Human herpesvirus 6A nuclear matrix protein U37 interacts with heat shock transcription factor 1 and activates the heat shock response.

Nascent nucleocapsids of herpesviruses acquire a primary envelope during their nuclear export by budding through the inner nuclear membrane into the perinuclear space between the inner and outer nuclear membranes. This process is mediated by a conserved viral heterodimeric complex designated the nuclear egress complex, which consists of the nuclear matrix protein and the nuclear membrane protein. In addition to its essential roles during nuclear egress, the nuclear matrix protein has been shown to interact with intracellular signaling pathway molecules including NF-κB and IFN-ß to affect viral or cellular gene expression. The human herpesvirus 6A (HHV-6A) U37 gene encodes a nuclear matrix protein, the role of which has not been analyzed. Here, we show that HHV-6A U37 activates the heat shock element promoter and induces the accumulation of the molecular chaperone Hsp90. Mechanistically, HHV-6A U37 interacts with heat shock transcription factor 1 (HSF1) and induces its phosphorylation at Ser-326. We report that pharmacological inhibition of HSF1, Hsp70, or Hsp90 decreases viral protein accumulation and viral replication. Taken together, our results lead us to propose a model in which HHV-6A U37 activates the heat shock response to support viral gene expression and replication. IMPORTANCE Human herpesvirus 6A (HHV-6A) is a dsDNA virus belonging to the Roseolovirus genus within the Betaherpesvirinae subfamily. It is frequently found in patients with neuroinflammatory disease, although its pathogenetic role, if any, awaits elucidation. The heat shock response is important for cell survival under stressful conditions that disrupt homeostasis. Our results indicate that HHV-6A U37 activates the heat shock element promoter and leads to the accumulation of heat shock proteins. Next, we show that the heat shock response is important for viral replication. Overall, our findings provide new insights into the function of HHV-6A U37 in host cell signaling and identify potential cellular targets involved in HHV-6A pathogenesis and replication.

Extragenic suppression of an HSV-1 UL34 nuclear egress mutant reveals role for pUS9 as an inhibitor of epithelial cell-to-cell spread.

IMPORTANCE: Herpesviruses are able to disseminate in infected hosts despite development of a strong immune response. Their ability to do this relies on a specialized process called cell-to-cell spread in which newly assembled virus particles are trafficked to plasma membrane surfaces that abut adjacent uninfected cells. The mechanism of cell-to-cell spread is obscure, and little is known about whether or how it is regulated in different cells. We show here that a viral protein with a well-characterized role in promoting spread from neurons has an opposite, inhibitory role in other cells.

'Getting Better'-Is It a Feasible Strategy of Broad Pan-Antiherpesviral Drug Targeting by Using the Nuclear Egress-Directed Mechanism?

The herpesviral nuclear egress represents an essential step of viral replication efficiency in host cells, as it defines the nucleocytoplasmic release of viral capsids. Due to the size limitation of the nuclear pores, viral nuclear capsids are unable to traverse the nuclear envelope without a destabilization of this natural host-specific barrier. To this end, herpesviruses evolved the regulatory nuclear egress complex (NEC), composed of a heterodimer unit of two conserved viral NEC proteins (core NEC) and a large-size extension of this complex including various viral and cellular NEC-associated proteins (multicomponent NEC). Notably, the NEC harbors the pronounced ability to oligomerize (core NEC hexamers and lattices), to multimerize into higher-order complexes, and, ultimately, to closely interact with the migrating nuclear capsids. Moreover, most, if not all, of these NEC proteins comprise regulatory modifications by phosphorylation, so that the responsible kinases, and additional enzymatic activities, are part of the multicomponent NEC. This sophisticated basis of NEC-specific structural and functional interactions offers a variety of different modes of antiviral interference by pharmacological or nonconventional inhibitors. Since the multifaceted combination of NEC activities represents a highly conserved key regulatory stage of herpesviral replication, it may provide a unique opportunity towards a broad, pan-antiherpesviral mechanism of drug targeting. This review presents an update on chances, challenges, and current achievements in the development of NEC-directed antiherpesviral strategies.

The crystal structure of the varicella-zoster Orf24-Orf27 nuclear egress complex spotlights multiple determinants of herpesvirus subfamily specificity.

Varicella-zoster virus (VZV) is a human pathogen from the α-subfamily of herpesviruses. The VZV Orf24-Orf27 complex represents the essential viral core nuclear egress complex (NEC) that orchestrates the egress of the preassembled virus capsids from the nucleus. While previous studies have primarily emphasized that the architecture of core NEC complexes is highly conserved among herpesviruses, the present report focuses on subfamily-specific structural and functional features that help explain the differences in the autologous versus nonautologous interaction patterns observed for NEC formation across herpesviruses. Here, we describe the crystal structure of the Orf24-Orf27 complex at 2.1 Å resolution. Coimmunoprecipitation and confocal imaging data show that Orf24-Orf27 complex formation displays some promiscuity in a herpesvirus subfamily-restricted manner. At the same time, analysis of thermodynamic parameters of NEC formation of three prototypical α-, ß-, and γ herpesviruses, i.e., VZV, human cytomegalovirus (HCMV), and Epstein-Barr virus (EBV), revealed highly similar binding affinities for the autologous interaction with specific differences in enthalpy and entropy. Computational alanine scanning, structural comparisons, and mutational data highlight intermolecular interactions shared among α-herpesviruses that are clearly distinct from those seen in ß- and γ-herpesviruses, including a salt bridge formed between Orf24-Arg167 and Orf27-Asp126. This interaction is located outside of the hook-into-groove interface and contributes significantly to the free energy of complex formation. Combined, these data explain distinct properties of specificity and permissivity so far observed in herpesviral NEC interactions. These findings will prove valuable in attempting to target multiple herpesvirus core NECs with selective or broad-acting drug candidates.

Role of the Arginine Cluster in the Disordered Domain of Herpes Simplex Virus 1 UL34 for the Recruitment of ESCRT-III for Viral Primary Envelopment.

During the nuclear export of nascent nucleocapsids of herpesviruses, the nucleocapsids bud through the inner nuclear membrane (INM) by acquiring the INM as a primary envelope (primary envelopment). We recently reported that herpes simplex virus 1 (HSV-1) nuclear egress complex (NEC), which consists of UL34 and UL31, interacts with an endosomal sorting complex required for transport III (ESCRT-III) adaptor ALIX and recruits ESCRT-III machinery to the INM for efficient primary envelopment. In this study, we identified a cluster of six arginine residues in the disordered domain of UL34 as a minimal region required for the interaction with ALIX, as well as the recruitment of ALIX and an ESCRT-III protein CHMP4B to the INM in HSV-1-infected cells. Mutations in the arginine cluster exhibited phenotypes similar to those with ESCRT-III inhibition reported previously, including the mislocalization of NEC, induction of membranous invagination structures containing enveloped virions, aberrant accumulation of enveloped virions in the invaginations and perinuclear space, and reduction of viral replication. We also showed that the effect of the arginine cluster in UL34 on HSV-1 replication was dependent primarily on ALIX. These results indicated that the arginine cluster in the disordered domain of UL34 was required for the interaction with ALIX and the recruitment of ESCRT-III machinery to the INM to promote primary envelopment. IMPORTANCE Herpesvirus UL34 homologs contain conserved amino-terminal domains that mediate vesicle formation through interactions with UL31 homologs during primary envelopment. UL34 homologs also comprise other domains adjacent to their membrane-anchoring regions, which differ in length, are variable in herpesviruses, and do not form distinguished secondary structures. However, the role of these disordered domains in infected cells remains to be elucidated. In this study, we present data suggesting that the arginine cluster in the disordered domain of HSV-1 UL34 mediates the interaction with ALIX, thereby leading to the recruitment of ESCRT-III machinery to the INM for efficient primary envelopment. This is the first study to report the role of the disordered domain of a UL34 homolog in herpesvirus infections.

Role of the Orphan Transporter SLC35E1 in the Nuclear Egress of Herpes Simplex Virus 1.

This study developed a system consisting of two rounds of screening cellular proteins involved in the nuclear egress of herpes simplex virus 1 (HSV-1). Using this system, we first screened cellular proteins that interacted with the HSV-1 nuclear egress complex (NEC) consisting of UL34 and UL31 in HSV-1-infected cells, which are critical for the nuclear egress of HSV-1, by tandem affinity purification coupled with mass spectrometry-based proteomics technology. Next, we performed CRISPR/Cas9-based screening of live HSV-1-infected reporter cells under fluorescence microscopy using single guide RNAs targeting the cellular proteins identified in the first proteomic screening to detect the mislocalization of the lamin-associated protein emerin, which is a phenotype for defects in HSV-1 nuclear egress. This study focused on a cellular orphan transporter SLC35E1, one of the cellular proteins identified by the screening system. Knockout of SLC35E1 reduced HSV-1 replication and induced membranous invaginations containing perinuclear enveloped virions (PEVs) adjacent to the nuclear membrane (NM), aberrant accumulation of PEVs in the perinuclear space between the inner and outer NMs and the invagination structures, and mislocalization of the NEC. These effects were similar to those of previously reported mutation(s) in HSV-1 proteins and depletion of cellular proteins that are important for HSV-1 de-envelopment, one of the steps required for HSV-1 nuclear egress. Our newly established screening system enabled us to identify a novel cellular protein required for efficient HSV-1 de-envelopment. IMPORTANCE The identification of cellular protein(s) that interact with viral effector proteins and function in important viral procedures is necessary for enhancing our understanding of the mechanics of various viral processes. In this study, we established a new system consisting of interactome screening for the herpes simplex virus 1 (HSV-1) nuclear egress complex (NEC), followed by loss-of-function screening to target the identified putative NEC-interacting cellular proteins to detect a defect in HSV-1 nuclear egress. This newly established system identified SLC35E1, an orphan transporter, as a novel cellular protein required for efficient HSV-1 de-envelopment, providing an insight into the mechanisms involved in this viral procedure.

Redundant and Specific Roles of A-Type Lamins and Lamin B Receptor in Herpes Simplex Virus 1 Infection.

We investigated whether A-type lamins (lamin A/C) and lamin B receptor (LBR) are redundant during herpes simplex virus 1 (HSV-1) infection in HeLa cells expressing lamin A/C and LBR. Lamin A/C and LBR double knockout (KO) in HSV-1-infected HeLa cells significantly impaired expressions of HSV-1 early and late genes, maturation of replication compartments, marginalization of host chromatin to the nuclear periphery, enlargement of host cell nuclei, and viral DNA replication. Phenotypes of HSV-1-infected HeLa cells were restored by the ectopic expression of lamin A/C or LBR in lamin A/C and LBR double KO cells. Of note, lamin A/C single KO, but not LBR single KO, promoted the aberrant accumulation of virus particles outside the inner nuclear membrane (INM) and viral replication, as well as decreasing the frequency of virus particles inside the INM without affecting viral gene expression and DNA replication, time-spatial organization of replication compartments and host chromatin, and nuclear enlargement. These results indicated that lamin A/C and LBR had redundant and specific roles during HSV-1 infection. Thus, lamin A/C and LBR redundantly regulated the dynamics of the nuclear architecture, including the time-spatial organization of replication compartments and host chromatin, as well as promoting nuclear enlargement for efficient HSV-1 gene expression and DNA replication. In contrast, lamin A/C inhibited HSV-1 nuclear export through the INM during viral nuclear egress, which is a unique property of lamin A/C. IMPORTANCE This study demonstrated that lamin A/C and LBR had redundant functions associated with HSV-1 gene expression and DNA replication by regulating the dynamics of the nuclear architecture during HSV-1 infection. This is the first report to demonstrate the redundant roles of lamin A/C and LBR as well as the involvement of LBR in the regulation of these viral and cellular features in HSV-1-infected cells. These findings provide evidence for the specific property of lamin A/C to inhibit HSV-1 nuclear egress, which has long been considered but without direct proof.

Cell Culture Evolution of a Herpes Simplex Virus 1 (HSV-1)/Varicella-Zoster Virus (VZV) UL34/ORF24 Chimeric Virus Reveals Novel Functions for HSV Genes in Capsid Nuclear Egress.

Herpes simplex virus (HSV) and varicella-zoster virus (VZV) are both members of the alphaherpesvirus subfamily but belong to different genera. Substitution of the HSV-1 UL34 coding sequence with that of its VZV homolog, open reading frame 24 (ORF24), results in a virus that has defects in viral growth, spread, capsid egress, and nuclear lamina disruption very similar to those seen in a UL34-null virus despite normal interaction between ORF24 protein and HSV pUL31 and proper localization of the nuclear egress complex at the nuclear envelope. Minimal selection for growth in cell culture resulted in viruses that grew and spread much more efficiently that the parental chimeric virus. These viruses varied in their ability to support nuclear lamina disruption, normal nuclear egress complex localization, and capsid de-envelopment. Single mutations that suppress the growth defect were mapped to the coding sequences of ORF24, ICP22, and ICP4, and one virus carried single mutations in each of the ICP22 and US3 coding sequences. The phenotypes of these viruses support a role for ICP22 in nuclear lamina disruption and a completely unexpected role for the major transcriptional regulator, ICP4, in capsid nuclear egress. IMPORTANCE Interactions among virus proteins are critical for assembly and egress of virus particles, and such interactions are attractive targets for antiviral therapy. Identification of critical functional interactions can be slow and tedious. Capsid nuclear egress of herpesviruses is a critical event in the assembly and egress pathway and is mediated by two proteins, pUL31 and pUL34, that are conserved among herpesviruses. Here, we describe a cell culture evolution approach to identify other viral gene products that functionally interact with pUL34.

Herpes Simplex Virus 1 UL34 Mutants That Affect Membrane Budding Regulation and Nuclear Lamina Disruption.

Nuclear envelope budding in herpesvirus nuclear egress may be negatively regulated, since the pUL31/pUL34 nuclear egress complex heterodimer can induce membrane budding without capsids when expressed ectopically or on artificial membranes in vitro, but not in the infected cell. We have previously described a pUL34 mutant that contained alanine substitutions at R158 and R161 and that showed impaired growth, impaired pUL31/pUL34 interaction, and unregulated budding. Here, we determine the phenotypic contributions of the individual substitutions to these phenotypes. Neither substitution alone was able to reproduce the impaired growth or nuclear egress complex (NEC) interaction phenotypes. Either substitution, however, could fully reproduce the unregulated budding phenotype, suggesting that misregulated budding may not substantially impair virus replication. In addition, the R158A substitution caused relocalization of the NEC to intranuclear punctate structures and recruited lamin A/C to these structures, suggesting that this residue might be important for recruitment of kinases for dispersal of nuclear lamins. IMPORTANCE Herpesvirus nuclear egress is a complex, regulated process coordinated by two virus proteins that are conserved among the herpesviruses that form a heterodimeric nuclear egress complex (NEC). The NEC drives budding of capsids at the inner nuclear membrane and recruits other viral and host cell proteins for disruption of the nuclear lamina, membrane scission, and fusion. The structural basis of individual activities of the NEC, apart from membrane budding, are not clear, nor is the basis of the regulation of membrane budding. Here, we explore the properties of NEC mutants that have an unregulated budding phenotype, determine the significance of that regulation for virus replication, and also characterize a structural requirement for nuclear lamina disruption.

High-resolution crystal structures of two prototypical ß- and γ-herpesviral nuclear egress complexes unravel the determinants of subfamily specificity.

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 ß- 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 ß- 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.