Herpes Simplex Virus Type 1 Proteins Research Articles

Identification of a novel neurovirulence factor encoded by the cryptic orphan gene UL31.6 of herpes simplex virus 1.

Although the herpes simplex virus type 1 (HSV-1) genome was thought to contain approximately 80 different protein coding sequences (CDSs), recent multi-omics analyses reported HSV-1 encodes more than 200 potential CDSs. However, few of the newly identified CDSs were confirmed to be expressed at the peptide or protein level in HSV-1-infected cells. Furthermore, the impact of the proteins they encode on HSV-1 infection is largely unknown. This study focused on a newly identified CDS, UL31.6. Re-analyzation of our previous chemical proteomics data verified that UL31.6 was expressed at the peptide level in HSV-1-infected cells. Antisera raised against a viral protein encoded by UL31.6 (pUL31.6) reacted with a protein with an approximate molecular mass of 37 kDa in lysates of Vero cells infected with each of three HSV-1 strains. pUL31.6 was efficiently dissociated from virions in high-salt solution. A UL31.6-null mutation had a minimal effect on HSV-1 gene expression, replication, cell-to-cell spread, and morphogenesis in Vero cells; in contrast, it significantly reduced HSV-1 cell-to-cell spread in three neural cells but not in four non-neural cells including Vero cells. The UL31.6-null mutation also significantly reduced the mortality and viral replication in the brains of mice after intracranial infection, but had minimal effects on pathogenic manifestations in and around the eyes, and viral replication detected in the tear films of mice after ocular infection. These results indicated that pUL31.6 was a tegument protein and specifically acted as a neurovirulence factor by potentially promoting viral transmission between neuronal cells in the central nervous system.IMPORTANCERecent multi-omics analyses reported the herpes simplex virus type 1 (HSV-1) genome encodes an additional number of potential coding sequences (CDSs). However, the expressions of these CDSs at the peptide or protein levels and the biological effects of these CDSs on HSV-1 infection remain largely unknown. This study annotated a cryptic orphan CDS, termed UL31.6, an HSV-1 gene that encodes a tegument protein with an approximate molecular mass of 37 kDa, which specifically acts as a neurovirulence factor. Our study indicates that HSV-1 proteins important for viral pathogenesis remain to be identified and a comprehensive understanding of the pathogenesis of HSV-1 will require not only the identification of cryptic orphan CDSs using emerging technologies but also step-by-step and in-depth analyses of each of the cryptic orphan CDSs.

HSV-2 triggers upregulation of MALAT1 in CD4+ T cells and promotes HIV latency reversal.

Herpes simplex virus type 2 (HSV-2) coinfection is associated with increased HIV-1 viral loads and expanded tissue reservoirs, but the mechanisms are not well defined. HSV-2 recurrences result in an influx of activated CD4+ T cells to sites of viral replication and an increase in activated CD4+ T cells in peripheral blood. We hypothesized that HSV-2 induces changes in these cells that facilitate HIV-1 reactivation and replication and tested this hypothesis in human CD4+ T cells and 2D10 cells, a model of HIV-1 latency. HSV-2 promoted latency reversal in HSV-2-infected and bystander 2D10 cells. Bulk and single-cell RNA-Seq studies of activated primary human CD4+ T cells identified decreased expression of HIV-1 restriction factors and increased expression of transcripts including MALAT1 that could drive HIV replication in both the HSV-2-infected and bystander cells. Transfection of 2D10 cells with VP16, an HSV-2 protein that regulates transcription, significantly upregulated MALAT1 expression, decreased trimethylation of lysine 27 on histone H3 protein, and triggered HIV latency reversal. Knockout of MALAT1 from 2D10 cells abrogated the response to VP16 and reduced the response to HSV-2 infection. These results demonstrate that HSV-2 contributes to HIV-1 reactivation through diverse mechanisms, including upregulation of MALAT1 to release epigenetic silencing.

Inhibitory activity and mechanism of silver nanoparticles against herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) is a common pathogen that infects 50-90% of the world's population and causes a variety of diseases, some of which can be life-threatening. Silver nanoparticles (AgNPs) have been shown to have broad-spectrum antiviral activity. In this study, we investigated the activity of AgNPs against HSV-1 and found that AgNPs effectively inhibited plaque formation and HSV-1 progeny production, reduced the genomic load, and interfered with HSV-1 mRNA expression and protein synthesis. Transmission electron microscopy showed that AgNPs interacted with HSV-1 and altered the shape of the viral particles. Furthermore, AgNPs affected the entry of HSV-1 into cells as well as their release and cell-to-cell spread. AgNPs were also found to downregulate the expression of pro-inflammatory cytokines upon HSV-1 infection. Combined treatment with AgNPs and acyclovir (ACV) confirmed that AgNPs significantly enhanced the inhibitory effect of ACV against HSV-1. Our findings may contribute to an understanding of the mechanism of the antiviral effect of AgNPs against HSV-1 and help to provide a theoretical basis for their clinical application.

Herpes simplex virus 1 proteins can induce skin inflammation in an atopic dermatitis-like mouse model.

Herpes simplex virus type 1 (HSV-1) can induce in certain individuals with atopic dermatitis (AD) severe cutaneous infections that can spread throughout the entire body, a condition named as AD complicated by eczema herpeticum (ADEH). It has been recently found that ADEH patients can produce specific IgE against HSV-1 proteins, which may contribute to lower protection against HSV-1. However, little is known about the capacity of these HSV-1 proteins to produce an inflammatory response at the skin level. In this study, using a mouse model of AD-like dermatitis, three HSV-1 proteins (glycoprotein D -gD-, glycoprotein B -gB- and VP22) were applied on tape-stripped back skin mice in three exposures periods. Ovalbumin (OVA) and 0.9% NaCl were used as positive and negative controls, respectively. Skin samples were obtained for analysis of specific cell components of skin infiltration. The results showed that the viral protein gD induced a statistically significant increase in the number of dermal infiltrating CD3+, CD4+ cells and mast cells compared with the negative control group. gD was also able to induce epidermal thickening and epidermal infiltration of T cells closely related to the one produced in mice sensitized with OVA. However, VP22 and gB contributed to a lesser extent to skin inflammation. These results showed that proteins from HSV-1, especially gD, can have per se an important T cell and mast cell-driven inflammatory potential at the skin level.

Prediction and analysis of human‐herpes simplex virus type 1 protein‐protein interactions by integrating multiple methods

BackgroundHerpes simplex virus type 1 (HSV‐1) is a ubiquitous infectious pathogen that widely affects human health. To decipher the complicated human‐HSV‐1 interactions, a comprehensive protein‐protein interaction (PPI) network between human and HSV‐1 is highly demanded.MethodsTo complement the experimental identification of human‐HSV‐1 PPIs, an integrative strategy to predict proteome‐wide PPIs between human and HSV‐1 was developed. For each human‐HSV‐1 protein pair, four popular PPI inference methods, including interolog mapping, the domain‐domain interaction‐based method, the domain‐motif interaction‐based method, and the machine learning‐based method, were optimally implemented to generate four interaction probability scores, which were further integrated into a final probability score.ResultsAs a result, a comprehensive high‐confidence PPI network between human and HSV‐1 was established, covering 10,432 interactions between 4,546 human proteins and 72 HSV‐1 proteins. Functional and network analyses of the HSV‐1 targeting proteins in the context of human interactome can recapitulate the known knowledge regarding the HSV‐1 replication cycle, supporting the overall reliability of the predicted PPI network. Considering that HSV‐1 infections are implicated in encephalitis and neurodegenerative diseases, we focused on exploring the biological significance of the brain‐specific human‐HSV‐1 PPIs. In particular, the predicted interactions between HSV‐1 proteins and Alzheimer’s‐disease‐related proteins were intensively investigated.ConclusionThe current work can provide testable hypotheses to assist in the mechanistic understanding of the human‐HSV‐1 relationship and the anti‐HSV‐1 pharmaceutical target discovery. To make the predicted PPI network and the datasets freely accessible to the scientific community, a user‐friendly database browser was released at http://www.zzdlab.com/HintHSV/index.php.

Herpes Simplex Virus Type 1 and Host Antiviral Immune Responses: An Update.

Herpes simplex virus type 1 (HSV-1) infection activates a rapid stimulation of host innate immune responses and a delicate interplay between virus and host immune elements regulates the whole events. Although host immune elements play well in limiting the HSV-1 infection by interfering viral replication, they are still unable to remove the virus completely, because HSV-1 proteins are efficient enough to bypass the host antiviral immune responses and virus succeed to reactivate again from latency at opportune time. Type 1 interferon signaling pathway is the central point of innate immunity along with some of the activated neutrophils, monocytes, macrophages, and dendritic cells, and some natural killer cells play role, while the CD8+ T cells are crucial in adaptive immunity. In this review, the current knowledge of host and HSV-1 interaction has been described that how the host antiviral immune responses occur and what are the mechanisms of viral evasion adapted by virus to counteract with both arms of immunity.

Combined PD-1 and LAG-3 Blockade Together with HLA-A*0201 Epitopes Immunotherapy Restore the Function of CD8+ TRM Cells and Reduce UV-B Induced Recurrent Ocular Herpes in HSV-1 Infected “Humanized” HLA Transgenic Mice

Abstract Exhaustion of herpes simplex virus type 1 (HSV-1)-specific CD8+ T cells is a notable cause that contribute to the frequency and severity of recurrent herpes. In the present study, we found that functionally exhausted HSV-specific CD8+ T cells, expressing both the programmed death-1 (PD-1) and lymphocyte activation gene-3 (LAG-3), two immune checkpoint receptors that promotes T cell exhaustion, were frequent in symptomatic (SYMP) patients with a history of numerous episodes of recurrent corneal herpetic disease. Similarly, using a novel “humanized” HLA-A*0201 transgenic mouse model of UV-B induced recurrent corneal herpetic disease, we demonstrated that SYMP mice with increased virus shedding and severe recurrent corneal herpetic disease had more exhausted HSV-specific tissue resident PD1+LAG-3+CD8+ TRM cells in both trigeminal ganglia (TG) and cornea. Dual blockade of both PD-1 and LAG-3 pathways restored the number and function of antiviral CD8+ TRM cells and appeared to be synergistically superior to blockade of either pathway alone. Moreover, blockade of PD-1/LAG-3 immune checkpoints in combination with therapeutic vaccination with a mixture of HLA-A*0201-restricted CD8+ T cell epitopes UL9196–204, UL25572–580, and UL44400–408, recently identified from HSV-1 proteins, significantly improved protection against UV-B induced recurrent herpes. This pre-clinical study indicate that dual blockade of Immune checkpoints combined with therapeutic vaccination with human epitopes identified from HSV-1 proteins is a promising strategy to protection against recurrent herpes in human.

Herpes Simplex Virus Type 1 Enhances Expression of the Synaptic Protein Arc for Its Own Benefit.

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus able to reach the central nervous system (CNS) after primary infection in oronasal mucosa. HSV-1 establishes latency inside neurons due the repression of its gene expression process, which is related to periodic reactivations in response to cellular stress conditions, constituting a risk factor for neurodegenerative diseases such as Alzheimer’s disease (AD). The immediate-early gene Arc plays an essential role in neuronal morphology, synaptic plasticity and memory formation. Arc acts as a hub protein, interacting with components of the endocytic machinery required for AMPA receptor (AMPAR) recycling as well as with proteins of the post-synaptic density and actin cytoskeleton. However, to date, no studies have evaluated whether persistent neurotropic HSV-1 infection modulates the expression or function of Arc protein in brain tissue. Here, we report that neuronal in vivo and in vitro infection of HSV-1 significantly increases Arc protein levels, showing a robust perinuclear distribution in neuronal cell lines, a process that is dependent on an active HSV-1 replication cycle. Finally, we found that silencing Arc protein caused a decrease in HSV-1 proteins and viral progeny, suggesting that Arc is involved in the lifecycle of HSV-1. Our studies strongly suggest that pathogenicity of HSV-1 neuronal reactivations in humans could be mediated in part by Arc neuronal upregulation and its potential role in endocytic trafficking and AMPA-neuronal function impairment. Further studies are necessary to define whether this phenomenon could have repercussions in cognition and learning processes in infected individuals.

Viral Genetics Modulate Orolabial Herpes Simplex Virus Type 1 Shedding in Humans.

Orolabial herpes simplex virus type 1 (HSV-1) infection has a wide spectrum of severity in immunocompetent persons. To study the role of viral genotype and host immunity, we characterized oral HSV-1 shedding rates and host cellular response, and genotyped viral strains, in monozygotic (MZ) and dizygotic (DZ) twins. A total of 29 MZ and 22 DZ HSV-1-seropositive twin pairs were evaluated for oral HSV-1 shedding for 60 days. HSV-1 strains from twins were genotyped as identical or different. CD4+ T-cell responses to HSV-1 proteins were studied. The median per person oral HSV shedding rate was 9% of days that a swab was obtained (mean, 10.2% of days). A positive correlation between shedding rates was observed within all twin pairs, and in the MZ and DZ twins. In twin subsets with sufficient HSV-1 DNA to genotype, 15 had the same strain and 14 had different strains. Viral shedding rates were correlated for those with the same but not different strains. The median number of HSV-1 open reading frames recognized per person was 16. The agreement in the CD4+ T-cell response to specific HSV-1 open reading frames was greater between MZ twins than between unrelated persons (P = .002). Viral strain characteristics likely contribute to oral HSV-1 shedding rates.

Photodynamic Inactivation of Herpes Simplex Viruses.

Herpes simplex virus (HSV) infections can be treated with direct acting antivirals like acyclovir and foscarnet, but long-term use can lead to drug resistance, which motivates research into broadly-acting antivirals that can provide a greater genetic barrier to resistance. Photodynamic inactivation (PDI) employs a photosensitizer, light, and oxygen to create a local burst of reactive oxygen species that inactivate microorganisms. The botanical plant extract OrthoquinTM is a powerful photosensitizer with antimicrobial properties. Here we report that Orthoquin also has antiviral properties. Photoactivated Orthoquin inhibited herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) infection of target cells in a dose-dependent manner across a broad range of sub-cytotoxic concentrations. HSV inactivation required direct contact between Orthoquin and the inoculum, whereas pre-treatment of target cells had no effect. Orthoquin did not cause appreciable damage to viral capsids or premature release of viral genomes, as measured by qPCR for the HSV-1 genome. By contrast, immunoblotting for HSV-1 antigens in purified virion preparations suggested that higher doses of Orthoquin had a physical impact on certain HSV-1 proteins that altered protein mobility or antigen detection. Orthoquin PDI also inhibited the non-enveloped adenovirus (AdV) in a dose-dependent manner, whereas Orthoquin-mediated inhibition of the enveloped vesicular stomatitis virus (VSV) was light-independent. Together, these findings suggest that the broad antiviral effects of Orthoquin-mediated PDI may stem from damage to viral attachment proteins.

Assessment of Herpes Simplex Virus Type 1 and 2 by IHC in Association with CD14 Antigen in Placental Tissues from Women with Miscarriage

Among many viral causes of miscarriage, maternal infections caused by herpes simplex virus type -1 (HSV-1) and Herpes simplex virus type-2 (HSV-2) infections are important causes. CD14 was implicated in immune mechanisms, and might be involved in pregnancy loss. The aim of this study was to detect the possible occurrence of two HSV-1 and HSV-2 infections as well as the immunological factor cd14 in placental tissues from patients with spontaneous abortion using immunohistochemistry techniques. Immunohistochemistry technique assay was used to detect placental infection with HSV-1& HSV-2 as well as CD14 in 40 women with spontaneous miscarriage and in 40 healthy deliveries in Baghdad/Iraq. The IHC- expressions of HSV-1, HSV-2, and CD14 proteins were detected in 4(10%), 15(37.5%), 15(37.5%) in placental tissues from miscarriage women, respectively. The association between CD14 with HSV-1 and HSV-2 among the placental tissues from miscarriage patients was constituting (5%), (20%) with HSV-1 & HSV-2, respectively. The considerable proportion of HSV-1, HSV-2, and infections found in the placental tissues from miscarriage women could drag the attention of obstetricians to implicate these viruses as possible targets in the etiology of spontaneous abortion case and there is possible association of CD14 with HSV-1 and HSV-2 in women with abortion.

The lysosome system is severely impaired in a cellular model of neurodegeneration induced by HSV-1 and oxidative stress.

The causal agent(s) and molecular mechanisms of Alzheimer's disease (AD) remain unclear. Mounting evidence suggests that herpes simplex virus type 1 (HSV-1) infection is involved in the AD pathogenesis. Oxidative stress (OS) may also be crucial in the AD development. Our group previously reported that both HSV-1 and OS trigger the appearance of AD-type neurodegeneration markers. The main aim of the present study was to identify the mechanisms involved in this triggering. Expression studies revealed the involvement of a set of OS-regulated genes in HSV-1-infected cells and in cells harboring the Swedish mutation of the amyloid beta precursor protein gene. Functional annotation of these genes revealed the lysosome system to be impaired, suggesting that the interaction of OS with both HSV-1 and amyloid beta precursor protein mutations affects lysosomal function. Functional studies revealed HSV-1 infection and OS to increase the lysosome load, reduce the activity of lysosomal hydrolases, affect cathepsin maturation, and inhibit the endocytosis-mediated degradation of the epidermal growth factor receptor. These findings suggest alterations in the lysosome system to be involved in different forms of AD.

DETECTION OF SPECIFIC ANTIBODIES TO HSV-1 WITH THE OPTICAL BIOSENSOR

Aim. To investigate the possibility of using the optoelectronic biosensor «Plasmon-6» based on surface plasmon resonance (SPR) for detection of specific antibodies to herpes simplex virus type 1 (HSV-1). Methods. HSV-1 was propagated in MDBK (Madin-Darby Bovine Kidney) cell culture and was purified by differential centrifugation in cesium chloride density gradient. The presence of viral proteins was tested by polyacrylamide gel electrophoresis. Viral proteins were applied on the biochip surface pre-modified by dextran. Selection of human blood sera was performed by ELISA using the test systems «HSV-1 IgG ELISA» (GenWay, USA) and «HSV-1 IgG ELISA» (Vector-Best, Russia). Biosensor analysis was carried out on the optoelectronic spectrometer «Plasmon-6». Results. Concentrated preparation of HSV-1 proteins was obtained. It was shown that application of viral proteins at 8x10-5 mg / mm2 was optimal for detection of antibodies against HSV-1. 10 negative to HSV-1 blood sera of donors have been tested on biochips to determine the limits of positive and negative response. The comparative analysis of 25 blood sera of the patients for detection the antibodies to HSV-1 was carried out by ELISA and SPR methods. The coincidence of the results of SPR-analysis and ELISA was in 24 of 25 cases. Conclusion. As a result of carried out investigations there were shown that immunosensor analysis using the device «Plasmon-6» as well as traditional approaches allowed to detect specific antibodies to HSV-1 in blood sera.

Broad-spectrum non-nucleoside inhibitors of human herpesviruses

Herpesvirus infections cause considerable morbidity and mortality through lifelong recurrent cycles of lytic and latent infection in several tissues, including the human nervous system. Acyclovir (ACV) and its prodrug, the current antivirals of choice for herpes simplex virus (HSV) and, to some extent, varicella zoster virus (VZV) infections are nucleoside analogues that inhibit viral DNA replication. Rising viral resistance and the need for more effective second-line drugs have motivated searches for additional antiviral agents, particularly non-nucleoside based agents. We evaluated the antiviral activity of five compounds with predicted lysosomotropic activity using conventional and human induced pluripotent stem cell-derived neuronal (iPSC-neurons) cultures. Their potency and toxicity were compared with ACV and the lysosomotropic agents chloroquine and bafilomycin A1. Out of five compounds tested, micromolar concentrations of 30N12, 16F19, and 4F17 showed antiviral activity comparable to ACV (50μM) during lytic herpes simplex virus type 1 (HSV-1) infections, reduced viral DNA copy number, and reduced selected HSV-1 protein levels. These compounds also inhibited the reactivation of ‘quiescent’ HSV-1 infection established in iPSC-neurons, but did not inhibit viral entry into host cells. The same compounds had greater potency than ACV against lytic VZV infection; they also inhibited replication of human cytomegalovirus. The anti-herpetic effects of these non-nucleoside agents merit further evaluation in vivo.

Targeting HSV-1 protein ICP6 by RIP1 and RIP3 initiates necroptosis to restrict HSV-1 propagation in mice (VIR9P.1159)

Abstract Necroptosis is a type of programmed necrosis that participates in host defense to eliminate certain pathogens. We show that receptor interacting protein 3 (RIP3 or RIPK3) restricts herpes simplex virus type 1 (HSV-1) propagation in mouse cells and in mice by mediating necroptosis of infected cells. ICP6 protein of HSV-1 triggers necroptosis by direct interaction with RIP1 and RIP3 through its RIP homotypic interaction motif (RHIM). ICP6 can be dimerized or oliogmerized by its C-terminal R1 homology domain. This leads to RIP1-RIP3 hetero- and RIP3-RIP3 homo-interaction and subsequent execution of necroptosis in HSV-1 infected cells. We show that both pro-necroptotic and anti-necroptotic viral RHIM exist since the RHIM in viral inhibitor of RIP activation (vIRA) of murine cytomegalovirus (MCMV) is known to inhibit necroptosis. Our data demonstrated that RIP1 and RIP3 can directly sense certain RHIM like sequences encoded by some viruses to initiate cellular responses for pathogen clearance.

Development of a novel cell-based assay to monitor the transactivation activity of the HSV-1 protein ICP0

The herpes simplex virus type 1 (HSV-1) immediate-early phosphoprotein infected cell protein 0 (ICP0) is a potent transcriptional activator of viral genes and is required for efficient viral replication and reactivation from latency. However, it is largely unknown what role specific cellular factors play in the transactivator function of ICP0. With the long-term goal of identifying these factors, we developed a cell-based assay in a 96-well format to measure this activity of ICP0. We designed a system using a set of HSV-1 GFP reporter viruses in which the expression of GFP is potently induced by ICP0 in cell culture. The initial feasibility of this system was confirmed over a 24-h period by fluorescence microscopy. We adapted this assay to a 96-well plate format, quantifying GFP expression with a fluorescence scanner. Our results indicate that the cell-based assay we developed is a valid and effective method for examining the transactivating activity of ICP0. This assay can be used to identify cellular factors that regulate the transactivating activity of ICP0.

Herpes simplex virus type 1 and Alzheimer's disease: increasing evidence for a major role of the virus.

Herpes simplex virus type 1 (HSV1), when present in brain of carriers of the type 4 allele of the apolipoprotein E gene (APOE), has been implicated as a major factor in Alzheimer’s disease (AD). It is proposed that virus is normally latent in many elderly brains but reactivates periodically (as in the peripheral nervous system) under certain conditions, for example stress, immunosuppression, and peripheral infection, causing cumulative damage and eventually development of AD. Diverse approaches have provided data that explicitly support, directly or indirectly, these concepts. Several have confirmed HSV1 DNA presence in human brains, and the HSV1-APOE-ε4 association in AD. Further, studies on HSV1-infected APOE-transgenic mice have shown that APOE-e4 animals display a greater potential for viral damage. Reactivated HSV1 can cause direct and inflammatory damage, probably involving increased formation of beta amyloid (Aβ) and of AD-like tau (P-tau)—changes found to occur in HSV1-infected cell cultures. Implicating HSV1 further in AD is the discovery that HSV1 DNA is specifically localized in amyloid plaques in AD. Other relevant, harmful effects of infection include the following: dynamic interactions between HSV1 and amyloid precursor protein (APP), which would affect both viral and APP transport; induction of toll-like receptors (TLRs) in HSV1-infected astrocyte cultures, which has been linked to the likely effects of reactivation of the virus in brain. Several epidemiological studies have shown, using serological data, an association between systemic infections and cognitive decline, with HSV1 particularly implicated. Genetic studies too have linked various pathways in AD with those occurring on HSV1 infection. In relation to the potential usage of antivirals to treat AD patients, acyclovir (ACV) is effective in reducing HSV1-induced AD-like changes in cell cultures, and valacyclovir, the bioactive form of ACV, might be most effective if combined with an antiviral that acts by a different mechanism, such as intravenous immunoglobulin (IVIG).

Persistence of mucosal T-cell responses to herpes simplex virus type 2 in the female genital tract.

Relatively little is known about the human T cell response to HSV-2 in the female genital tract, a major site of heterosexual HSV-2 acquisition, transmission and reactivation. In order to understand the role of local mucosal immunity in HSV-2 infection, T cell lines were expanded from serial cervical cytobrush samples from 30 HSV-2 infected women and examined for reactivity to HSV-2. Approximately 3% of the CD3+ T cells isolated from the cervix were HSV-2 specific and of these, a median of 91.3% were CD4+ while a median of 3.9% were CD8+. HSV-2 specific CD4+ T cells expanded from the cervix were not only more frequent than CD8+ T cells but also exhibited greater breadth in terms of antigenic reactivity. T cells directed at the same HSV-2 protein were often detected in serial cervical cytobrush samples and in blood. Thus, broad and persistent mucosal T cell responses to HSV-2 were detected in the female genital tract of HSV-2+ women suggesting that these cells are resident at the site of HSV-2 infection. Understanding the role of these T cells at this biologically relevant site will be central to the elucidation of adaptive immune mechanisms involved in controlling HSV-2 disease.

Local CD4 and CD8 T-Cell Reactivity to HSV-1 Antigens Documents Broad Viral Protein Expression and Immune Competence in Latently Infected Human Trigeminal Ganglia

Herpes simplex virus type 1 (HSV-1) infection results in lifelong chronic infection of trigeminal ganglion (TG) neurons, also referred to as neuronal HSV-1 latency, with periodic reactivation leading to recrudescent herpetic disease in some persons. HSV-1 proteins are expressed in a temporally coordinated fashion during lytic infection, but their expression pattern during latent infection is largely unknown. Selective retention of HSV-1 reactive T-cells in human TG suggests their role in controlling reactivation by recognizing locally expressed HSV-1 proteins. We characterized the HSV-1 proteins recognized by virus-specific CD4 and CD8 T-cells recovered from human HSV-1–infected TG. T-cell clusters, consisting of both CD4 and CD8 T-cells, surrounded neurons and expressed mRNAs and proteins consistent with in situ antigen recognition and antiviral function. HSV-1 proteome-wide scans revealed that intra-TG T-cell responses included both CD4 and CD8 T-cells directed to one to three HSV-1 proteins per person. HSV-1 protein ICP6 was targeted by CD8 T-cells in 4 of 8 HLA-discordant donors. In situ tetramer staining demonstrated HSV-1-specific CD8 T-cells juxtaposed to TG neurons. Intra-TG retention of virus-specific CD4 T-cells, validated to the HSV-1 peptide level, implies trafficking of viral proteins from neurons to HLA class II-expressing non-neuronal cells for antigen presentation. The diversity of viral proteins targeted by TG T-cells across all kinetic and functional classes of viral proteins suggests broad HSV-1 protein expression, and viral antigen processing and presentation, in latently infected human TG. Collectively, the human TG represents an immunocompetent environment for both CD4 and CD8 T-cell recognition of HSV-1 proteins expressed during latent infection. HSV-1 proteins recognized by TG-resident T-cells, particularly ICP6 and VP16, are potential HSV-1 vaccine candidates.

Proteomics Analysis of Herpes Simplex Virus Type 1-Infected Cells Reveals Dynamic Changes of Viral Protein Expression, Ubiquitylation, and Phosphorylation

Herpesviruses are among the most complex and widespread human viruses and cause a number of diseases ranging from cold sores to genital infections and encephalitis. While the composition of viral particles has been studied, less is known about the expression of the whole viral proteome in infected cells. Here, we analyzed the proteome of the prototypical Herpes Simplex Virus type 1 (HSV1) in infected cells by mass spectrometry. Using a high sensitivity LTQ-Orbitrap, we achieved a very high level of protein coverage and identified a total of 67 structural and nonstructural viral proteins. We also identified 90 novel phosphorylation sites and 10 novel ubiquitylation sites on different viral proteins. Ubiquitylation was observed on nine HSV1 proteins. We identified phosphorylation sites on about half of the detected viral proteins; many of the highly phosphorylated ones are known to regulate gene expression. Treatment with inhibitors of DNA replication induced changes of both viral protein abundance and modifications, highlighting the interdependence of viral proteins during the life cycle. Given the importance of expression dynamics, ubiquitylation, and phosphorylation for protein function, these findings will serve as important tools for future studies on herpesvirus biology.