Sharma Mayuri, Kamil Jeremy P, Coughlin Margaret, Reim Natalia I, Coen Donald M
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.
J Virol. 2014 Jan;88(1):249-62. doi: 10.1128/JVI.02358-13. Epub 2013 Oct 23.
Herpesvirus nucleocapsids traverse the nuclear envelope into the cytoplasm in a process called nuclear egress that includes disruption of the nuclear lamina. In several herpesviruses, a key player in nuclear egress is a complex of two proteins, whose homologs in human cytomegalovirus (HCMV) are UL50 and UL53. However, their roles in nuclear egress during HCMV infection have not been shown. Based largely on transfection studies, UL50 and UL53 have been proposed to facilitate disruption of the nuclear lamina by recruiting cellular protein kinase C (PKC), as occurs with certain other herpesviruses, and/or the viral protein kinase UL97 to phosphorylate lamins. To investigate these issues during HCMV infection, we generated viral mutants null for UL50 or UL53. Correlative light electron microscopic analysis of null mutant-infected cells showed the presence of intranuclear nucleocapsids and the absence of cytoplasmic nucleocapsids. Confocal immunofluorescence microscopy revealed that UL50 and UL53 are required for disruption of the nuclear lamina. A subpopulation of UL97 colocalized with the nuclear rim, and this was dependent on UL50 and, to a lesser extent, UL53. However, PKC was not recruited to the nuclear rim, and its localization was not affected by the absence of UL50 or UL53. Immunoprecipitation from cells infected with HCMV expressing tagged UL53 detected UL97 but not PKC. In summary, HCMV UL50 and UL53 are required for nuclear egress and disruption of nuclear lamina during HCMV infection, and they recruit UL97, not PKC, for these processes. Thus, despite the strong conservation of herpesvirus nuclear egress complexes, a key function can differ among them.
疱疹病毒核衣壳通过一个称为核出芽的过程穿过核膜进入细胞质，该过程包括核纤层的破坏。在几种疱疹病毒中，核出芽的一个关键因子是由两种蛋白质组成的复合物，其在人巨细胞病毒（HCMV）中的同源物是UL50和UL53。然而，它们在HCMV感染期间核出芽中的作用尚未得到证实。主要基于转染研究，有人提出UL50和UL53通过招募细胞蛋白激酶C（PKC）来促进核纤层的破坏，就像某些其他疱疹病毒那样，和/或通过病毒蛋白激酶UL97使核纤层蛋白磷酸化。为了在HCMV感染期间研究这些问题，我们构建了缺失UL50或UL53的病毒突变体。对缺失突变体感染细胞的相关光电子显微镜分析显示存在核内核衣壳而不存在细胞质核衣壳。共聚焦免疫荧光显微镜检查表明，UL50和UL53是核纤层破坏所必需的。UL97的一个亚群与核边缘共定位，这依赖于UL50，在较小程度上依赖于UL53。然而，PKC没有被招募到核边缘，其定位不受UL50或UL53缺失的影响。从感染了表达带标签UL53的HCMV的细胞中进行免疫沉淀，检测到了UL97但未检测到PKC。总之，HCMV UL50和UL53是HCMV感染期间核出芽和核纤层破坏所必需的，并且它们在这些过程中招募UL97而不是PKC。因此，尽管疱疹病毒核出芽复合物有很强的保守性，但它们之间的一个关键功能可能有所不同。
