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

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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.