Major Virion Tegument Protein VP22 Targets Nuclear Matrix and Chromatin upon Entry into Cells during Productive Herpes Simplex Virus 1 Infection.

HSV-1 major tegument protein VP22 is present in multiple subcellular locations in the late stages of productive viral infection. We initially performed a detailed time course experiment and observed that VP22 was detected in nuclear and nuclear matrix fractions as early as 4 hpi. The goal was to determine the fate of virion-derived incoming VP22, and we report the following: (i) VP22 was detected in nuclear matrix fractions 1 hpi. (ii) In the presence of cycloheximide (CHX), VP22 was present in the nuclear matrix 1-6 hpi, demonstrating the stability of the protein. (iii) The nuclear matrix targeting of VP22 occurred in infected Vero, HEp-2, and human mammary epithelial (HME) cells and following synchronized infection. Based on these results, we conclude that (iv) VP22 targets the nuclear matrix and chromatin upon entry into cells during productive HSV-1 infection.

The impact of the National Science Foundation's Innovation Corps (I-Corps) on academic innovation and entrepreneurship.

Abstract: In 2011, the U.S. National Science Foundation created the Innovation Corps (I-Corps) program in an effort to explore ways to translate the results of the academic research the agency has funded into new products, processes, devices, or services and move them to the marketplace. The agency established a 3-tier structure to support the implementation of the I-Corps concept. Selected I-Corps teams consisting of the principal investigator, an entrepreneurial lead, and an industry mentor participate in a 7-week accelerated version of the Lean Launchpad methodology that was first developed by Steve Blank at Stanford University. Participating teams engage in talking to potential customers, partners, and competitors and address the challenges and the uncertainty of creating successful ventures. I-Corps sites were set up to promote selected aspects of innovation and entrepreneurship ecosystems at the grantee institutions. I-Corps Regional Nodes were charged with recruiting I-Corps teams in a larger geographical area as well as stimulating a new culture of academic entrepreneurship in the institutions in their area of influence. This Topical Review describes the experiences and the impact of the New York City Regional Innovation Node, which is led by the City University of New York, in partnership with New York University and Columbia University.

Comparison of HEp-2 and Vero Cell Responses Reveal Unique Proapoptotic Activities of the Herpes Simplex Virus Type 1 α0 Gene Transcript and Product.

Previous studies have provided evidence suggesting a role for apoptosis in the control of Herpes Simplex Virus 1 (HSV-1) latency. HSV-1 induces and then later blocks apoptosis in infected cells. The immediate early viral gene α0, which synthesizes the ICP0 protein, is necessary and sufficient for HSV-1-induced apoptosis in human epithelial (HEp-2) cells. While previous research showed that ICP0 protein synthesis is not necessary for HSV-1-induced apoptosis in infected HEp-2 cells, circumstantial evidence suggested that it might be needed in infected African green monkey kidney (Vero) cells. In this study, we determined the specific aspects of α0 needed to trigger apoptosis in these two cell types. HEp-2 cells transfected with α0 expressing plasmids that generated either full-length, truncated, or no detectable (multiple stop codons) ICP0 protein died through apoptosis. This indicates that ICP0 protein is not necessary for α0-induced apoptosis and that α0 mRNA alone has apoptotic induction properties in HEp-2 cells. We next investigated the primary structure of α0's mRNA to better define its proapoptotic ability. Since α0 is one of the few HSV-1 genes that are spliced, we transfected cells with a plasmid expressing ICP0 from cDNA copy, pcDNAICP0. The cells transfected with pcDNAICP0 underwent apoptosis at a level equivalent to those transfected with the genomic copy of α0, which indicates that neither splicing events nor introns are required for the apoptotic function of α0 in HEp-2 cells. Next, we studied the ability of α0 to cause apoptosis in Vero cells. Since HSV-1-induced apoptosis in Vero cells requires protein synthesis early in infection, proteins synthesized with immediate early kinetics may facilitate apoptosis. Vero cells were transfected with plasmids producing either full-length ICP0 or ICP0 truncated at codon 212. Full-length ICP0, but not truncated ICP0, induced apoptosis in Vero cells. Together, these results suggest that α0 gene expression triggers apoptosis, but ICP0 protein is needed to facilitate apoptosis in Vero cells. In addition, ICP0's facilitation activity may lie in its carboxyl-terminated domain. Thus, our results demonstrate that α0's mRNA and protein possess proapoptotic properties. The requirement for ICP0 protein during HSV-dependent apoptosis appears to be cell type specific.

Targeting Autophagy in Dendritic Cells as a Mechanism to Limit Immunopathogenesis in Herpetic Stromal Keratitis.

In a recent mBio article, Y. Jiang, X. Yin, P. M. Stuart, and D. A. Lieb [mBio 6(6):e01426-15, 2015, doi:10.1128/mBio.01426-15] presented an elegant set of experiments that utilized a transgenic, knockout strain of mice whose dendritic cells (DCs) are incapable of undergoing autophagy, to dissect out the aspects of the chronic inflammatory response following viral infection of corneal epithelial cells. The authors' results provide a potential proof of concept that the DC autophagy pathway may be a valid target for therapeutic drug design in certain inflammatory pathologies.

A truncation mutation of the neurovirulence ICP22 protein produced by a recombinant HSV-1 generated by bacterial artificial chromosome technology targets infected cell nuclei.

The major regulatory protein ICP22 is unique among the immediate early proteins of herpes simplex virus. Viruses deleted for ICP22 replicate well in actively dividing cells, but not in quiescent cells or certain rodent lines. Accordingly, ICP22 represents an understudied herpes simplex virus (HSV) neurovirulence marker which is absolutely essential for viral neurogrowth. We utilized the bacterial artificial chromosome methodology to create a novel ICP22 truncation mutant, termed HSV-1(BACX). The integrity of HSV-1(BACX) was confirmed by detailed polymerase chain reaction analyses and immunoblotting using anti-ICP22 antibody. HSV-1(BACX) showed a reduced replication capacity in rabbit skin cells, consistent with previous studies using ICP22-null viruses. Importantly, HSV-1(BACX) localized to nuclei of infected primate Vero cells in a manner similar to wild-type ICP22. Thus, HSV-1(BACX) will serve as a useful tool to decipher the unusual biological properties and functions of the ICP22 protein.

The virion host shutoff protein of herpes simplex virus 1 blocks the replication-independent activation of NF-κB in dendritic cells in the absence of type I interferon signaling.

Immune evasion is a defining feature of the virus-host relationship. During infection, herpes simplex virus type 1 (HSV-1) utilizes multiple proteins to manipulate the host immune response. In the present study, we investigated the mechanism by which the virion host shutoff (vhs) protein blocks the activation of dendritic cells (DCs). Previously, we found that coinfection of wild-type HSV-1 with a panel of RNA viruses resulted in a block to DC activation that was attributable to vhs. These observations led us to hypothesize that the vhs-mediated inhibition was dependent on signaling through the RIG-I-like receptor (RLR) signaling pathway. By examining DCs generated from MAVS (IPS-1) knockout (KO) mice, we determined that RLR/MAVS signaling is not essential for the DC response to HSV-1. We also evaluated the requirement for the type I interferon (IFN) signaling pathway in DC activation following infection with HSV-1 and found that stimulation of DCs with wild-type HSV-1 required intact type I IFN signaling for the production of cytokines, whereas the vhs deletion (vhs(-)) mutant virus activated DCs without the need for exogenous IFN signaling. Comparisons of transcription factor activation in DCs infected with wild-type HSV and the vhs(-) mutant virus revealed that NF-κB activation was inhibited by vhs in the early phase of the infection. In contrast, IRF3 activation was not influenced by vhs. In these studies, measurement of proinflammatory cytokines and type I IFN release from the infected DCs reflected the activation status of these transcription factors. Taken together, the work presented here (i) describes a novel role for the vhs protein as an inhibitor of the early activation of NF-κB during HSV-1 infection of DCs and (ii) offers a mechanistic explanation of how this protein interferes with DC activation.

The virion host shut-off (vhs) protein blocks a TLR-independent pathway of herpes simplex virus type 1 recognition in human and mouse dendritic cells.

Molecular pathways underlying the activation of dendritic cells (DCs) in response to Herpes Simplex Virus type 1 (HSV-1) are poorly understood. Removal of the HSV virion host shut-off (vhs) protein relieves a block to DC activation observed during wild-type infection. In this study, we utilized a potent DC stimulatory HSV-1 recombinant virus lacking vhs as a tool to investigate the mechanisms involved in the activation of DCs by HSV-1. We report that the release of pro-inflammatory cytokines by conventional DC (cDC) during HSV-1 infection is triggered by both virus replication-dependent and replication-independent pathways. Interestingly, while vhs is capable of inhibiting the release of cytokines during infection of human and mouse cDCs, the secretion of cytokines by plasmacytoid DC (pDC) is not affected by vhs. These data prompted us to postulate that infection of cDCs by HSV triggers a TLR independent pathway for cDC activation that is susceptible to blockage by the vhs protein. Using cDCs isolated from mice deficient in both the TLR adaptor protein MyD88 and TLR3, we show that HSV-1 and the vhs-deleted virus can activate cDCs independently of TLR signaling. In addition, virion-associated vhs fails to block cDC activation in response to treatment with TLR agonists, but it efficiently blocked cDC activation triggered by the paramyxoviruses Sendai Virus (SeV) and Newcastle Disease Virus (NDV). This block to SeV- and NDV-induced activation of cDC resulted in elevated SeV and NDV viral gene expression indicating that infection with HSV-1 enhances the cell's susceptibility to other pathogens through the action of vhs. Our results demonstrate for the first time that a viral protein contained in the tegument of HSV-1 can block the induction of DC activation by TLR-independent pathways of viral recognition.

Genital herpes simplex virus type 1 in women: detection in cervicovaginal specimens from gynecological practices in the United States.

Herpes simplex virus types 1 and 2 (HSV-1 and -2) are significant human pathogens causing clinically indistinguishable facial and genital lesions. Recently, the number of reported genital herpes cases caused by type 1 virus has increased. Identifying the HSV type is of clinical importance to determine proper treatment, as there is no licensed vaccine or cure. We assessed, by PCR, the frequency of HSV-1 and HSV-2 present in more than 60,000 clinical cervicovaginal specimens derived from samples originating from 43 continental U.S. states. Fourteen percent were positive for HSV-1 and/or HSV-2. This likely represents subclinal shedding. It was not a measurement of the prevalence of HSV infection. While the majority were HSV-2, 32% were HSV-1. The distribution of HSV types varied between the states with the largest number of specimens, New Jersey, Florida, and Texas. Specimens from women under the age of 24 had an HSV-1 positivity rate of 47 percent. Importantly, in New Jersey, an observed age effect was the disproportionately high prevalence of genital HSV-1 in young women. This represents the largest analysis of HSV types reported and has important public health implications, particularly for younger women.

Oncoapoptosis: a novel molecular therapeutic for cancer treatment.

Many cancer cells refractory to radiation treatment and chemotherapy proliferate due to loss of intrinsic programmed cell death (apoptosis) regulation. Consequently, the resolution of these cancers are many times outside the management capabilities of conventional therapeutics. We have developed a replication defective herpes simplex virus system which triggers apoptosis specifically in transformed human cells, termed oncoapoptosis. Susceptibility to virus induced cell death is dependent on the p53 protein status in the tumor cells, indicating specific targeting of the treatment. Primary cells which produce functional p53 are resistant to oncoapoptotic killing but not to apoptosis induced by nonviral environmental factors. Thus, induction of apoptosis by nonreplicating virus is a feasible molecular therapeutic approach for killing human cancer cells. Our findings have important implications in designing novel virus-based anticancer strategies.

Association of the herpes simplex virus major tegument structural protein VP22 with chromatin.

The abundant tegument protein VP22 has homologs throughout the alphaherpesvirus family. Numerous groups have been involved in the examination of these homologs, trying to decipher their multiple functions. Investigations early on indicated that VP22 associates with chromatin and further evidence has accumulated in recent years, correlating this protein with chromatin and its components. Ongoing and future studies will determine whether this association with chromatin is specific and whether it serves a function during infection in a physiologically relevant setting.

Role of viral chromatin structure in the regulation of herpes simplex virus 1 gene expression and replication.

Herpes simplex virus 1 initially infects epithelial cells during the lytic phase of its infectious cycle, followed by establishment of the latent phase within neuronal cells. The two different phases of infection are characterized by distinct gene-expression profiles, involving a temporal gene-expression pattern during the lytic phase succeeded by a complete shutdown of all gene expression, except for one abundant transcript, during the latent phase. The mechanisms controlling these varying degrees of gene expression appear to involve regulation of the viral chromatin structure, presumably using many of the same tactics employed by the host cell.

Detection of herpes simplex virus dependent apoptosis.

Subversion of the host response to virus infection is a universal theme of virology and viral immunology. Multiple mechanisms are in place to limit virus spread on behalf of the host, yet through evolution, viruses have adapted to either weaken or eliminate the effects of these host factors. Cell death or apoptosis is one such example of a host response to viral infection. As such, experimental techniques that enable analysis of viruses (and viral genes) involved in triggering, blocking, or perhaps augmenting this process represent important tools for virologists, immunologists, and cell biologists. Presented here are a series of techniques developed in our lab for the analysis of apoptosis that occurs as a consequence of herpes simplex virus type 1 infection.

Rapid isolation of HSV-1 and HSV-2 from OneSwab cervicovaginal specimens.

Herpes simplex viruses are significant human pathogens which remain latent in their hosts for life and reactivate to cause disease at or near the initial site of infection. Since HSV infections are associated with high morbidity, rapid detection and diagnosis of these viruses is imperative. While PCR technology for the diagnosis of HSV is now almost utilized universally, virus isolation from mucosa and skin in shell cultures is still applied in many clinical settings. Virus growth in cultured cells alone has the disadvantage that it cannot distinguish between HSV-1 and HSV-2. The aim of this study was to combine these two disparate methods. A protocol was developed to isolate clinical strains directly from the medium used for as the standard source of extraction to isolate DNA. Both HSV-1 and HSV-2 strains were isolated successfully from cervicovaginal swabs transported in OneSwab devices. It was found that the titers of these viruses did not correlate with the C(T) scores determined for the samples using real-time PCR. HSV-2 strains generally had lower titers than those of HSV-1, suggesting that the HSV-2 may be more sensitive to the sample handling procedures. These studies represent the first report of the culture and isolation of HSV from clinical samples using OneSwab intended exclusively for molecular diagnostic analyses.

A conserved carboxy-terminal domain in the major tegument structural protein VP22 facilitates virion packaging of a chimeric protein during productive herpes simplex virus 1 infection.

Recombinant virus HSV-1(RF177) was previously generated to examine tegument protein VP22 function by inserting the GFP gene into the gene encoding VP22. During a detailed analysis of this virus, we discovered that RF177 produces a novel fusion protein between the last 15 amino acids of VP22 and GFP, termed GCT-VP22. Thus, the VP22 carboxy-terminal specific antibody 22-3 and two anti-GFP antibodies reacted with an approximately 28 kDa protein from RF177-infected Vero cells. GCT-VP22 was detected at 1 and 3 hpi. Examination of purified virions indicated that GCT-VP22 was incorporated into RF177 virus particles. These observations imply that at least a portion of the information required for virion targeting is located in this domain of VP22. Indirect immunofluorescence analyses showed that GCT-VP22 also localized to areas of marginalized chromatin during RF177 infection. These results indicate that the last fifteen amino acids of VP22 participate in virion targeting during HSV-1 infection.

Cellular players in the herpes simplex virus dependent apoptosis balancing act.

Apoptosis is triggered as an intrinsic defense against numerous viral infections. Almost every virus encodes apoptotic modulators, and the herpes simplex viruses (HSV) are no exception. During HSV infection, there is an intricate balance between pro- and anti-apoptotic factors that delays apoptotic death until the virus has replicated. Perturbations in the apoptotic balance can cause premature cell death and have the potential to dramatically alter the outcome of infection. Recently, certain cellular genes have been shown to regulate sensitivity to HSV-dependent apoptosis. This review summarizes current knowledge of the cellular genes that impact the apoptotic balance during HSV infection.

Herpes simplex virus downregulates secretory leukocyte protease inhibitor: a novel immune evasion mechanism.

Secretory leukocyte protease inhibitor (SLPI), an anti-inflammatory mediator of mucosal immunity, inhibits human immunodeficiency virus (HIV) and herpes simplex virus (HSV) in cell culture. Epidemiological studies demonstrate that higher concentrations of SLPI in mucosal secretions are associated with a reduced risk of HIV transmission. The current studies were designed to test the hypothesis that HSV triggers a loss of SLPI to evade innate immunity and that this response may contribute to the increased risk of HIV infection in the setting of HSV infection. Exposure of human cervical epithelial cells to HSV-1 or HSV-2, but not HIV or vesicular stomatitis virus, triggered a significant and sustained reduction in SLPI levels. The reduction persisted when cells were infected in the presence of acyclovir but not following infection with UV-inactivated virus, indicating that viral gene expression, but not replication, is required. Reverse transcriptase PCR studies demonstrated that the loss of SLPI is mediated by downregulation of gene expression. SLPI downregulation was associated with activation of NF-kappaB signaling pathways and upregulation of proinflammatory cytokines, consistent with the known inhibitor effects of SLPI on NF-kappaB pathways. The downregulation mapped to viral early-gene expression, as variants impaired in expression of the ICP4 or ICP0 immediate-early gene failed to downregulate SLPI or activate NF-kappaB. Together, these results identify a novel role for HSV immediate-early-gene expression in regulating mucosal immune responses.

The major tegument structural protein VP22 targets areas of dispersed nucleolin and marginalized chromatin during productive herpes simplex virus 1 infection.

The herpes simplex virus (HSV) major tegument structural protein VP22 resides in multiple subcellular regions during productive infection. During an analysis of the molecular determinants of these localizations, we observed that a transfected fusion of the C-terminal portion of VP22, containing its pat4 nuclear localization signal, with GFP lacked nucleolar sparing compared to GFP alone. Thus, the initial goal was to determine whether VP22 associates with nucleoli. Using an optimized indirect immunofluorescence system to visualize nucleolin and viral proteins, we observed that VP22 present in VP22-expressing Vero (V49) cells "surrounded" nucleolin. These two initial findings implied that VP22 might associate directly with nucleoli. We next analyzed HSV-infected cells and observed that at late times, anti-nucleolin immune reactivity was dispersed throughout the nuclei while it retained uniform, circular staining in mock-infected cells. Time course infection experiments indicated that nucleolin initiated its transition from uniform to dispersed structures between 2 and 4 hpi. Comparison of Hoechst stained nuclei showed bright anti-nucleolin staining localized to regions of marginalized chromatin. These effects required de novo infected cell protein synthesis. A portion of VP22 detected in nuclei at 4 and 6 hpi localized to these areas of altered nucleolin and marginalized chromatin. VP22 was excluded from viral replication compartments containing the viral regulatory protein ICP22. Finally, altered nucleolin and marginalized chromatin were detected with a VP22-null virus, indicating that VP22 was not responsible for these nuclear architecture alterations. Thus, we conclude that nuclear VP22 targets unique subnuclear structures early (<6hpi) during herpes simplex virus 1 (HSV-1) infection.

Reconsideration of viral protein immunoblotting for differentiation of human herpes simplex viruses.

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are ubiquitous human pathogens that infect their hosts for life and reactivate to cause disease at or near the initial site of infection. As the incidence of genital HSV-1 infections increase, there is an increased demand for valid viral typing diagnostics. In this report, we reconsidered and developed a triple-phase immune-typing procedure that compares differences in electrophoretic mobilities of viral ICP4, ICP27, and VP22 proteins between HSV-1 and HSV-2 strains. We isolated and immunotyped 5 primary HSV-1 strains derived from orofacial, ocular, and genital areas along with 2 primary HSV-2 strains from the genital area. Advantages of this methodology include its general technical simplicity, sensitivity, and ability to definitively type HSV. It is anticipated that this methodology will be useful in distinguishing viruses obtained in clinical cultures.

Nuclear translocation of NF-kappaB precedes apoptotic poly(ADP-ribose) polymerase cleavage during productive HSV-1 replication in corneal epithelial cells.

PURPOSE: Herpes simplex virus (HSV)-1 infections of the human cornea range in severity from uncomplicated episodes that readily resolve to severe, recurring disease that invades the stroma, having a devastating permanent effect on vision. Recent published data implicate an apoptotic component to stromal HSV-1 infection. In a prior study, it was found that wild type (wt) HSV-1 infection induces, then blocks, apoptosis in epithelial cells derived from skin and that this block requires infected cell proteins (ICPs) synthesized between 3 and 6 hours post infection (hpi). This inhibition of apoptosis is in part dependent on the activation of inducible nuclear transcription factor kappaB (NF-kappaB). METHODS: HSV-1-dependent apoptosis in rabbit corneal epithelial (SIRC) cells was compared with that in infected human epithelial (HEp-2) cells. RESULTS: SIRC cells were sensitive to apoptotic cell death induced by environmental treatment with tumor necrosis factor (TNF)-alpha plus cycloheximide (CHX). HSV-1 stimulated the degradation of regulatory IkappaBalpha protein, resulting in nuclear translocation of NF-kappaB. This phenomenon was dependent on ICP synthesis. Neither wt nor apoptotic HSV-1 infection resulted in apoptosis in these cells. However, wt HSV-1-infected cells produced detectable levels of cleaved poly(ADP-ribose) (PARP). Inhibition of SIRC cell protein synthesis with CHX during wt HSV-1 infection led to a reduction in the amount of PARP cleavage. Whereas PARP cleavage defined cell death in most other cell types, its processing in SIRC cells was a reproducible characteristic of wt HSV-1 infection. CONCLUSIONS: This is the first report of such an effect, and it suggests that in corneal epithelial cells, activation of apoptotic pathways may be necessary for productive viral replication. Thus, efficient replication of HSV-1 in the corneal milieu proceeds via a different mechanism than it does in skin. However, it appears that NF-kappaB participates in inhibiting apoptosis during HSV-1 infection in both systems.