
单纯疱疹病毒1型（HSV-1）的UL12蛋白（pUL12）是一种核酸酶，对体外病毒复制和体内神经毒性至关重要。在本研究中，对pUL12进行质谱分析以及磷酸亲和SDS聚丙烯酰胺凝胶电泳分析，确定pUL12第371位残基的酪氨酸（Tyr-371）为pUL12的磷酸化位点：Tyr-371在疱疹病毒科所有亚科的疱疹病毒pUL12同源物中保守。将pUL12中的酪氨酸371（Y371F）替换为苯丙氨酸后，（i）在HSV-1感染的Vero、HEL和A549细胞中消除了其核酸外切酶活性，（ii）以细胞类型依赖的方式降低了感染细胞中的病毒复制、细胞间传播和pUL12表达，（iii）以细胞类型依赖的方式导致感染细胞中pUL12出现异常亚细胞定位，（iv）降低了HSV-1在小鼠中的神经毒性。pUL12 Y371F突变在细胞培养物和小鼠中的作用与pUL12中的核酸酶失活双突变相似，尽管Y371F突变以细胞类型和感染复数依赖的方式比核酸酶失活双突变更能降低病毒复制数倍。用模拟组成型磷酸化的谷氨酸替换Tyr-371，可在细胞培养物和小鼠中恢复野生型表型。这些结果表明，pUL12 Tyr-371的磷酸化对于pUL12在HSV-1感染细胞中表达其核酸酶活性至关重要，并且这种磷酸化主要通过上调pUL12核酸酶活性以及部分通过调节HSV-1感染细胞中pUL12的亚细胞定位和表达，促进了细胞培养物中的病毒复制和细胞间传播以及小鼠中的神经毒性。
疱疹病毒编码大量用于其复制的酶。与细胞酶一样，病毒酶在感染细胞中需要得到适当调节。尽管疱疹病毒酶的功能方面已逐渐阐明，但关于这些酶在感染细胞中如何被调节的信息却很缺乏。在本研究中，我们报告单纯疱疹病毒1型碱性核酸酶pUL12的酶活性在感染细胞中受pUL12 Tyr-371磷酸化的调节，并且这种磷酸化主要通过上调pUL12核酸酶活性，促进了细胞培养物中的病毒复制和细胞间传播以及小鼠中的神经毒性。有趣的是，pUL12以及pUL12第371位残基的酪氨酸似乎在疱疹病毒科的所有疱疹病毒中都保守，这增加了疱疹病毒pUL12同源物也可能受保守酪氨酸残基磷酸化调节的可能性。
