The order Herpesvirales contains three families, Orthoherpesviridae, Alloherpesviridae, and Malacoherpesviridae. The time since divergence of families from the common ancestor makes protein primary sequence comparison an insensitive tool for identifying common genes. Comparison of three-dimensional protein structures can reveal similarities that are not evident in primary sequences. Salmonid herpesvirus 1 (SalHV-1) is an alloherpesvirus. Complete sequencing of SalHV-1 VR-868 strain Winthrop by a combination of short- and long-read methods revealed 120 putative open reading frames (ORFs). BLAST search for similar protein sequences discovered five ORFs that encoded proteins with homologs in the orthoherpesviruses, including the major capsid protein, capsid triplex subunit 2, the catalytic subunit of the DNA polymerase, the helicase subunit of the helicase/primase complex, and the terminase ATPase subunit. An annotation of the ORFs of SalHV-1 was performed in which ORFs of SalHV-1 were modeled using AlphaFold3, and the models were used as prompts for structural similarity search using DALI and FoldSeek. Completion of this search strategy for the entire genome expanded the set of genes shared among the Herpesvirales to include additional proteins related to DNA replication and genome integrity, capsid assembly and genome packaging, and capsid nuclear egress. No homologs for any tegument proteins or proteins of the conserved entry apparatus of the Herpesviridae (gB, gH or gL) were discovered.

Nuclear egress is an essential process in herpesviral replication whereby nascent capsids translocate from the nucleus to the cytoplasm. This initial step of nuclear egress—budding at the inner nuclear membrane—is coordinated by the nuclear egress complex (NEC). Composed of the viral proteins UL31 and UL34, NEC deforms the membrane around the capsid as the latter buds into the perinuclear space. NEC oligomerization into a hexagonal membrane-bound lattice is essential for budding because mutations designed to perturb lattice interfaces reduce its budding ability.

Nuclear egress is an essential process in herpesvirus replication whereby nascent capsids translocate from the nucleus to the cytoplasm. This initial step of nuclear egress-budding at the inner nuclear membrane-is coordinated by the nuclear egress complex (NEC). Composed of the viral proteins UL31 and UL34, NEC deforms the membrane around the capsid as the latter buds into the perinuclear space. NEC oligomerization into a hexagonal membrane-bound lattice is essential for budding because NEC mutants designed to perturb lattice interfaces reduce its budding ability. Previously, we identified an NEC suppressor mutation capable of restoring budding to a mutant with a weakened hexagonal lattice. Using an established in-vitro budding assay and HSV-1 infected cell experiments, we show that the suppressor mutation can restore budding to a broad range of budding-deficient NEC mutants thereby acting as a universal suppressor. Cryogenic electron tomography of the suppressor NEC mutant lattice revealed a hexagonal lattice reminiscent of wild-type NEC lattice instead of an alternative lattice. Further investigation using x-ray crystallography showed that the suppressor mutation promoted the formation of new contacts between the NEC hexamers that, ostensibly, stabilized the hexagonal lattice. This stabilization strategy is powerful enough to override the otherwise deleterious effects of mutations that destabilize the NEC lattice by different mechanisms, resulting in a functional NEC hexagonal lattice and restoration of membrane budding.

ABSTRACT Herpesvirus assembly requires the cytoplasmic association of large macromolecular and membrane structures that derive from both the nucleus and cytoplasmic membrane systems. Results from the study of human cytomegalovirus (HCMV) in cells where it organizes a perinuclear cytoplasmic virus assembly compartment (cVAC) show a clear requirement for the minus-end-directed microtubule motor, dynein, for virus assembly. In contrast, the assembly of herpes simplex virus -1 (HSV-1) in epithelial cells where it forms multiple dispersed, peripheral assembly sites is only mildly inhibited by the microtubule-depolymerizing agent, nocodazole. Here, we make use of a neuronal cell line system in which HSV-1 forms a single cVAC and show that dynein and its co-factor dynactin localize to the cVAC, and dynactin is associated with membranes that contain the virion tegument protein pUL11. We also show that the virus membrane-associated structural proteins pUL51 and the viral envelope glycoprotein gE arrive at the cVAC by different routes. Specifically, gE arrives at the cVAC after retrieval from the plasma membrane, suggesting the need for an intact retrograde transport system. Finally, we demonstrate that inhibition of dynactin function profoundly inhibits cVAC formation and virus production during the cytoplasmic assembly phase of infection. IMPORTANCE Many viruses reorganize cytoplasmic membrane systems and macromolecular transport systems to promote the production of progeny virions. Clarifying the mechanisms by which they accomplish this may reveal novel therapeutic strategies and illustrate mechanisms that are critical for normal cellular organization. Here, we explore the mechanism by which HSV-1 moves macromolecular and membrane cargo to generate a virus assembly compartment in the infected cell. We find that the virus makes use of a well-characterized, microtubule-based transport system that is stabilized against drugs that disrupt microtubules.

Herpesviruses transport nucleocapsids from the nucleus to the cytoplasm by capsid envelopment into the inner nuclear membrane and de-envelopment from the outer nuclear membrane, a process that is coordinated by nuclear egress complex (NEC) proteins, pUL34, and pUL31. Both pUL31 and pUL34 are phosphorylated by the virus-encoded protein kinase, pUS3, and phosphorylation of pUL31 regulates NEC localization at the nuclear rim. pUS3 also controls apoptosis and many other viral and cellular functions in addition to nuclear egress, and the regulation of these various activities in infected cells is not well understood. It has been previously proposed that pUS3 activity is selectively regulated by another viral protein kinase, pUL13 such that its activity in nuclear egress is pUL13-dependent, but apoptosis regulation is not, suggesting that pUL13 might regulate pUS3 activity on specific substrates. We compared HSV-1 UL13 kinase-dead and US3 kinase-dead mutant infections and found that pUL13 kinase activity does not regulate the substrate choice of pUS3 in any defined classes of pUS3 substrates and that pUL13 kinase activity is not important for promoting de-envelopment during nuclear egress. We also find that mutation of all pUL13 phosphorylation motifs in pUS3, individually or in aggregate, does not affect the localization of the NEC, suggesting that pUL13 regulates NEC localization independent of pUS3. Finally, we show that pUL13 co-localizes with pUL31 inside the nucleus in large aggregates, further suggesting a direct effect of pUL13 on the NEC and suggesting a novel mechanism for both UL31 and UL13 in the DNA damage response pathway. IMPORTANCE Herpes simplex virus infections are regulated by two virus-encoded protein kinases, pUS3 and pUL13, which each regulate multiple processes in the infected cell, including capsid transport from the nucleus to the cytoplasm. Regulation of the activity of these kinases on their various substrates is poorly understood, but importantly, kinases are attractive targets for the generation of inhibitors. It has been previously suggested that pUS3 activity on specific substrates is differentially regulated by pUL13 and, specifically, that pUL13 regulates capsid egress from the nucleus by phosphorylation of pUS3. In this study, we determined that pUL13 and pUS3 have different effects on nuclear egress and that pUL13 may interact directly with the nuclear egress apparatus with implications both for virus assembly and egress and, possibly, the host cell DNA- damage response.

ABSTRACT The herpes simplex virus pUL34 component of the nuclear egress complex functions in both nuclear egress and cell-to-cell spread. A R158A/R161A double substitution in pUL34 has been shown to strongly inhibit both nuclear egress and spread. Selection for extragenic suppressors of UL34 R158A/R161A on the background of a virus that expresses monomeric red fluorescent protein-tagged pUL35 yielded two viruses , SUP7 and SUP9, that both replicated and spread much better than the parental virus. The two viruses carried both unique and common genetic changes. The most striking unique genetic change was a large rearrangement of the US region of SUP7 resulting in duplication of ICP22 and deletion of part of US3 and all of US9-12. The two common genetic changes were a frame shift that eliminated expression of pUL35, and mutations that either eliminated or diminished expression of pUS9. The mutations in pUL35 significantly improved replication but had only a small effect on spread. We observed that mutations that diminish or eliminate detectable pUS9 expression significantly improved spread on Vero cells of viruses that were impaired in the spread function of either pUL34 or pUL51, but did not enhance spread of an otherwise wild-type virus. A similar enhancement was observed on U2OS, but not HaCaT cells, suggesting that the spread-inhibitory function of pUS9 differs between cell lines. 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.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals.

Dynein motors are microtubule associated protein complexes that mediate multiple essential cellular processes, such as long-distance cargo trafficking and stabilization of the microtubule organization center. Most of these functions and their regulations depend on the dynein motor subunit dynactin. By using an infection-inducible system, we disrupted dynein motor function after HSV entry by overexpressing a dominant-negative inhibitor of dynein, resulting in a 5-fold growth defect in Vero cells and 1000-fold growth defect in CAD neuronal cells. Also, we found that in infected CAD cells, the dynein complex was recruited to viral assembly sites regardless of microtubule polymerization. Based on these observations, we then identified a novel interaction between conserved HSV-1 tegument protein pUL51 and p150Glued. pUL51 is a palmitoylated Golgi membrane-associated protein that is required for efficient virus assembly and spread. Overexpression of pUL51 alone was sufficient to recruit p150Glued to Golgi membranes. Sequences that are important and sufficient for pUL51-p150Glued interaction were mapped to residues 90 to 124 in pUL51 and residues 548 to 911 in p150Glued. Deletion of a.a 90-124 in pUL51 resulted in a moderate viral growth defect, a profound spread defect, and failure to accumulate both dynactin and the viral spread factor glycoprotein E (gE) at cell-cell junctions. A synthetic peptide that contains pUL51 a.a 90-125 could also inhibit viral growth and spread in pUL51-dependent manner. Taken together, our results suggest that the proper function of pUL51 in efficient viral assembly and spread depends on its interaction with p150Glued. Author Summary During their life-long infections, herpesviruses can spread within the human body in spite of a powerful immune response that includes antibodies that inactivate the virus. The virus accomplishes this by targeting its newly made infectious particles to junctions where infected cells meet their uninfected neighbors. Junctional targeting of virus particles requires that the virus hijack normal cellular machinery used for moving large cargoes within the cell, a process that is only partially understood. In this study, we have discovered a critical interaction between a virus protein that is present in all herpesviruses and a host cell cargo-moving protein that is required for virus spread between cells and shown that this interaction is common to several medically important human herpesviruses. We have also identified a short piece of this virus protein that is responsible for the interaction and shown that this short piece can be used to interrupt the interaction and prevent spread of herpes simplex virus (HSV) in cultured cells. Our study provides prove-of-concept evidence that the spread pathway of HSV, and possibly other herpesviruses, can be targeted for therapeutic purposes.

Herpesvirus capsids are assembled in the nucleus and undergo a two-step process to cross the nuclear envelope. Capsids bud into the inner nuclear membrane (INM) aided by the nuclear egress complex (NEC) proteins UL31/34. At that stage of egress, enveloped virions are found for a short time in the perinuclear space. In the second step of nuclear egress, perinuclear enveloped virions (PEVs) fuse with the outer nuclear membrane (ONM) delivering capsids into the cytoplasm. Once in the cytoplasm, capsids undergo re-envelopment in the Golgi/trans-Golgi apparatus producing mature virions. This second step of nuclear egress is known as de-envelopment and is the focus of this review. Compared with herpesvirus envelopment at the INM, much less is known about de-envelopment. We propose a model in which de-envelopment involves two phases: (i) fusion of the PEV membrane with the ONM and (ii) expansion of the fusion pore leading to release of the viral capsid into the cytoplasm. The first phase of de-envelopment, membrane fusion, involves four herpes simplex virus (HSV) proteins: gB, gH/gL, gK and UL20. gB is the viral fusion protein and appears to act to perturb membranes and promote fusion. gH/gL may also have similar properties and appears to be able to act in de-envelopment without gB. gK and UL20 negatively regulate these fusion proteins. In the second phase of de-envelopment (pore expansion and capsid release), an alpha-herpesvirus protein kinase, US3, acts to phosphorylate NEC proteins, which normally produce membrane curvature during envelopment. Phosphorylation of NEC proteins reverses tight membrane curvature, causing expansion of the membrane fusion pore and promoting release of capsids into the cytoplasm.

Herpes simplex virus capsid envelopment at the nuclear membrane is coordinated by nuclear egress complex (NEC) proteins, pUL34 and pUL31, and is accompanied by alteration in the nuclear architecture and local disruption of nuclear lamina. Here, we examined the role of capsid envelopment in the changes of the nuclear architecture by characterizing HSV-1 recombinants that do not form capsids. Typical changes in nuclear architecture and disruption of the lamina were observed in the absence of capsids, suggesting that disruption of the nuclear lamina occurs prior to capsid envelopment. Surprisingly, in the absence of capsid envelopment, lamin A/C becomes concentrated at the nuclear envelope in a pUL34-independent and cell type-specific manner, suggesting that ongoing nuclear egress may be required for the dispersal of lamins observed in wild-type infection. Mutation of virus-encoded protein kinase, pUS3, on a wild-type virus background has been shown to cause accumulation of perinuclear enveloped capsids, formation of NEC aggregates, and exacerbated lamina disruption. We observed that mutation of US3 in the absence of capsids results in identical NEC aggregation and lamina disruption phenotypes, suggesting that they do not result from accumulation of perinuclear virions. TEM analysis revealed that, in the absence of capsids, NEC aggregates correspond to multi-folded nuclear membrane structures, suggesting that pUS3 may control NEC self-association and membrane deformation. To determine the significance of the pUS3 nuclear egress function for virus growth, the replication of single and double UL34 and US3 mutants was measured, showing that the significance of pUS3 nuclear egress function is cell-type specific.ImportanceThe nuclear lamina is an important player in infection by viruses that replicate in the nucleus. Herpesviruses alter the structure of the nuclear lamina to facilitate transport of capsids from the nucleus to the cytoplasm and use both viral and cellular effectors to disrupt the protein-protein interactions that maintain the lamina. Here we explore the role of capsid envelopment and the virus-encoded protein kinase, pUS3, in the disruption of lamina structure. We show that capsid envelopment is not necessary for the lamina disruption, or for US3 mutant phenotypes, including exaggerated lamina disruption, that accompany nuclear egress. These results clarify the mechanisms behind alteration of nuclear lamina structure and support a function for pUS3 in regulating the aggregation state of the nuclear egress machinery.

Atopic dermatitis (AD) is a condition affecting 30 million persons in the United States. AD patients are heavily infected with Staphylococcus aureus on the skin. A particularly severe form of AD is eczema herpeticum (ADEH), where the patients' AD is complicated by S. aureus and herpes simplex virus (HSV) infection. This study examined the S. aureus strains from 15 ADEH patients, provided blinded, and showed a high association of ADEH with strains that produce toxic shock syndrome toxin-1 (TSST-1; 73%) compared to 10% production by typical AD isolates from patients without EH and those from another unrelated condition, cystic fibrosis. The ADEH isolates produced the superantigens associated with TSS (TSST-1 and staphylococcal enterotoxins A, B, and C). This association may in part explain the potential severity of ADEH. We also examined the effect of TSST-1 and HSV-1 on human epithelial cells and keratinocytes. TSST-1 used CD40 as its receptor on epithelial cells, and HSV-1 either directly or indirectly interacted with CD40. The consequence of these interactions was chemokine production, which is capable of causing harmful inflammation, with epidermal/keratinocyte barrier disruption. Human epithelial cells treated first with TSST-1 and then HSV-1 resulted in enhanced chemokine production. Finally, we showed that TSST-1 modestly increased HSV-1 replication but did not increase viral plaque size. Our data suggest that ADEH is associated with production of the major TSS-associated superantigens, together with HSV reactivation. The superantigens plus HSV may damage the skin barrier by causing harmful inflammation, thereby leading to increased symptoms. IMPORTANCE Atopic dermatitis (eczema, AD) with concurrent herpes simplex virus infection (eczema herpeticum, ADEH) is a severe form of AD. We show that ADEH patients are colonized with Staphylococcus aureus that primarily produces the superantigen toxic shock syndrome toxin-1 (TSST-1); however, significantly but to a lesser extent the superantigens staphylococcal enterotoxins A, B, and C are also represented in ADEH. Our studies showed that TSST-1 uses the immune costimulatory molecule CD40 as its epithelial cell receptor. Herpes simplex virus (HSV) also interacted directly or indirectly with CD40 on epithelial cells. Treatment of epithelial cells with TSST-1 and then HSV-1 resulted in enhanced chemokine production. We propose that this combination of exposures (TSST-1 and then HSV) leads to opening of epithelial and skin barriers to facilitate potentially serious ADEH.

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.

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 (HSV) is a neuroinvasive virus that has been used as a model organism for studying common properties of all herpesviruses. HSV induces host organelle rearrangement and forms dispersed assembly compartments in epithelial cells, which complicates the study of HSV assembly. In this study, we show that HSV forms a visually distinct unitary cytoplasmic viral assembly center (cVAC) in both cancerous and primary neuronal cells that concentrates viral structural proteins and is the site of capsid envelopment. The HSV cVAC also concentrates host membranes that are important for viral assembly, such as Golgi- and recycling endosome-derived membranes. Lastly, we show that HSV cVAC formation and/or maintenance depends on an intact microtubule network and a viral tegument protein, pUL51. Our observations suggest that the neuronal cVAC is a uniquely useful model to study common herpesvirus assembly pathways, and cell-specific pathways for membrane reorganization.

Herpes simplex virus (HSV) is a neuroinvasive virus that has been used as a model organism for studying common properties of all herpesviruses. HSV induces host organelle rearrangement and forms multiple, dispersed assembly compartments in epithelial cells, which complicates the study of HSV assembly. In this study, we show that HSV forms a visually distinct unitary cytoplasmic viral assembly center (cVAC) in both cancerous and primary neuronal cells that concentrates viral structural proteins and is a major site of capsid envelopment. The HSV cVAC also concentrates host membranes that are important for viral assembly, such as Golgiand recycling endosome-derived membranes. Finally, we show that HSV cVAC formation and/or maintenance depends on an intact microtubule network and a viral tegument protein, pUL51. Our observations suggest that the neuronal cVAC is a uniquely useful model to study common herpesvirus assembly pathways and cell specific pathways for membrane reorganization. IMPORTANCE Herpesvirus particles are complex and contain many different proteins that must come together in an organized and coordinated fashion. Many viruses solve this coordination problem by creating a specialized assembly factory in the host cell, and the formation of such factories provides a promising target for interfering with virus production. Herpes simplex virus 1 (HSV-1) infects several types of cells, including neurons, but has not previously been shown to form such an organized factory in the nonneuronal cells in which its assembly has been best studied. Here, we show that HSV-1 forms an organized assembly factory in neuronal cells, and we identify some of the viral and host cell factors that are important for its formation.

Herpes simplex virus spread between epithelial cells is mediated by virus tegument and envelope protein complexes including gE/gI and pUL51/pUL7. pUL51 interacts with both pUL7 and gE/gI in infected cells. We show that amino acids 30-90 of pUL51 mediate interaction with pUL7. We also show that deletion of amino acids 167-244 of pUL51, or ablation of pUL7 expression both result in failure of gE to concentrate at junctional surfaces of Vero cells. We also tested the hypothesis that gE and pUL51 function on the same pathway for cell-to-cell spread by analyzing the phenotype of a double gE/UL51 mutant. In HaCaT cells, pUL51 and gE function on the same spread pathway, whereas in Vero cells they function on different pathways. Deletion of the gE gene strongly enhanced virus release to the medium in Vero cells, suggesting that the gE-dependent spread pathway may compete with virion release to the medium.

Cellular proteins that support influenza virus infection represent potential therapeutic targets. Cytoplasmic egress intermediates of influenza A/WSN/33 were isolated and shown to be associated with the cellular enzymes peroxiredoxins-1 (Prdx-1) during glycerol gradient fractionation. Prdx-1 also co-localizes with influenza NP at the cell periphery late in infection. Knock-down or knockout of Prdx-1 expression inhibit influenza A replication. Inhibition of replication is not correlated with defects in initiation of infection or mRNA expression, but is correlated with inhibition of accumulation of viral proteins and vRNAs.

Herpesviruses assemble and package their genomes into capsids in the nucleus, but complete final assembly of the mature virion in the cell cytoplasm. This requires passage of the genome-containing capsid across the double-membrane nuclear envelope. Herpesviruses have evolved a mechanism that relies on a pair of conserved viral gene products to shuttle the capsids from the nucleus to the cytoplasm by way of envelopment and de-envelopment at the inner and outer nuclear membranes, respectively. This complex process requires orchestration of the activities of viral and cellular factors to alter the architecture of the nuclear membrane, select capsids at the appropriate stage for egress, and accomplish efficient membrane budding and fusion events. The last few years have seen major advances in our understanding of the membrane budding mechanism and helped clarify the roles of viral and cellular proteins in the other, more mysterious steps. Here, we summarize and place into context this recent research and, hopefully, clarify both the major advances and major gaps in our understanding.

ABSTRACT Herpes simplex virus 1 (HSV-1) remodels nuclear membranes during virus egress. Although the UL31 and UL34 proteins control nucleocapsid transit in infected cells, the molecular interactions required for their function are unclear. Here we report that the γ 1 34.5 gene product of HSV-1 facilitates nucleocapsid release to the cytoplasm through bridging the UL31/UL34 complex, cellular p32, and protein kinase C. Unlike wild-type virus, an HSV mutant devoid of γ 1 34.5 or its amino terminus is crippled for viral growth and release. This is attributable to a defect in virus nuclear egress. In infected cells, wild-type virus recruits protein kinase C to the nuclear membrane and triggers its activation, whereas the γ 1 34.5 mutants fail to exert such an effect. Accordingly, the γ 1 34.5 mutants are unable to induce phosphorylation and reorganization of lamin A/C. When expressed in host cells γ 1 34.5 targets p32 and protein kinase C. Meanwhile, it communicates with the UL31/UL34 complex through UL31. Deletion of the amino terminus from γ 1 34.5 disrupts its activity. These results suggest that disintegration of the nuclear lamina mediated by γ 1 34.5 promotes HSV replication. IMPORTANCE HSV nuclear egress is a key step that determines the outcome of viral infection. While the nuclear egress complex mediates capsid transit across the nuclear membrane, the regulatory components are not clearly defined in virus-infected cells. We report that the γ 1 34.5 gene product, a virulence factor of HSV-1, facilitates nuclear egress cooperatively with cellular p32, protein kinase C, and the nuclear egress complex. This work highlights a viral mechanism that may contribute to the pathogenesis of HSV infection.

ABSTRACT Nuclear egress of herpesviruses is accompanied by changes in the architecture of the nuclear membrane and nuclear lamina that are thought to facilitate capsid access to the inner nuclear membrane (INM) and curvature of patches of the INM around the capsid during budding. Here we report the properties of a point mutant of pUL34 (Q163A) that fails to induce gross changes in nuclear architecture or redistribution of lamin A/C. The UL34(Q163A) mutant shows a roughly 100-fold defect in single-step growth, and it forms small plaques. This mutant has a defect in nuclear egress, and furthermore, it fails to disrupt nuclear shape or cause observable displacement of lamin A/C despite retaining the ability to recruit the pUS3 and PKC protein kinases and to mediate phosphorylation of emerin. Extragenic suppressors of the UL34(Q163A) phenotype were isolated, and all of them carry a single mutation of arginine 229 to leucine in UL31. Surprisingly, although this UL31 mutation largely restores virus replication, it does not correct the lamina disruption defect, suggesting that, in Vero cells, changes in nuclear shape and gross displacements of lamin A/C may facilitate but are unnecessary for nuclear egress. IMPORTANCE Herpesvirus nuclear egress is an essential and conserved process that requires close association of the viral capsid with the inner nuclear membrane and budding of the capsid into that membrane. Access to the nuclear membrane and tight curvature of that membrane are thought to require disruption of the nuclear lamina that underlies the inner nuclear membrane, and consistent with this idea, herpesvirus infection induces biochemical and architectural changes at the nuclear membrane. The significance of the nuclear membrane architectural changes is poorly characterized. The results presented here address that deficiency in our understanding and show that a combination of mutations in two of the viral nuclear egress factors results in a failure to accomplish at least two components of lamina disruption while still allowing relatively efficient viral replication, suggesting that changes in nuclear shape and displacement of lamins are not necessary for herpes simplex virus 1 (HSV-1) nuclear egress.