Role of the human cytomegalovirus UL77 protein in capsid assembly and maturation

人类巨细胞病毒(HCMV)是导致出生缺陷的主要病毒原因，也是免疫功能低下患者发病甚至死亡的主要原因。有一种尚未得到满足的医学需求，即开发针对HCMV的新型抗病毒药物，以克服目前药物的局限性(不良反应，出现耐药株)，特别是设想联合疗法，这是一种成功地应用于治疗艾滋病毒和丙型肝炎病毒感染的策略。识别药物靶点需要详细了解病毒复制周期所必需的蛋白质-蛋白质相互作用。HCMV衣壳的组装是一个复杂的多步骤过程，其特征是大量的蛋白质相互作用，因此在这方面引起了人们的主要兴趣。在拟议的项目中，我们专注于HCMV UL77蛋白，它是病毒基因组包装中的关键角色。我们以前的结果表明，pUL77的功能与其α-和γ-疱疹病毒同源基因有一定程度的不同，因为它是切割dna所必需的，并且在所有衣壳类型上都存在相同的量。与这些差异相一致的是，疱疹病毒不同亚科之间的衣壳结合也不同，因为在最近的人巨细胞病毒衣壳的冷冻-EM重建中，没有观察到人巨细胞病毒pUL77在五角体周围的密度，就像α-和γ-疱疹病毒pUL77同源基因所显示的那样。取而代之的是，β疱疹病毒特异性被膜蛋白pp150将五角体和六角体紧紧地包裹在一起，没有留下容纳PUL77的空间。然而，我们最近对pUL77及其同源基因的比较结构分析显示，疱疹病毒亚家族之间存在显著的结构保守性，表明其功能高度保守。正如最近报道的α和γ疱疹病毒一样，这种功能可能是形成五聚体门静脉帽来封闭充满DNA的衣壳，预计在β疱疹病毒中也是如此。我们的目的是通过描述关键的蛋白质-蛋白质相互作用，更好地了解pUL77在HCMV衣壳组装过程中的重要作用。特别是，我们将研究(I)可能的pUL77寡聚，(Ii)使用体内文库产生感染性后代所必需的pUL77接口，(Iii)pUL77与pUL93(假定为pUL77相互作用伙伴)以及与最内侧的HCMV被膜蛋白pp150和pUL48的相互作用，(Iv)pUL77/pUL93与HCMV门户以及终止酶之间的关系，以揭示pUL77在HCMV基因组封装中的作用。我们的结果将为了解pUL77在HCMV衣壳组装过程中的功能和相互作用提供新的见解，并阐明不同疱疹病毒亚家族衣壳组装的进化差异和相似性。重要的是，我们的结果将定义新的靶点，可能有助于合理设计HCMV感染的抑制剂。

Human cytomegalovirus (HCMV) is the leading viral cause of birth defects and a major cause of morbidity and even mortality in immunocompromised patients. There is an unmet medical need to develop novel antivirals against HCMV to overcome the limitations of current medication (adverse effects, emergence of drug-resistant strains) and in particular to envisage combination therapy, a strategy successfully applied to treat HIV and HCV infection. Identification of drug targets requires a detailed comprehension of protein-protein interactions essential for the viral replication cycle. HCMV capsid assembly is a complex multi-step process characterized by numerous protein-protein interactions, and thus of major interest in this respect.In the proposed project, we focus on the HCMV UL77 protein, a key player in viral genome packaging. Our previous results indicate that the pUL77 function differs to some extent from that of its α- and γ-herpesvirus orthologs, as it is required for DNA cleavage and is present in similar amounts on all capsid types. In line with these differences, also capsid association differs between herpesvirus subfamilies, since in recent cryo-EM reconstructions of HCMV capsids no density for HCMV pUL77 was observed around the pentons, as shown for α- and γ-herpesvirus pUL77 orthologs. Instead, the β-herpesvirus-specific tegument protein pp150 tightly encloses pentons and hexons, leaving no space to incorporate pUL77. However, our recent comparative structural analysis of pUL77 and its orthologs revealed a striking structural conservation across herpesvirus subfamilies, pointing to a highly conserved function. Such a function could be the formation of the pentameric portal cap to seal DNA-filled capsids, as recently reported for α- and γ-herpesviruses, and expected to be conserved also in β-herpesviruses.Our aim is to better understand the essential role of pUL77 during HCMV capsid assembly via the characterization of key protein-protein interactions using 1) suitable HCMV BAC mutants to study pUL77 in the context of infection and 2) a baculovirus-based HCMV capsid assembly assay. In particular, we will investigate (i) putative pUL77 oligomerization, (ii) pUL77 interfaces essential for the production of infectious progeny using an intrabody library, (iii) the interplay of pUL77 with pUL93 (a presumptive pUL77 interaction partner) and with the innermost HCMV tegument proteins pp150 and pUL48, (iv) the relationship between pUL77/pUL93 and the HCMV portal as well as the terminase to uncover the role of pUL77 in HCMV genome encapsidation. Our results will provide novel insights into pUL77 function and interactions during HCMV capsid assembly, and shed light on the evolutionary differences and similarities of capsid assembly across herpesviral subfamilies. Not least, our results will define novel targets that may facilitate the rational design of inhibitors of HCMV infection.