Protein interactions and consensus clustering analysis uncover insights into herpesvirus virion structure and function relationships.

Infections with human herpesviruses are ubiquitous and a public health concern worldwide. Current treatments reduce the severity of some symptoms associated to herpetic infections but neither remove the viral reservoir from the infected host nor protect from the recurrent symptom outbreaks that characterise herpetic infections. The difficulty in therapeutically tackling these viral systems stems in part from their remarkably large proteomes and the complex networks of physical and functional associations that they tailor. This study presents our efforts to unravel the complexity of the interactome of herpes simplex virus type 1 (HSV1), the prototypical herpesvirus species. Inspired by our previous work, we present an improved and more integrative computational pipeline for the protein-protein interaction (PPI) network reconstruction in HSV1, together with a newly developed consensus clustering framework, which allowed us to extend the analysis beyond binary physical interactions and revealed a system-level layout of higher-order functional associations in the virion proteome. Additionally, the analysis provided new functional annotation for the currently undercharacterised protein pUS10. In-depth bioinformatics sequence analysis unravelled structural features in pUS10 reminiscent of those observed in some capsid-associated proteins in tailed bacteriophages, with which herpesviruses are believed to share a common ancestry. Using immunoaffinity purification (IP)-mass spectrometry (MS), we obtained additional support for our bioinformatically predicted interaction between pUS10 and the inner tegument protein pUL37, which binds cytosolic capsids, contributing to initial tegumentation and eventually virion maturation. In summary, this study unveils new, to our knowledge, insights at both the system and molecular levels that can help us better understand the complexity behind herpesvirus infections.

Two distinct trimeric conformations of natively membrane-anchored full-length herpes simplex virus 1 glycoprotein B.

Many viruses are enveloped by a lipid bilayer acquired during assembly, which is typically studded with one or two types of glycoproteins. These viral surface proteins act as the primary interface between the virus and the host. Entry of enveloped viruses relies on specialized fusogen proteins to help merge the virus membrane with the host membrane. In the multicomponent herpesvirus fusion machinery, glycoprotein B (gB) acts as this fusogen. Although the structure of the gB ectodomain postfusion conformation has been determined, any other conformations (e.g., prefusion, intermediate conformations) have so far remained elusive, thus restricting efforts to develop antiviral treatments and prophylactic vaccines. Here, we have characterized the full-length herpes simplex virus 1 gB in a native membrane by displaying it on cell-derived vesicles and using electron cryotomography. Alongside the known postfusion conformation, a novel one was identified. Its structure, in the context of the membrane, was determined by subvolume averaging and found to be trimeric like the postfusion conformation, but appeared more condensed. Hierarchical constrained density-fitting of domains unexpectedly revealed the fusion loops in this conformation to be apart and pointing away from the anchoring membrane. This vital observation is a substantial step forward in understanding the complex herpesvirus fusion mechanism, and opens up new opportunities for more targeted intervention of herpesvirus entry.

Conserved Central Intraviral Protein Interactome of the Herpesviridae Family.

Protein interactions are major driving forces behind the functional phenotypes of biological processes. As such, evolutionary footprints are reflected in system-level collections of protein-protein interactions (PPIs), i.e., protein interactomes. We conducted a comparative analysis of intraviral protein interactomes for representative species of each of the three subfamilies of herpesviruses (herpes simplex virus 1, human cytomegalovirus, and Epstein-Barr virus), which are highly prevalent etiologic agents of important human diseases. The intraviral interactomes were reconstructed by combining experimentally supported and computationally predicted protein-protein interactions. Using cross-species network comparison, we then identified family-wise conserved interactions and protein complexes, which we defined as a herpesviral "central" intraviral protein interactome. A large number of widely accepted conserved herpesviral protein complexes are present in this central intraviral interactome, encouragingly supporting the biological coherence of our results. Importantly, these protein complexes represent most, if not all, of the essential steps required during a productive life cycle. Hence the central intraviral protein interactome could plausibly represent a minimal infectious interactome of the herpesvirus family across a variety of hosts. Our data, which have been integrated into our herpesvirus interactomics database, HVint2.0, could assist in creating comprehensive system-level computational models of this viral lineage.IMPORTANCE Herpesviruses are an important socioeconomic burden for both humans and livestock. Throughout their long evolutionary history, individual herpesvirus species have developed remarkable host specificity, while collectively the Herpesviridae family has evolved to infect a large variety of eukaryotic hosts. The development of approaches to fight herpesvirus infections has been hampered by the complexity of herpesviruses' genomes, proteomes, and structural features. The data and insights generated by our study add to the understanding of the functional organization of herpesvirus-encoded proteins, specifically of family-wise conserved features defining essential components required for a productive infectious cycle across different hosts, which can contribute toward the conceptualization of antiherpetic infection strategies with an effect on a broader range of target species. All of the generated data have been made freely available through our HVint2.0 database, a dedicated resource of curated herpesvirus interactomics purposely created to promote and assist future studies in the field.