Division of Viral Infection, Department of Infectious Disease Control, International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
J Virol. 2011 Sep;85(18):9599-613. doi: 10.1128/JVI.00845-11. Epub 2011 Jul 6.
Us3 is a serine-threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). We have identified UL47, a major virion protein, as a novel physiological substrate of Us3. In vitro kinase assays and systematic analysis of mutations at putative Us3 phosphorylation sites near the nuclear localization signal of UL47 showed that serine at residue 77 (Ser-77) was required for Us3 phosphorylation of UL47. Replacement of UL47 Ser-77 by alanine produced aberrant accumulation of UL47 at the nuclear rim and impaired the nuclear localization of UL47 in a significant fraction of infected cells. The same defect in UL47 localization was produced by an amino acid substitution in Us3 that inactivated its protein kinase activity. In contrast, a phosphomimetic mutation at UL47 Ser-77 restored wild-type nuclear localization. The UL47 S77A mutation also reduced viral replication in the mouse cornea and the development of herpes stromal keratitis in mice. In addition, UL47 formed a stable complex with Us3 in infected cells, and nuclear localization of Us3 was significantly impaired in the absence of UL47. These results suggested that Us3 phosphorylation of UL47 Ser-77 promoted the nuclear localization of UL47 in cell cultures and played a critical role in viral replication and pathogenesis in vivo. Furthermore, UL47 appeared to be required for efficient nuclear localization of Us3 in infected cells. Therefore, Us3 protein kinase and its substrate UL47 demonstrated a unique regulatory feature in that they reciprocally regulated their subcellular localization in infected cells.
Us3 是单纯疱疹病毒 1（HSV-1）编码的丝氨酸/苏氨酸蛋白激酶。我们已经鉴定出 UL47，一种主要的病毒蛋白，是 Us3 的一种新型生理底物。在体外激酶测定和对 UL47 核定位信号附近假定的 Us3 磷酸化位点的突变的系统分析表明，残基 77 处的丝氨酸（Ser-77）是 Us3 磷酸化 UL47 所必需的。用丙氨酸取代 UL47 的 Ser-77 会导致 UL47 在核边缘异常积累，并使相当一部分感染细胞中的 UL47 核定位受损。在 Us3 中产生失活其蛋白激酶活性的氨基酸取代也会产生 UL47 定位的相同缺陷。相比之下，UL47 Ser-77 的磷酸模拟突变恢复了野生型核定位。UL47 S77A 突变还降低了小鼠角膜中的病毒复制和小鼠疱疹性基质性角膜炎的发展。此外，UL47 在感染细胞中与 Us3 形成稳定复合物，并且在没有 UL47 的情况下，Us3 的核定位明显受损。这些结果表明，Us3 对 UL47 Ser-77 的磷酸化促进了 UL47 在细胞培养中的核定位，并在体内病毒复制和发病机制中发挥了关键作用。此外，UL47 似乎是 Us3 在感染细胞中有效核定位所必需的。因此，Us3 蛋白激酶及其底物 UL47 表现出独特的调节特征，它们在感染细胞中相互调节其亚细胞定位。
