Role of the human cytomegalovirus UL77 protein in capsid assembly and maturation
Final Report Abstract
Human cytomegalovirus (HCMV) is a widespread pathogen with seroprevalences reaching up to 90% in the human population. Although being mostly asymptomatic or causing only mild flu-like symptoms in healthy individuals, HCMV is a major risk factor for immunecompromised persons such as transplant recipients. Moreover, HCMV is the leading viral cause for malformations in newborns. For decades, antiviral therapy targeted mainly viral DNA replication, and only recently the packaging of viral genomes into capsids has emerged as an auspicious drug target as it is a highly virus-specific process that has no counterpart in eukaryotic cells. Cleavage-packaging of HCMV genomes requires several essential viral proteins whose functions are only partially understood so far. In this project, we have focused mainly on three HCMV encapsidation proteins, namely pUL77, pUL52, and the small capsid protein SCP. We analyzed the role of pUL77 and especially of SCP in capsid assembly, and identified viral proteins interacting with pUL52 in HCMV-infected cells. While pUL77 was dispensable for capsid formation, SCP exerted a major effect by stabilizing the major capsid protein MCP. In the absence of SCP, MCP levels were strongly reduced and no capsids could be observed. We also pinned down the region in SCP mediating MCP stabilization to a C- terminal helix, in agreement with the previously shown ability to mediate MCP-binding. To gain insight into pUL52 function, we combined state-of-the-art proteomic analyses with affinity purification and chemical cross-linking, and verified the identified pUL52 interacting proteins by immunoblotting. The most important pUL52 interactors were the terminase proteins pUL56 and pUL89, and to a lesser extent the portal protein pUL104. Cross-linking experiments indicated direct binding of pUL52 to both pUL56 and pUL89, while interaction with pUL104 appears to be more indirect. These findings argue for a role of pUL52 at the portal vertex and suggest that pUL52 is essential for HCMV genome cleavage-packaging because it is needed to connect the terminase with viral DNA, thereby initiating genome encapsidation. In parallel, we identified several pUL77-specific antibodies that serve as tools to better understand HCMV capsid binding of pUL77. We have identified several antigenic regions on the surface of pUL77 that are targeted by individual sybodies. Epitope mapping was attempted by complex crystallization without success; therefore, we switched to the analysis of molecular interfaces by structure-based mutagenesis that is currently in progress. Eventually a careful analysis of the impact of individual sybodies on HCMV infection has been carried out by lentiviral generation of stably sybody-expressing HFF cell lines. Plaque number, plaque size and a time-resolved life-cell imaging revealed the impact of individual sybodies on HCMV infection and identified a highly potent sybody that is affecting plaque size as well as plaque number and colocalizes with pUL77 in virus-infected cells. In summary, our results contribute to the understanding of the roles of different HCMV proteins essential for capsid assembly and viral genome cleavage-packaging, knowledge that may be exploited in future studies to develop novel antiviral strategies.
Publications
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Assembly of infectious Kaposi’s sarcoma-associated herpesvirus progeny requires formation of a pORF19 pentamer. PLOS Biology, 19(11), e3001423.
Naniima, Peter; Naimo, Eleonora; Koch, Sandra; Curth, Ute; Alkharsah, Khaled R.; Ströh, Luisa J.; Binz, Anne; Beneke, Jan-Marc; Vollmer, Benjamin; Böning, Heike; Borst, Eva Maria; Desai, Prashant; Bohne, Jens; Messerle, Martin; Bauerfeind, Rudolf; Legrand, Pierre; Sodeik, Beate; Schulz, Thomas F. & Krey, Thomas
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A Unique Role of the Human Cytomegalovirus Small Capsid Protein in Capsid Assembly. mBio, 13(5).
Borst, Eva Maria; Harmening, Sarah; Sanders, Saskia; Caragliano, Enrico; Wagner, Karen; Lenac, Roviš Tihana; Jonjić, Stipan; Bosse, Jens Bernhard & Messerle, Martin
