The wild-type U _L 31, U _L 34, and U _S 3 proteins localized on nuclear membranes and perinuclear virions; the U _S 3 protein was also on cytoplasmic membranes and extranuclear virions. The U _L 31 and U _L 34 proteins were not detected in extracellular virions. U _S 3 deletion caused (i) virion accumulation in nuclear membrane invaginations, (ii) delayed virus production onset, and (iii) reduced peak virus titers. These data support the herpes simplex virus type 1 deenvelopment-reenvelopment model of virion egress and suggest that the U _S 3 protein plays an important, but nonessential, role in the egress pathway.

We report that herpes simplex virus type 1 (HSV-1) infection leads to the recruitment of protein kinase C (PKC) to the nuclear rim. In HEp-2 cells, PKC recruitment to the nuclear rim was initiated between 8 h and 12 h postinfection. PKCδ, a proapoptotic kinase, was completely recruited to the nuclear rim upon infection with HSV-1. PKCα was less dramatically relocalized mostly at the nuclear rim upon infection, although some PKCα remained in the cytoplasm. PKCζ-specific immunofluorescence was not significantly relocated to the nuclear rim. The U _L 34 and U _L 31 proteins, as well as their association, were each required for PKC recruitment to the nuclear rim. The HSV-1 U _S 3 protein product, a kinase which regulates the phosphorylation state and localization of U _L 34, was not required for PKC recruitment to the nuclear rim; however, it was required for proper localization along the nuclear rim, as PKC appeared unevenly distributed along the nuclear rim of cells infected with U _S 3 null and kinase-dead mutants. HSV-1 infection induced the phosphorylation of both lamin B and PKC. Elevated lamin B phosphorylation in HSV-1-infected cells was partially reduced by inhibitors of PKC. The data suggest a model in which kinases that normally disassemble the nuclear lamina during apoptosis are recruited to the nuclear membrane through functions requiring U _L 31 and U _L 34. We hypothesize that the recruitment of PKC functions to phosphorylate lamin B to help modify the nuclear lamina and promote budding of nucleocapsids at the inner nuclear membrane.

Infection with herpes simplex virus (HSV) types 1 and 2 is ubiquitous in the human population. Most commonly, virus replication is limited to the epithelia and establishes latency in enervating sensory neurons, reactivating periodically to produce localized recurrent lesions. However, these viruses can also cause severe disease such as recurrent keratitis leading potentially to blindness, as well as encephalitis, and systemic disease in neonates and immunocompromised patients. Although antiviral therapy has allowed continual and substantial improvement in the management of both primary and recurrent infections, resistance to currently available drugs and long-term toxicity pose a current and future threat that should be addressed through the development of new antiviral compounds directed against new targets. The development of several promising HSV vaccines has been terminated recently because of modest or controversial therapeutic effects in humans. Nevertheless, several exciting vaccine candidates remain in the pipeline and are effective in animal models; these must also be tested in humans for sufficient therapeutic effects to warrant continued development. Approaches using compounds that modulate the chromatin state of the viral genome to suppress infection and reactivation or induce enhanced antiviral immunity have potential. In addition, technologies such as CRISPR/Cas9 have the potential to edit latent viral DNA in sensory neurons, potentially curing the neuron and patient of latent infection. It is hoped that development on all three fronts—antivirals, vaccines, and gene editing—will lead to substantially less HSV morbidity in the future.

The herpes simplex virus type 1 (HSV-1) U _S 3 gene encodes a serine/threonine kinase that, when inactivated, causes capsids to aggregate aberrantly between the inner and outer nuclear membranes (INM and ONM, respectively) within evaginations/extensions of the perinuclear space. In both Hep2 cells and an engineered cell line derived from Hep2 cells expressing lamin A/C fused to enhanced green fluorescent protein (eGFP-lamin A/C), lamin A/C localized mostly in a reticular pattern with small regions of the INM devoid of eGFP-lamin A/C when they were either mock infected or infected with wild-type HSV-1(F). Cells infected with HSV-1(F) also contained some larger diffuse regions lacking lamin A/C. Proteins U _L 31 and U _L 34, markers of potential envelopment sites at the INM and perinuclear virions, localized within the regions devoid of lamin A/C and also in regions containing lamin A/C. Similar to previous observations with Vero cells (S. L. Bjerke and R. J. Roller, Virology 347:261-276, 2006), the proteins U _L 34 and U _L 31 localized exclusively in very discrete regions of the nuclear lamina lacking lamin A/C in the absence of U _S 3 kinase activity. To determine how U _S 3 alters lamin A/C distribution, U _S 3 was purified and shown to phosphorylate lamin A/C at multiple sites in vitro, despite the presence of only one putative U _S 3 kinase consensus site in the lamin A/C sequence. U _S 3 kinase activity was also sufficient to invoke partial solubilization of lamin A/C from permeabilized Hep2 cell nuclei in an ATP-dependent manner. Two-dimensional electrophoretic analyses of lamin A/C revealed that lamin A/C is phosphorylated in HSV-infected cells, and the full spectrum of phosphorylation requires U _S 3 kinase activity. These data suggest that U _S 3 kinase activity regulates HSV-1 capsid nuclear egress at least in part by phosphorylation of lamin A/C.

The herpes simplex virus type 1 (HSV-1) U _L 31 and U _L 34 proteins are dependent on each other for proper targeting to the nuclear membrane and are required for efficient envelopment of nucleocapsids at the inner nuclear membrane. In this work, we show that whereas the solubility of lamins A and C (lamin A/C) was not markedly increased, HSV induced conformational changes in the nuclear lamina of infected cells, as viewed after staining with three different lamin A/C-specific antibodies. In one case, reactivity with a monoclonal antibody that recognizes an epitope in the lamin tail domain was greatly reduced in HSV-infected cells. This apparent HSV-induced epitope masking required both U _L 31 and U _L 34, but these proteins were not sufficient to mask the epitope in uninfected cells, indicating that other HSV proteins are also required. In the second case, staining with a rabbit polyclonal antibody that primarily recognizes epitopes in the lamin A/C rod domain revealed that U _L 34 is required for HSV-induced decreased availability of epitopes for reaction with the antibody, whereas U _L 31 protein was dispensable for this effect. Still another polyclonal antibody indicated virtually no difference in lamin A/C staining in infected versus uninfected cells, indicating that the HSV-induced changes are more conformational than the result of lamin depletion at the nuclear rim. Further evidence supporting an interaction between the nuclear lamina and the U _L 31/U _L 34 protein complex includes the observations that (i) overexpression of the U _L 31 protein in uninfected cells was sufficient to relocalize lamin A/C from the nuclear rim into nucleoplasmic aggregates, (ii) overexpression of U _L 34 was sufficient to relocalize some lamin A/C into the cytoplasm, and (iii) both U _L 31 and U _L 34 could directly bind lamin A/C in vitro. These studies suggest that the U _L 31 and U _L 34 proteins modify the conformation of the nuclear lamina in infected cells, possibly by direct interaction with lamin A/C, and that other proteins are also likely involved. Given that the nuclear lamina potentially excludes nucleocapsids from envelopment sites at the inner nuclear membrane, the lamina alteration may reflect a role of the U _L 31/U _L 34 protein complex in perturbing the lamina to promote nucleocapsid egress from the nucleus. Alternatively, the data are compatible with a role of the lamina in targeting the U _L 31/U _L 34 protein complex to the nuclear membrane.

Herpes simplex virus 1 nucleocapsids bud through the inner nuclear membrane (INM) into the perinuclear space to obtain a primary viral envelope. This process requires a protein complex at the INM composed of the U _L 31 and U _L 34 gene products. While it is clear that the viral kinase encoded by the U _S 3 gene regulates the localization of pU _L 31/pU _L 34 within the INM, the molecular mechanism by which this is accomplished remains enigmatic. Here, we have determined the following. (i) The N terminus of pU _L 31 is indispensable for the protein's normal function and contains up to six serines that are phosphorylated by the U _S 3 kinase during infection. (ii) Phosphorylation at these six serines was not essential for a productive infection but was required for optimal viral growth kinetics. (iii) In the presence of active U _S 3 kinase, changing the serines to alanine caused the pU _L 31/pU _L 34 complex to aggregate at the nuclear rim and caused some virions to accumulate aberrantly in herniations of the nuclear membrane, much as in cells infected with a U _S 3 kinase-dead mutant. (iv) The replacement of the six serines of pU _L 31 with glutamic acid largely restored the smooth distribution of pU _L 34/pU _L 31 at the nuclear membrane and precluded the accumulation of virions in herniations whether or not U _S 3 kinase was active but also precluded the optimal primary envelopment of nucleocapsids. These observations indicate that the phosphorylation of pU _L 31 by pU _S 3 represents an important regulatory event in the virion egress pathway that can account for much of pU _S 3's role in nuclear egress. The data also suggest that the dynamics of pU _L 31 phosphorylation modulate both the primary envelopment and the subsequent fusion of the nascent virion envelope with the outer nuclear membrane.