Division of Molecular Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan.
Department of Infectious Disease Control, International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan.
J Virol. 2018 Aug 29;92(18). doi: 10.1128/JVI.01035-18. Print 2018 Sep 15.
Herpes simplex virus 1 (HSV-1) UL51 is a phosphoprotein that functions in the final envelopment in the cytoplasm and viral cell-cell spread, leading to efficient viral replication in cell cultures. To clarify the mechanism by which UL51 is regulated in HSV-1-infected cells, we focused on the phosphorylation of UL51. Mass spectrometry analysis of purified UL51 identified five phosphorylation sites in UL51. Alanine replacement of one of the identified phosphorylation sites in UL51, serine 184 (Ser-184), but not the other identified phosphorylation sites, significantly reduced viral replication and cell-cell spread in HaCaT cells. This mutation induced membranous invaginations adjacent to the nuclear membrane, the accumulation of primary enveloped virions in the invaginations and perinuclear space, and mislocalized UL34 and UL31 in punctate structures at the nuclear membrane; however, it had no effect on final envelopment in the cytoplasm of HaCaT cells. Of note, the alanine mutation in UL51 Ser-184 significantly reduced the mortality of mice following ocular infection. Phosphomimetic mutation in UL51 Ser-184 partly restored the wild-type phenotype in cell cultures and in mice. Based on these results, we concluded that some UL51 functions are specifically regulated by phosphorylation at Ser-184 and that this regulation is critical for HSV-1 replication in cell cultures and pathogenicity HSV-1 UL51 is conserved in all members of the family. This viral protein is phosphorylated and functions in viral cell-cell spread and cytoplasmic virion maturation in HSV-1-infected cells. Although the downstream effects of HSV-1 UL51 have been clarified, there is a lack of information on how this viral protein is regulated as well as the significance of the phosphorylation of this protein in HSV-1-infected cells. In this study, we show that the phosphorylation of UL51 at Ser-184 promotes viral replication, cell-cell spread, and nuclear egress in cell cultures and viral pathogenicity in mice. This is the first report to identify the mechanism by which UL51 is regulated as well as the significance of UL51 phosphorylation in HSV-1 infection. Our study may provide insights into the regulatory mechanisms of other herpesviral UL51 homologs.
单纯疱疹病毒 1（HSV-1）UL51 是一种磷酸化蛋白，在细胞质中完成最后的包膜和病毒细胞间传播，从而在细胞培养物中实现高效的病毒复制。为了阐明 HSV-1 感染细胞中 UL51 的调节机制，我们专注于 UL51 的磷酸化。对纯化的 UL51 进行质谱分析，鉴定出 UL51 中有五个磷酸化位点。UL51 中一个鉴定出的磷酸化位点丝氨酸 184（Ser-184）的丙氨酸替换，而不是其他鉴定出的磷酸化位点，显著降低了 HaCaT 细胞中的病毒复制和细胞间传播。这种突变诱导核膜附近的膜内陷，初级包膜病毒粒子在这些内陷和核周空间中积累，并使 UL34 和 UL31 在核膜上的点状结构中定位错误；然而，它对 HaCaT 细胞细胞质中的最终包膜没有影响。值得注意的是，UL51 Ser-184 的丙氨酸突变显著降低了眼部感染后小鼠的死亡率。UL51 Ser-184 的磷酸模拟突变在细胞培养物和小鼠中部分恢复了野生型表型。基于这些结果，我们得出结论，UL51 的一些功能是通过 Ser-184 的磷酸化特异性调节的，这种调节对 HSV-1 在细胞培养物中的复制和致病性至关重要。HSV-1 UL51 在疱疹病毒家族的所有成员中都保守。这种病毒蛋白在 HSV-1 感染细胞中的病毒细胞间传播和细胞质病毒粒子成熟中被磷酸化并发挥作用。尽管已经阐明了 HSV-1 UL51 的下游效应，但关于这种病毒蛋白如何被调节以及该蛋白在 HSV-1 感染细胞中的磷酸化的意义，我们知之甚少。在这项研究中，我们表明 UL51 在 Ser-184 处的磷酸化促进了病毒在细胞培养物中的复制、细胞间传播和核输出以及在小鼠中的病毒致病性。这是第一个报道鉴定 UL51 调节机制以及 UL51 磷酸化在 HSV-1 感染中的意义的报告。我们的研究可能为其他疱疹病毒 UL51 同源物的调节机制提供启示。
