Interaction of Human Cytomegalovirus pUL52 with major components of the viral DNA encapsidation network underlines its essential role in genome cleavage-packaging

Cleavage of human cytomegalovirus (HCMV) genomes and their packaging into capsids requires at least seven essential viral proteins, yet it is not completely understood how these proteins cooperate to accomplish this task. Besides the portal protein pUL104 and the terminase subunits pUL51, pUL56, and pUL89, the UL52 protein is also necessary for HCMV genome encapsidation, however, knowledge about pUL52 is scant. In the absence of pUL52, viral concatemers are not cleaved into unit-length genomes and no DNA-filled capsids are observed, yet no viral or cellular proteins interacting with pUL52 have been identified that would explain how pUL52 exerts its essential role in the HCMV infection cycle. In this study, we aimed at a comprehensive definition of pUL52-interacting proteins in infected cells. Using suitable HCMV mutants, we employed three complementary state-of-the-art proteomic approaches, namely biotin ligase-dependent proximity labeling, affinity purification and cross-linking mass spectrometry. These experiments, combined with thorough validation by immunoblotting, pointed to several viral DNA-associated proteins and key players pivotal for genome encapsidation as interactors of pUL52. The most noticeable direct pUL52 interaction partners were the terminase subunits pUL56 and pUL89 as well as the portal protein pUL104. Hence, we suggest a model of pUL52 function in which pUL52 mediates association of HCMV genomes with the terminase subunits and the capsid portal. Taken together, our data contribute to the understanding of an essential viral process previously recognized as a prominent antiviral target. Disturbing the identified pUL52 interactions may provide a starting point to develop novel antiviral medication.

人巨细胞病毒（human cytomegalovirus, HCMV）基因组的切割与衣壳包装过程，至少需要七种必需病毒蛋白参与，但目前学界尚未完全阐明这些蛋白如何协同完成该任务。除门户蛋白pUL104与末端酶亚基pUL51、pUL56、pUL89外，UL52蛋白同样是HCMV基因组衣壳化的必需蛋白，但目前关于pUL52的研究资料仍十分匮乏。当pUL52缺失时，病毒多联体基因组无法被切割为单位长度的基因组，且未观察到装载病毒DNA的衣壳；但目前尚未发现任何可与pUL52相互作用的病毒或宿主蛋白，以此解释pUL52在HCMV感染周期中发挥必需功能的具体机制。本研究旨在全面鉴定感染细胞中与pUL52相互作用的蛋白。我们通过构建合适的HCMV突变体，采用了三种互补的前沿蛋白质组学方法：依赖生物素连接酶的邻近标记技术、亲和纯化技术以及交联质谱技术。上述实验结合免疫印迹法的严格验证，鉴定出多种病毒DNA相关蛋白以及基因组衣壳化过程中的关键调控因子作为pUL52的互作蛋白。其中，与pUL52直接互作最显著的蛋白为末端酶亚基pUL56、pUL89以及门户蛋白pUL104。据此，我们提出pUL52的功能模型：pUL52介导HCMV基因组与末端酶亚基及衣壳门户蛋白的结合。综上，本研究数据有助于学界进一步阐明这一此前被视为重要抗病毒靶点的必需病毒生命过程。靶向所鉴定出的pUL52相互作用通路，或可为新型抗病毒药物的研发提供全新切入点。