Herpesviruses uniquely express two essential nuclear egress-regulating proteins forming a heterodimeric basic structure of the nuclear egress complex (core NEC). These core NECs serve as a hexameric lattice-structured platform for capsid docking and recruit viral and cellular NEC-associated factors that jointly exert nuclear lamina- and membrane-rearranging functions (multicomponent NEC). Here, we report the X-ray structures of β- and γ-herpesvirus core NECs obtained through an innovative recombinant expression strategy based on NEC-hook::NEC-groove protein fusion constructs. This approach yielded the first structure of γ-herpesviral core NEC, namely the 1.56 Å structure of Epstein-Barr virus (EBV) BFRF1–BFLF2, as well as an increased resolution 1.48 Å structure of human cytomegalovirus (HCMV) pUL50-pUL53. Detailed analysis of these structures revealed that the prominent hook segment is absolutely required for core NEC formation and contributes approximately 80% of the interaction surface of the globular domains of NEC proteins. Moreover, using HCMV::EBV hook domain swap constructs, computational prediction of the roles of individual hook residues for binding, and quantitative binding assays with synthetic peptides presenting the HCMV- and EBV-specific NEC hook sequences, we characterized the unique hook-into-groove NEC interaction at various levels. Although the overall physicochemical characteristics of the protein interfaces differ considerably in these β- and γ-herpesvirus NECs, the binding free energy contributions of residues displayed from identical positions are similar. In summary, the results of our study reveal critical details of the molecular mechanism of herpesviral NEC interactions and highlight their potential as an antiviral drug target.

Herpesviruses uniquely express two essential nuclear egress-regulating proteins forming a heterodimeric nuclear egress complex (core NEC). These core NECs serve as hexameric lattice-structured platforms for capsid docking and recruit viral and cellular NEC-associated factors that jointly exert nuclear lamina as well as membrane-rearranging functions (multicomponent NEC). The regulation of nuclear egress has been profoundly analyzed for murine and human cytomegaloviruses (CMVs) on a mechanistic basis, followed by the description of core NEC crystal structures, first for HCMV, then HSV-1, PRV and EBV. Interestingly, the highly conserved structural domains of these proteins stand in contrast to a very limited sequence conservation of the key amino acids within core NEC-binding interfaces. Even more surprising, although a high functional consistency was found when regarding the basic role of NECs in nuclear egress, a clear specification was identified regarding the limited, subfamily-spanning binding properties of core NEC pairs and NEC multicomponent proteins. This review summarizes the evolving picture of the relationship between sequence coevolution, structural conservation and properties of NEC interaction, comparing HCMV to α-, β- and γ-herpesviruses. Since NECs represent substantially important elements of herpesviral replication that are considered as drug-accessible targets, their putative translational use for antiviral strategies is discussed.

Herpesviral nuclear egress is a regulated process shared by all family members, ensuring the efficient cytoplasmic release of viral capsids. In the case of human cytomegalovirus (HCMV), the core of the nuclear egress complex (NEC) consists of the pUL50-pUL53 heterodimer that builds hexameric lattices for capsid binding and multicomponent interaction, including NEC-associated host factors. A characteristic feature of NEC interaction is the N-terminal hook structure of pUL53 that binds to an alpha-helical groove of pUL50, thus termed as hook-into-groove interaction. This central regulatory element is essential for viral replication and shows structural–functional conservation, which has been postulated as a next-generation target of antiviral strategies. However, a solid validation of this concept has been missing. In the present study, we focused on the properties of oligomeric HCMV core NEC interaction and the antiviral activity of specifically targeted prototype inhibitors. Our data suggest the following: (i) transiently expressed, variably tagged versions of HCMV NEC proteins exert hook-into-groove complexes, putatively in oligomeric assemblies that are distinguishable from heterodimers, as shown by in vitro assembly and coimmunoprecipitation approaches; (ii) this postulated oligomeric binding pattern was further supported by the use of a pUL50::pUL53 fusion construct also showing a pronounced multi-interaction potency; (iii) using confocal imaging cellular NEC-associated proteins were found partly colocalized with the tagged core NECs; (iv) a small inhibitory molecule, recently identified by an in vitro binding inhibition assay, was likewise active in blocking pUL50–pUL53 oligomeric assembly and in exerting antiviral activity in HCMV-infected fibroblasts. In summary, the findings refine the previous concept of HCMV core NEC formation and nominate this drug-accessible complex as a validated antiviral drug target.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Nuclear egress is an essential process in the replication of human cytomegalovirus (HCMV), as it enables the migration of newly formed viral capsids from the nucleus into the cytoplasm. Inhibition of the HCMV core nuclear egress complex (core NEC), composed of viral proteins pUL50 and pUL53, has been proposed as a potential new target for the treatment of HCMV infection and disease. Here, we present a new type of small molecule inhibitors of HCMV core NEC formation, which inhibit the pUL50-pUL53 interaction at nanomolar concentrations. These inhibitors, i.e., verteporfin and merbromin, were identified through the screening of the Prestwick Chemical Library® of approved drug compounds. The inhibitory effect of merbromin is both compound- and target-specific, as no inhibition was seen for other mercury-organic compounds. Furthermore, merbromin does not inhibit an unrelated protein–protein interaction either. More importantly, merbromin was found to inhibit HCMV infection of cells in three different assays, as well as to disrupt HCMV NEC nuclear rim formation. Thus, while not being an ideal drug candidate by itself, merbromin may serve as a blueprint for small molecules with high HCMV core NEC inhibitory potential, as candidates for novel anti-herpesviral drugs.