Morrison E E, Wang Y F, Meredith D M
Molecular Medicine Unit, University of Leeds, St. James University Hospital, Leeds LS9 7TF, United Kingdom.
J Virol. 1998 Sep;72(9):7108-14. doi: 10.1128/JVI.72.9.7108-7114.1998.
The role of phosphorylation in the dissociation of structural components of the herpes simplex virus type 1 (HSV-1) tegument was investigated, using an in vitro assay. Addition of physiological concentrations of ATP and magnesium to wild-type virions in the presence of detergent promoted the release of VP13/14 and VP22. VP1/2 and the UL13 protein kinase were not significantly solubilized. However, using a virus with an inactivated UL13 protein, we found that the release of VP22 was severely impaired. Addition of casein kinase II (CKII) to UL13 mutant virions promoted VP22 release. Heat inactivation of virions or addition of phosphatase inhibited the release of both proteins. Incorporation of radiolabeled ATP into the assay demonstrated the phosphorylation of VP1/2, VP13/14, VP16, and VP22. Incubation of detergent-purified, heat-inactivated capsid-tegument with recombinant kinases showed VP1/2 phosphorylation by CKII, VP13/14 phosphorylation by CKII, protein kinase A (PKA), and PKC, VP16 phosphorylation by PKA, and VP22 phosphorylation by CKII and PKC. Proteolytic mapping and phosphoamino acid analysis of phosphorylated VP22 correlated with previously published work. The phosphorylation of virion-associated VP13/14, VP16, and VP22 was demonstrated in cells infected in the presence of cycloheximide. Use of equine herpesvirus 1 in the in vitro release assay resulted in the enhanced release of VP10, the homolog of HSV-1 VP13/14. These results suggest that the dissociation of major tegument proteins from alphaherpesvirus virions in infected cells may be initiated by phosphorylation events mediated by both virion-associated and cellular kinases.
利用体外试验研究了磷酸化在1型单纯疱疹病毒（HSV-1）被膜结构成分解离中的作用。在去污剂存在的情况下，向野生型病毒粒子中添加生理浓度的ATP和镁可促进VP13/14和VP22的释放。VP1/2和UL13蛋白激酶未显著溶解。然而，使用具有失活UL13蛋白的病毒，我们发现VP22的释放严重受损。向UL13突变体病毒粒子中添加酪蛋白激酶II（CKII）可促进VP22的释放。病毒粒子的热灭活或磷酸酶的添加会抑制这两种蛋白质的释放。将放射性标记的ATP掺入试验表明VP1/2、VP13/14、VP16和VP22发生了磷酸化。用重组激酶孵育经去污剂纯化、热灭活的衣壳-被膜，结果显示CKII使VP1/2磷酸化，CKII、蛋白激酶A（PKA）和蛋白激酶C（PKC）使VP13/14磷酸化，PKA使VP16磷酸化，CKII和PKC使VP22磷酸化。磷酸化VP22的蛋白水解图谱和磷酸氨基酸分析与先前发表的工作相关。在存在环己酰亚胺的情况下感染的细胞中证实了病毒粒子相关的VP13/14、VP16和VP22的磷酸化。在体外释放试验中使用马疱疹病毒1导致HSV-1 VP13/14同源物VP10的释放增强。这些结果表明，感染细胞中α疱疹病毒病毒粒子主要被膜蛋白的解离可能由病毒粒子相关激酶和细胞激酶介导的磷酸化事件引发。
