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

人巨细胞病毒 UL77 蛋白在衣壳组装和成熟中的作用

• 批准号: 441233738
• 负责人: Dr. Eva Maria Borst
• 金额: --
• 依托单位: Institut für Virologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2020
• 资助国家: 德国
• 起止时间: 2019-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/441233738?language=en
• 关键词: Role; human; cytomegalovirus; UL77; protein

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.

人巨细胞病毒（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。开发针对HCMV的新型抗病毒药物以克服当前药物治疗的局限性（不良反应、耐药菌株的出现），特别是设想联合治疗，这是一种成功应用于治疗HIV和HCV感染的策略，存在未满足的医疗需求。药物靶点的鉴定需要对病毒复制周期所必需的蛋白质-蛋白质相互作用的详细理解。HCMV衣壳组装是一个复杂的多步骤过程，其特征在于许多蛋白质-蛋白质相互作用，因此在这方面的主要兴趣。我们先前的结果表明，pUL 77的功能在某种程度上不同于其α-和γ-疱疹病毒直系同源物的功能，因为它是DNA切割所需的，并且以相似的量存在于所有衣壳类型上。与这些差异一致，疱疹病毒亚科之间的衣壳缔合也不同，因为在最近的HCMV衣壳的冷冻-EM重建中，在五邻体周围没有观察到HCMV pUL 77的密度，如α-和γ-疱疹病毒pUL 77直系同源物所示。相反，β-疱疹病毒特异性被膜蛋白pp 150紧密包围五邻体和六邻体，没有留下空间来掺入pUL 77。然而，我们最近对pUL 77及其直系同源物的比较结构分析揭示了疱疹病毒亚科之间惊人的结构保守性，指出了高度保守的功能。这种功能可能是形成五聚体门户帽以密封DNA填充的衣壳，如最近报道的α和γ疱疹病毒，我们的目的是通过使用1）通过表征关键蛋白质-蛋白质相互作用来更好地理解pUL 77在HCMV衣壳组装过程中的重要作用。合适的HCMV BAC突变体以在感染的情况下研究pUL 77和2）基于杆状病毒的HCMV衣壳组装测定。特别地，我们将研究（i）推定的pUL 77寡聚化，（ii）使用胞内抗体文库产生感染性子代所必需的pUL 77界面，（iii）pUL 77与pUL 93的相互作用（一种假定的pUL 77相互作用配偶体）和最里面的HCMV被膜蛋白pp 150和pUL 48，（iv）pUL 77/pUL 93与HCMV入口和末端酶的关系，以揭示pUL 77在HCMV基因组终止中的作用。我们的研究结果将提供新的见解pUL 77的功能和相互作用在HCMV衣壳组装，并阐明了进化的差异和相似性的衣壳组装疱疹病毒亚科。尤其重要的是，我们的研究结果将确定新的目标，可能有助于合理设计的抑制剂的HCMV感染。