MYBBP1A is required for efficient replication and gene expression of herpes simplex virus 1.

More than 100 different herpes simplex virus 1 (HSV-1) genes belong to three major classes, and their expression is coordinately regulated and sequentially ordered in a cascade. This complex HSV-1 gene expression is thought to be regulated by various viral and host cellular proteins. A host cellular protein, Myb-binding protein 1A (MYBBP1A), has been reported to be associated with HSV-1 viral genomes in conjunction with viral and cellular proteins critical for DNA replication, repair, and transcription within infected cells. However, the role(s) of MYBBP1A in HSV-1 infections remains unclear. In this study, we examined the effects of MYBBP1A depletion on HSV-1 infection and found that MYBBP1A depletion significantly reduced HSV-1 replication, as well as the accumulation of several viral proteins. These results suggest that MYBBP1A is an important host cellular factor that contributes to HSV-1 replication, plausibly by promoting viral gene expression.

Dual impacts of a glycan shield on the envelope glycoprotein B of HSV-1: evasion from human antibodies in vivo and neurovirulence.

Identification of the mechanisms of viral evasion from human antibodies is crucial both for understanding viral pathogenesis and for designing effective vaccines. Here we show in cell cultures that an N-glycan shield on the herpes simplex virus 1 (HSV-1) envelope glycoprotein B (gB) mediated evasion from neutralization and antibody-dependent cellular cytotoxicity due to pooled γ-globulins derived from human blood. We also demonstrated that the presence of human γ-globulins in mice and immunity to HSV-1 induced by viral infection in mice significantly reduced replication in their eyes of a mutant virus lacking the glycosylation site but had little effect on the replication of its repaired virus. These results suggest that an N-glycan shield on a specific site of HSV-1 envelope gB mediated evasion from human antibodies in vivo and from HSV-1 immunity induced by viral infection in vivo. Notably, we also found that an N-glycan shield on a specific site of HSV-1 gB was significant for HSV-1 neurovirulence and replication in the central nervous system of naïve mice. Thus, we have identified a critical N-glycan shield on HSV-1 gB that has dual impacts, namely evasion from human antibodies in vivo and viral neurovirulence. IMPORTANCE Herpes simplex virus 1 (HSV-1) establishes lifelong latent and recurrent infections in humans. To produce recurrent infections that contribute to transmission of the virus to new human host(s), the virus must be able to evade the antibodies persisting in latently infected individuals. Here, we show that an N-glycan shield on the specific site of the envelope glycoprotein B (gB) of HSV-1 mediates evasion from pooled γ-globulins derived from human blood both in cell cultures and mice. Notably, the N-glycan shield on the specific site of gB was also significant for HSV-1 neurovirulence in naïve mice. Considering the clinical features of HSV-1 infection, these results suggest that the glycan shield not only facilitates recurrent HSV-1 infections in latently infected humans by evading antibodies but is also important for HSV-1 pathogenesis during the initial infection.

Establishment of a system to quantify wild-type herpes simplex virus-induced cell-cell fusion reveals a role of N-glycosylation of HSV-1 envelope glycoprotein B in cell-cell fusion.

Wild-type herpes simplex virus (HSV) strains infrequently mediate cell-cell fusion in cell cultures and barely induce large multinucleated cells. In this study, we established a system to quantify infrequent cell-cell fusion induced by wild-type HSV strains. The established system clarified that the HSV-1 envelope glycoprotein B and its N-glycosylation at asparagine at position 141 were required for efficient cell-cell fusion. This study provides a link between cell-cell fusion induced by wild-type HSV-1 and viral pathogenesis in vivo.

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.

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.

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 DNA Binding Activity of Herpes Simplex Virus 1 VP22 in Evading AIM2-Dependent Inflammasome Activation Induced by the Virus.

AIM2 is a cytosolic DNA sensor of the inflammasome, which induces critical innate immune responses against various invading pathogens. Earlier biochemical studies showed that the binding of AIM2 to DNA triggered the self-oligomerization of AIM2, which is essential for AIM2 inflammasome activation. We recently reported that VP22, a virion tegument protein of herpes simplex virus 1 (HSV-1), inhibited activation of the AIM2 inflammasome in HSV-1-infected cells by preventing AIM2 oligomerization. VP22 binds non-specifically to DNA; however, its role in HSV-1 replication is unclear. We investigated the role of VP22 DNA binding activity in the VP22-mediated inhibition of AIM2 inflammasome activation. We identified a VP22 domain encoded by amino acids 227 to 258 as the minimal domain required for its binding to DNA in vitro Consecutive alanine substitutions in this domain substantially impaired the DNA binding activity of VP22 in vitro and attenuated the inhibitory effect of VP22 on AIM2 inflammasome activation in an AIM2 inflammasome reconstitution system. The inhibitory effect of VP22 on AIM2 inflammasome activation was completely abolished in macrophages infected with a recombinant virus harboring VP22 with one of the consecutive alanine substitutions, similar to the effect of a VP22-null mutant virus. These results suggested that the DNA binding activity of VP22 is critical for VP22-mediated AIM2 inflammasome activation in HSV1-infected cells.IMPORTANCE VP22, a major component of the HSV-1 virion tegument, is conserved in alphaherpesviruses and has structural similarity to ORF52, a component of the virion tegument that is well-conserved in gammaherpesviruses. Although the potential DNA binding activity of VP22 was discovered decades ago, its significance in the HSV-1 life cycle is poorly understood. Here, we show that the DNA binding activity of VP22 is critical for the inhibition of AIM2 inflammasome activation induced in HSV-1-infected cells. This is the first report to show a role for the DNA binding activity of VP22 in the HSV-1 life cycle, allowing the virus to evade AIM2 inflammasome activation, which is critical for its replication in vivo.

Prohibitin-1 Contributes to Cell-to-Cell Transmission of Herpes Simplex Virus 1 via the MAPK/ERK Signaling Pathway.

Viral cell-to-cell spread, a method employed by several viral families for entrance via cell junctions, is highly relevant to the pathogenesis of various viral infections. Cell-to-cell spread of herpes simplex virus 1 (HSV-1) is known to depend greatly on envelope glycoprotein E (gE). However, the molecular mechanism by which gE acts in HSV-1 cell-to-cell spread and the mechanisms of cell-to-cell spread by other herpesviruses remain poorly understood. Here, we describe our identification of prohibitin-1 as a novel gE-interacting host cell protein. Ectopic expression of prohibitin-1 increased gE-dependent HSV-1 cell-to-cell spread. As observed with the gE-null mutation, decreased expression or pharmacological inhibition of prohibitin-1 reduced HSV-1 cell-to-cell spread without affecting the yield of virus progeny. Similar effects were produced by pharmacological inhibition of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, wherein prohibitin-1 acts as a protein scaffold and is required for induction of this pathway. Furthermore, artificial activation of the MAPK/ERK pathway restored HSV-1 cell-to-cell spread impaired by the gE-null mutation. Notably, pharmacological inhibition of prohibitins or the MAPK/ERK pathway reduced viral cell-to-cell spread of representative members in all herpesvirus subfamilies. Our results suggest that prohibitin-1 contributes to gE-dependent HSV-1 cell-to-cell spread via the MAPK/ERK pathway and that this mechanism is conserved throughout the Herpesviridae, whereas gE is conserved only in the Alphaherpesvirinae subfamily.IMPORTANCE Herpesviruses are ubiquitous pathogens of various animals, including humans. These viruses primarily pass through cell junctions to spread to uninfected cells. This method of cell-to-cell spread is an important pathogenic characteristic of these viruses. Here, we show that the host cell protein prohibitin-1 contributes to HSV-1 cell-to-cell spread via a downstream intracellular signaling cascade, the MAPK/ERK pathway. We also demonstrate that the role of the prohibitin-1-mediated MAPK/ERK pathway in viral cell-to-cell spread is conserved in representative members of every herpesvirus subfamily. This study has revealed a common molecular mechanism of the cell-to-cell spread of herpesviruses.

ESCRT-III controls nuclear envelope deformation induced by progerin.

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder, caused by mutation in the gene encoding lamin A/C, which produces a truncated protein called progerin. In cells from HGPS patients, progerin accumulates at the nuclear membrane (NM), where it causes NM deformations. In this study, we investigated whether progerin-induced NM deformation involved ESCRT-III, a protein complex that remodels nuclear and cytoplasmic membranes. The ESCRT-III protein CHMP4B was recruited to sites of aberrant NM proliferation in human cells ectopically expressing progerin and in patient-derived HGPS fibroblasts. Derepression of NM deformation in these cells was observed following depletion of CHMP4B or an ESCRT-III adaptor, ALIX. Treatment with rapamycin (which induce autophagic clearance of progerin and reverse progerin-induced cellular phenotypes) down-regulated progerin-induced NM deformation, whereas treatment with bafilomycin A1 (an inhibitor of autophagy and lysosome-based degradation) or CHMP4B depletion antagonized the effects of rapamycin. These results indicate that the ALIX-mediated ESCRT-III pathway plays a suppressive role in progerin-induced NM deformation and suggest that autophagy down-regulates progerin-induced NM deformation in a manner dependent on ESCRT-III machinery.

Role of Phosphatidylethanolamine Biosynthesis in Herpes Simplex Virus 1-Infected Cells in Progeny Virus Morphogenesis in the Cytoplasm and in Viral Pathogenicity In Vivo.

Glycerophospholipids are major components of cell membranes. Phosphatidylethanolamine (PE) is a glycerophospholipid that is involved in multiple cellular processes, such as membrane fusion, the cell cycle, autophagy, and apoptosis. In this study, we investigated the role of PE biosynthesis in herpes simplex virus 1 (HSV-1) infection by knocking out the host cell gene encoding phosphate cytidylyltransferase 2, ethanolamine (Pcyt2), which is a key rate-limiting enzyme in one of the two major pathways for PE biosynthesis. Pcyt2 knockout reduced HSV-1 replication and caused an accumulation of unenveloped and partially enveloped nucleocapsids in the cytoplasm of an HSV-1-infected cell culture. A similar phenotype was observed when infected cells were treated with meclizine, which is an inhibitor of Pcyt2. In addition, treatment of HSV-1-infected mice with meclizine significantly reduced HSV-1 replication in the mouse brains and improved their survival rates. These results indicated that PE biosynthesis mediated by Pcyt2 was required for efficient HSV-1 envelopment in the cytoplasm of infected cells and for viral replication and pathogenicity in vivo The results also identified the PE biosynthetic pathway as a possible novel target for antiviral therapy of HSV-associated diseases and raised an interesting possibility for meclizine repositioning for treatment of these diseases, since it is an over-the-counter drug that has been used for decades against nausea and vertigo in motion sickness.IMPORTANCE Glycerophospholipids in cell membranes and virus envelopes often affect viral entry and budding. However, the role of glycerophospholipids in membrane-associated events in viral replication in herpesvirus-infected cells has not been reported to date. In this study, we have presented data showing that cellular PE biosynthesis mediated by Pcyt2 is important for HSV-1 envelopment in the cytoplasm, as well as for viral replication and pathogenicity in vivo This is the first report showing the importance of PE biosynthesis in herpesvirus infections. Our results showed that inhibition of Pcyt2, a key cell enzyme for PE synthesis, significantly inhibited HSV-1 replication and pathogenicity in mice. This suggested that the PE biosynthetic pathway, as well as the HSV-1 virion maturation pathway, can be a target for the development of novel anti-HSV drugs.

Identification of a herpes simplex virus 1 gene encoding neurovirulence factor by chemical proteomics.

Identification of the complete set of translated genes of viruses is important to understand viral replication and pathogenesis as well as for therapeutic approaches to control viral infection. Here, we use chemical proteomics, integrating bio-orthogonal non-canonical amino acid tagging and high-resolution mass spectrometry, to characterize the newly synthesized herpes simplex virus 1 (HSV-1) proteome in infected cells. In these infected cells, host cellular protein synthesis is shut-off, increasing the chance to preferentially detect viral proteomes. We identify nine previously cryptic orphan protein coding sequences whose translated products are expressed in HSV-1-infected cells. Functional characterization of one identified protein, designated piUL49, shows that it is critical for HSV-1 neurovirulence in vivo by regulating the activity of virally encoded dUTPase, a key enzyme that maintains accurate DNA replication. Our results demonstrate that cryptic orphan protein coding genes of HSV-1, and probably other large DNA viruses, remain to be identified.

Phosphoregulation of a Conserved Herpesvirus Tegument Protein by a Virally Encoded Protein Kinase in Viral Pathogenicity and Potential Linkage between Its Evolution and Viral Phylogeny.

Us3 proteins of herpes simplex virus 1 (HSV-1) and HSV-2 are multifunctional serine-threonine protein kinases. Here, we identified an HSV-2 tegument protein, UL7, as a novel physiological substrate of HSV-2 Us3. Mutations in HSV-2 UL7, which precluded Us3 phosphorylation of the viral protein, significantly reduced mortality, viral replication in the vagina, and development of vaginal disease in mice following vaginal infection. These results indicated that Us3 phosphorylation of UL7 in HSV-2 was required for efficient viral replication and pathogenicity in vivo Of note, this phosphorylation was conserved in UL7 of chimpanzee herpesvirus (ChHV), which phylogenetically forms a monophyletic group with HSV-2 and the resurrected last common ancestral UL7 for HSV-2 and ChHV. In contrast, the phosphorylation was not conserved in UL7s of HSV-1, which belongs to a sister clade of the monophyletic group, the resurrected last common ancestor for HSV-1, HSV-2, and ChHV, and other members of the genus Simplexvirus that are phylogenetically close to these viruses. Thus, evolution of Us3 phosphorylation of UL7 coincided with the phylogeny of simplex viruses. Furthermore, artificially induced Us3 phosphorylation of UL7 in HSV-1, in contrast to phosphorylation in HSV-2, had no effect on viral replication and pathogenicity in mice. Our results suggest that HSV-2 and ChHV have acquired and maintained Us3 phosphoregulation of UL7 during their evolution because the phosphoregulation had an impact on viral fitness in vivo, whereas most other simplex viruses have not because the phosphorylation was not necessary for efficient fitness of the viruses in vivoIMPORTANCE It has been hypothesized that the evolution of protein phosphoregulation drives phenotypic diversity across species of organisms, which impacts fitness during their evolution. However, there is a lack of information regarding linkage between the evolution of viral phosphoregulation and the phylogeny of virus species. In this study, we clarified the novel HSV-2 Us3 phosphoregulation of UL7 in infected cells, which is important for viral replication and pathogenicity in vivo We also showed that the evolution of Us3 phosphoregulation of UL7 was linked to the phylogeny of viruses that are phylogenetically close to HSV-2 and to the phosphorylation requirements for the efficient in vivo viral fitness of HSV-2 and HSV-1, which are representative of viruses that have and have not evolved phosphoregulation, respectively. This study reports the first evidence showing that evolution of viral phosphoregulation coincides with phylogeny of virus species and supports the hypothesis regarding the evolution of viral phosphoregulation during viral evolution.

Influenza Virus-Induced Oxidized DNA Activates Inflammasomes.

Influenza virus M2 and PB1-F2 proteins have been proposed to activate the Nod-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome in macrophages by altering intracellular ionic balance or mitochondrial reactive oxygen species (ROS) production. However, the precise mechanism by which these viral proteins trigger the NLRP3 inflammasome activation remains unclear. Here we show that influenza virus stimulates oxidized DNA release from macrophages. Ion channel activity of the M2 protein or mitochondrial localization of the PB1-F2 protein was required for oxidized DNA release. The oxidized DNA enhanced influenza virus-induced IL-1ß secretion, whereas inhibition of mitochondrial ROS production by antioxidant Mito-TEMPO decreased the virus-induced IL-1ß secretion. In addition, we show that influenza virus stimulates IL-1ß secretion from macrophages in an AIM2-dependent manner. These results provide a missing link between influenza viral proteins and the NLRP3 inflammasome activation and reveal the importance of influenza virus-induced oxidized DNA in inflammasomes activation.

Identification of the Capsid Binding Site in the Herpes Simplex Virus 1 Nuclear Egress Complex and Its Role in Viral Primary Envelopment and Replication.

During nuclear egress of nascent progeny herpesvirus nucleocapsids, the nucleocapsids acquire a primary envelope by budding through the inner nuclear membrane of infected cells into the perinuclear space between the inner and outer nuclear membranes. Herpes simplex virus 1 (HSV-1) UL34 and UL31 proteins form a nuclear egress complex (NEC) and play critical roles in this budding process, designated primary envelopment. To clarify the role of NEC binding to progeny nucleocapsids in HSV-1 primary envelopment, we established an assay system for HSV-1 NEC binding to nucleocapsids and capsid proteins in vitro Using this assay system, we showed that HSV-1 NEC bound to nucleocapsids and to capsid protein UL25 but not to the other capsid proteins tested (i.e., VP5, VP23, and UL17) and that HSV-1 NEC binding of nucleocapsids was mediated by the interaction of NEC with UL25. UL31 residues arginine-281 (R281) and aspartic acid-282 (D282) were required for efficient NEC binding to nucleocapsids and UL25. We also showed that alanine substitution of UL31 R281 and D282 reduced HSV-1 replication, caused aberrant accumulation of capsids in the nucleus, and induced an accumulation of empty vesicles that were similar in size and morphology to primary envelopes in the perinuclear space. These results suggested that NEC binding via UL31 R281 and D282 to nucleocapsids, and probably to UL25 in the nucleocapsids, has an important role in HSV-1 replication by promoting the incorporation of nucleocapsids into vesicles during primary envelopment.IMPORTANCE Binding of HSV-1 NEC to nucleocapsids has been thought to promote nucleocapsid budding at the inner nuclear membrane and subsequent incorporation of nucleocapsids into vesicles during nuclear egress of nucleocapsids. However, data to directly support this hypothesis have not been reported thus far. In this study, we have present data showing that two amino acids in the membrane-distal face of the HSV-1 NEC, which contains the putative capsid binding site based on the solved NEC structure, were in fact required for efficient NEC binding to nucleocapsids and for efficient incorporation of nucleocapsids into vesicles during primary envelopment. This is the first report showing direct linkage between NEC binding to nucleocapsids and an increase in nucleocapsid incorporation into vesicles during herpesvirus primary envelopment.

Roles of the Interhexamer Contact Site for Hexagonal Lattice Formation of the Herpes Simplex Virus 1 Nuclear Egress Complex in Viral Primary Envelopment and Replication.

During the nuclear export of nascent nucleocapsids of herpes simplex virus 1 (HSV-1), the nucleocapsids acquire a primary envelope by budding through the inner nuclear membrane into the perinuclear space between the inner and outer nuclear membranes. This unique budding process, termed primary envelopment, is initiated by the nuclear egress complex (NEC), composed of the HSV-1 UL31 and UL34 proteins. Earlier biochemical approaches have shown that the NEC has an intrinsic ability to vesiculate membranes through the formation of a hexagonal lattice structure. The significance of intrahexamer interactions of the NEC in the primary envelopment of HSV-1-infected cells has been reported. In contrast, the contribution of lattice formation of the NEC hexamer to primary envelopment in HSV-1-infected cells remains to be elucidated. Therefore, we constructed and characterized a recombinant HSV-1 strain carrying an amino acid substitution in a UL31 residue that is an interhexamer contact site for the lattice formation of the NEC hexamer. This mutation was reported to destabilize the interhexamer interactions of the HSV-1 NEC. Here, we demonstrate that the mutation causes the aberrant accumulation of nucleocapsids in the nucleus and reduces viral replication in Vero and HeLa cells. Thus, the ability of HSV-1 to form the hexagonal lattice structure of the NEC was linked to an increase in primary envelopment and viral replication. Our results suggest that the lattice formation of the NEC hexamer has an important role in HSV-1 replication by regulating primary envelopment.IMPORTANCE The scaffolding proteins of several envelope viruses required for virion assembly form high-order lattice structures. However, information on the significance of their lattice formation in infected cells is limited. Herpesviruses acquire envelopes twice during their viral replication. The first envelop acquisition (primary envelopment) is one of the steps in the vesicle-mediated nucleocytoplasmic transport of nascent nucleocapsids, which is unique in biology. HSV-1 NEC, thought to be conserved in all members of the Herpesviridae family, is critical for primary envelopment and was shown to form a hexagonal lattice structure. Here, we investigated the significance of the interhexamer contact site for hexagonal lattice formation of the NEC in HSV-1-infected cells and present evidence suggesting that the lattice formation of the NEC hexamer has an important role in HSV-1 replication by regulating primary envelopment. Our results provide insights into the mechanisms of the envelopment of herpesviruses and other envelope viruses.

ESCRT-III mediates budding across the inner nuclear membrane and regulates its integrity.

Vesicle-mediated nucleocytoplasmic transport is a nuclear pore-independent mechanism for the nuclear export of macromolecular complexes, but the molecular basis for this transport remains largely unknown. Here we show that endosomal sorting complex required for transport-III (ESCRT-III) is recruited to the inner nuclear membrane (INM) during the nuclear export of herpes simplex virus 1 (HSV-1). Scission during HSV-1 budding through the INM is prevented by depletion of ESCRT-III proteins. Interestingly, in uninfected human cells, the depletion of ESCRT-III proteins induces aberrant INM proliferation. Our results show that HSV-1 expropriates the ESCRT-III machinery in infected cells for scission of the INM to produce vesicles containing progeny virus nucleocapsids. In uninfected cells, ESCRT-III regulates INM integrity by downregulating excess INM.

Roles of the Phosphorylation of Herpes Simplex Virus 1 UL51 at a Specific Site in Viral Replication and Pathogenicity.

Herpes simplex virus 1 (HSV-1) UL51 is a phosphoprotein that functions in the final envelopment in the cytoplasm and viral cell-cell spread, leading to efficient viral replication in cell cultures. To clarify the mechanism by which UL51 is regulated in HSV-1-infected cells, we focused on the phosphorylation of UL51. Mass spectrometry analysis of purified UL51 identified five phosphorylation sites in UL51. Alanine replacement of one of the identified phosphorylation sites in UL51, serine 184 (Ser-184), but not the other identified phosphorylation sites, significantly reduced viral replication and cell-cell spread in HaCaT cells. This mutation induced membranous invaginations adjacent to the nuclear membrane, the accumulation of primary enveloped virions in the invaginations and perinuclear space, and mislocalized UL34 and UL31 in punctate structures at the nuclear membrane; however, it had no effect on final envelopment in the cytoplasm of HaCaT cells. Of note, the alanine mutation in UL51 Ser-184 significantly reduced the mortality of mice following ocular infection. Phosphomimetic mutation in UL51 Ser-184 partly restored the wild-type phenotype in cell cultures and in mice. Based on these results, we concluded that some UL51 functions are specifically regulated by phosphorylation at Ser-184 and that this regulation is critical for HSV-1 replication in cell cultures and pathogenicity in vivoIMPORTANCE HSV-1 UL51 is conserved in all members of the Herpesviridae family. This viral protein is phosphorylated and functions in viral cell-cell spread and cytoplasmic virion maturation in HSV-1-infected cells. Although the downstream effects of HSV-1 UL51 have been clarified, there is a lack of information on how this viral protein is regulated as well as the significance of the phosphorylation of this protein in HSV-1-infected cells. In this study, we show that the phosphorylation of UL51 at Ser-184 promotes viral replication, cell-cell spread, and nuclear egress in cell cultures and viral pathogenicity in mice. This is the first report to identify the mechanism by which UL51 is regulated as well as the significance of UL51 phosphorylation in HSV-1 infection. Our study may provide insights into the regulatory mechanisms of other herpesviral UL51 homologs.

Regulation of Herpes Simplex Virus 2 Protein Kinase UL13 by Phosphorylation and Its Role in Viral Pathogenesis.

UL13 proteins are serine/threonine protein kinases encoded by herpes simplex virus 1 (HSV-1) and HSV-2. Although the downstream effects of the HSV protein kinases, mostly those of HSV-1 UL13, have been reported, there is a lack of information on how these viral protein kinases are regulated in HSV-infected cells. In this study, we used a large-scale phosphoproteomic analysis of HSV-2-infected cells to identify a physiological phosphorylation site in HSV-2 UL13 (i.e., Ser-18) and investigated the significance of phosphorylation of this site in HSV-2-infected cell cultures and mice. Our results were as follows. (i) An alanine substitution at UL13 Ser-18 (S18A) significantly reduced HSV-2 replication and cell-to-cell spread in U2OS cells to a level similar to those of the UL13-null and kinase-dead mutations. (ii) The UL13 S18A mutation significantly impaired phosphorylation of a cellular substrate of this viral protein kinase in HSV-2-infected U2OS cells. (iii) Following vaginal infection of mice, the UL13 S18A mutation significantly reduced mortality, HSV-2 replication in the vagina, and development of vaginal disease to levels similar to those of the UL13-null and the kinase-dead mutations. (iv) A phosphomimetic substitution at UL13 Ser-18 significantly restored the phenotype observed with the UL13 S18A mutation in U2OS cells and mice. Collectively, our results suggested that phosphorylation of UL13 Ser-18 regulated UL13 function in HSV-2-infected cells and that this regulation was critical for the functional activity of HSV-2 UL13 in vitro and in vivo and also for HSV-2 replication and pathogenesis.IMPORTANCE Based on studies on cellular protein kinases, it is obvious that the regulatory mechanisms of protein kinases are as crucial as their functional consequences. Herpesviruses each encode at least one protein kinase, but the mechanism by which these kinases are regulated in infected cells remains to be elucidated, with a few exceptions, although information on their functional effects has been accumulating. In this study, we have shown that phosphorylation of the HSV-2 UL13 protein kinase at Ser-18 regulated its function in infected cells, and this regulation was critical for HSV-2 replication and pathogenesis in vivo UL13 is conserved in all members of the family Herpesviridae, and this is the first report clarifying the regulatory mechanism of a conserved herpesvirus protein kinase that is involved in viral replication and pathogenesis in vivo Our study may provide insight into the regulatory mechanisms of the other conserved herpesvirus protein kinases.

Herpes Simplex Virus 1 VP22 Inhibits AIM2-Dependent Inflammasome Activation to Enable Efficient Viral Replication.

The AIM2 inflammasome is activated by DNA, leading to caspase-1 activation and release of pro-inflammatory cytokines interleukin 1ß (IL-1ß) and IL-18, which are critical mediators in host innate immune responses against various pathogens. Some viruses employ strategies to counteract inflammasome-mediated induction of pro-inflammatory cytokines, but their in vivo relevance is less well understood. Here we show that the herpes simplex virus 1 (HSV-1) tegument protein VP22 inhibits AIM2-dependent inflammasome activation. VP22 interacts with AIM2 and prevents its oligomerization, an initial step in AIM2 inflammasome activation. A mutant virus lacking VP22 (HSV-1ΔVP22) activates AIM2 and induces IL-1ß and IL-18 secretion, but these responses are lost in the absence of AIM2. Additionally, HSV-1ΔVP22 infection results in diminished viral yields in vivo, but HSV-1ΔVP22 replication is largely restored in AIM2-deficient mice. Collectively, these findings reveal a mechanism of HSV-1 evasion of the host immune response that enables efficient viral replication in vivo.

Herpes simplex virus-1 evasion of CD8+ T cell accumulation contributes to viral encephalitis.

Herpes simplex virus-1 (HSV-1) is the most common cause of sporadic viral encephalitis, which can be lethal or result in severe neurological defects even with antiviral therapy. While HSV-1 causes encephalitis in spite of HSV-1-specific humoral and cellular immunity, the mechanism by which HSV-1 evades the immune system in the central nervous system (CNS) remains unknown. Here we describe a strategy by which HSV-1 avoids immune targeting in the CNS. The HSV-1 UL13 kinase promotes evasion of HSV-1-specific CD8+ T cell accumulation in infection sites by downregulating expression of the CD8+ T cell attractant chemokine CXCL9 in the CNS of infected mice, leading to increased HSV-1 mortality due to encephalitis. Direct injection of CXCL9 into the CNS infection site enhanced HSV-1-specific CD8+ T cell accumulation, leading to marked improvements in the survival of infected mice. This previously uncharacterized strategy for HSV-1 evasion of CD8+ T cell accumulation in the CNS has important implications for understanding the pathogenesis and clinical treatment of HSV-1 encephalitis.