TRPV4 channel is involved in HSV-2 infection in human vaginal epithelial cells through triggering Ca2+ oscillation.

Herpes simplex virus (HSV) infection induces a rapid and transient increase in intracellular calcium concentration ([Ca2+]i), which plays a critical role in facilitating viral entry. T-type calcium channel blockers and EGTA, a chelate of extracellular Ca2+, suppress HSV-2 infection. But the cellular mechanisms mediating HSV infection-activated Ca2+ signaling have not been completely defined. In this study we investigated whether the TRPV4 channel was involved in HSV-2 infection in human vaginal epithelial cells. We showed that the TRPV4 channel was expressed in human vaginal epithelial cells (VK2/E6E7). Using distinct pharmacological tools, we demonstrated that activation of the TRPV4 channel induced Ca2+ influx, and the TRPV4 channel worked as a Ca2+-permeable channel in VK2/E6E7 cells. We detected a direct interaction between the TRPV4 channel protein and HSV-2 glycoprotein D in the plasma membrane of VK2/E6E7 cells and the vaginal tissues of HSV-2-infected mice as well as in phallic biopsies from genital herpes patients. Pretreatment with specific TRPV4 channel inhibitors, GSK2193874 (1-4 µM) and HC067047 (100 nM), or gene silence of the TRPV4 channel not only suppressed HSV-2 infectivity but also reduced HSV-2-induced cytokine and chemokine generation in VK2/E6E7 cells by blocking Ca2+ influx through TRPV4 channel. These results reveal that the TRPV4 channel works as a Ca2+-permeable channel to facilitate HSV-2 infection in host epithelial cells and suggest that the design and development of novel TRPV4 channel inhibitors may help to treat HSV-2 infections.

Herpes simplex virus type 2 inhibits TNF-α-induced NF-κB activation through viral protein ICP22-mediated interaction with p65.

Herpes simplex virus type 2 (HSV-2) is a prevalent human pathogen and the main cause of genital herpes. After initial infection, HSV-2 can establish lifelong latency within dorsal root ganglia by evading the innate immunity of the host. NF-κB has a crucial role in regulating cell proliferation, inflammation, apoptosis, and immune responses. It is known that inhibition of NF-κB activation by a virus could facilitate it to establish infection in the host. In the current study, we found that HSV-2 inhibited TNF-α-induced activation of NF-κB-responsive promoter in a dose-dependent manner, while UV-inactivated HSV-2 did not have such capability. We further identified the immediate early protein ICP22 of HSV-2 as a vital viral element in inhibiting the activation of NF-κB-responsive promoter. The role of ICP22 was confirmed in human cervical cell line HeLa and primary cervical fibroblasts in the context of HSV-2 infection, showing that ICP22 deficient HSV-2 largely lost the capability in suppressing NF-κB activation. HSV-2 ICP22 was further shown to suppress the activity of TNF receptor-associated factor 2 (TRAF2)-, IκB kinase α (IKK α)-, IKK ß-, IKK γ-, or p65-induced activation of NF-κB-responsive promoter. Mechanistically, HSV-2 ICP22 inhibited the phosphorylation and nuclear translocation of p65 by directly interacting with p65, resulting in the blockade of NF-κB activation. Furthermore, ICP22 from several alpha-herpesviruses could also inhibit NF-κB activation, suggesting the significance of ICP22 in herpesvirus immune evasion. Findings in this study highlight the importance of ICP22 in inhibiting NF-κB activation, revealing a novel mechanism by which HSV-2 evades the host antiviral responses.

The structural basis of herpesvirus entry.

Herpesviruses are ubiquitous, double-stranded DNA, enveloped viruses that establish lifelong infections and cause a range of diseases. Entry into host cells requires binding of the virus to specific receptors, followed by the coordinated action of multiple viral entry glycoproteins to trigger membrane fusion. Although the core fusion machinery is conserved for all herpesviruses, each species uses distinct receptors and receptor-binding glycoproteins. Structural studies of the prototypical herpesviruses herpes simplex virus 1 (HSV-1), HSV-2, human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) entry glycoproteins have defined the interaction sites for glycoprotein complexes and receptors, and have revealed conformational changes that occur on receptor binding. Recent crystallography and electron microscopy studies have refined our model of herpesvirus entry into cells, clarifying both the conserved features and the unique features. In this Review, we discuss recent insights into herpesvirus entry by analysing the structures of entry glycoproteins, including the diverse receptor-binding glycoproteins (HSV-1 glycoprotein D (gD), EBV glycoprotein 42 (gp42) and HCMV gH-gL-gO trimer and gH-gL-UL128-UL130-UL131A pentamer), as well gH-gL and the fusion protein gB, which are conserved in all herpesviruses.

Herpes Simplex Virus 2 Counteracts Neurite Outgrowth Repulsion during Infection in a Nerve Growth Factor-Dependent Manner.

During primary infection, herpes simplex virus 2 (HSV-2) replicates in epithelial cells and enters neurites to infect neurons of the peripheral nervous system. Growth factors and attractive and repulsive directional cues influence neurite outgrowth and neuronal survival. We hypothesized that HSV-2 modulates the activity of such cues to increase neurite outgrowth. To test this hypothesis, we exposed sensory neurons to nerve growth factor (NGF) and mock- or HSV-2-infected HEK-293T cells, since they express repellents of neurite outgrowth. We show that HEK-293T cells secrete factors that inhibit neurite outgrowth, while infection with HSV-2 strains MS and 333 reduces this repelling phenotype, increasing neurite numbers. The HSV-2-mediated restoration of neurite outgrowth required the activity of NGF. In the absence of infection, however, NGF did not overcome the repulsion mediated by HEK-293T cells. We previously showed that recombinant, soluble glycoprotein G of HSV-2 (rSgG2) binds and enhances NGF activity, increasing neurite outgrowth. However, the effect of gG2 during infection has not been investigated. Therefore, we addressed whether gG2 contributes to overcoming neurite outgrowth repulsion. To do so, we generated viruses lacking gG2 expression and complemented them by exogenous expression of gG2. Overall, our results suggest that HSV-2 infection of nonneuronal cells reduces their repelling effect on neurite outgrowth in an NGF-dependent manner. gG2 contributed to this phenotype, but it was not the only factor. The enhanced neurite outgrowth may facilitate HSV-2 spread from epithelial cells into neurons expressing NGF receptors and increase HSV-2-mediated pathogenesis.IMPORTANCE Herpes simplex virus 2 (HSV-2) is a prevalent human pathogen that establishes lifelong latency in neurons of the peripheral nervous system. Colonization of neurons is required for HSV-2 persistence and pathogenesis. The viral and cellular factors required for efficient infection of neurons are not fully understood. We show here that nonneuronal cells repel neurite outgrowth of sensory neurons, while HSV-2 infection overcomes this inhibition and, rather, stimulates neurite outgrowth. HSV-2 glycoprotein G and nerve growth factor contribute to this phenotype, which may attract neurites to sites of infection and facilitate virus spread to neurons. Understanding the mechanisms that modulate neurite outgrowth and facilitate HSV-2 infection of neurons might foster the development of therapeutics to reduce HSV-2 colonization of the nervous system and provide insights on neurite outgrowth and regeneration.

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

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

A Single-Cycle Glycoprotein D Deletion Viral Vaccine Candidate, ΔgD-2, Elicits Polyfunctional Antibodies That Protect against Ocular Herpes Simplex Virus.

Herpes simplex virus 1 (HSV-1) is a leading cause of infectious blindness, highlighting the need for effective vaccines. A single-cycle HSV-2 strain with the deletion of glycoprotein D, ΔgD-2, completely protected mice from HSV-1 and HSV-2 skin or vaginal disease and prevented latency following active or passive immunization in preclinical studies. The antibodies functioned primarily by activating Fc receptors to mediate antibody-dependent cellular cytotoxicity (ADCC). The ability of ADCC to protect the immune-privileged eye, however, may differ from skin or vaginal infections. Thus, the current studies were designed to compare active and passive immunization with ΔgD-2 versus an adjuvanted gD subunit vaccine (rgD-2) in a primary lethal ocular murine model. ΔgD-2 provided significantly greater protection than rgD-2 following a two-dose vaccine regimen, although both vaccines were protective compared to an uninfected cell lysate. However, only immune serum from ΔgD-2-vaccinated, but not rgD-2-vaccinated, mice provided significant protection against lethality in passive transfer studies. The significantly greater passive protection afforded by ΔgD-2 persisted after controlling for the total amount of HSV-specific IgG in the transferred serum. The antibodies elicited by rgD-2 had significantly higher neutralizing titers, whereas those elicited by ΔgD-2 had significantly more C1q binding and Fc gamma receptor activation, a surrogate for ADCC function. Together, the findings suggest ADCC is protective in the eye and that nonneutralizing antibodies elicited by ΔgD-2 provide greater protection than neutralizing antibodies elicited by rgD-2 against primary ocular HSV disease. The findings support advancement of vaccines, including ΔgD-2, that elicit polyfunctional antibody responses.IMPORTANCE Herpes simplex virus 1 is the leading cause of infectious corneal blindness in the United States and Europe. Developing vaccines to prevent ocular disease is challenging because the eye is a relatively immune-privileged site. In this study, we compared a single-cycle viral vaccine candidate, which is unique in that it elicits predominantly nonneutralizing antibodies that activate Fc receptors and bind complement, and a glycoprotein D subunit vaccine that elicits neutralizing but not Fc receptor-activating or complement-binding responses. Only the single-cycle vaccine provided both active and passive protection against a lethal ocular challenge. These findings greatly expand our understanding of the types of immune responses needed to protect the eye and will inform future prophylactic and therapeutic strategies.

Peculiar vegetative tumor-like genital herpes simplex nodules with brisk tissue eosinophilia in patients with human immunodeficiency virus infection.

Genital herpes simplex virus (HSV) infection in a human immunodeficiency virus (HIV) patient can present as a vegetative nodule. Clinical differential diagnoses of the nodule include condyloma latum, condyloma acuminatum, viral or fungal infection, and cutaneous neoplasms. Histological examination of herpetic nodules has been reported to show thick pseudoepitheliomatous hyperplasia with dense dermal lymphoplasmacytic infiltrate and multifocal multinucleated cells with herpetic viral cytopathic changes. We report two patients with HIV presenting with vegetative tumor-like HSV nodules with distinctive histopathologic pattern of inflammation that has not been described in the literature before. All samples displayed slightly acanthotic epidermis with focal ulceration, dense dermal sclerosis, scattered plasma cells, and a brisk lymphoeosinophilic infiltrate found dissecting between dense collagen bundles. This pattern of inflammation is an important clue that can guide the pathologist to look for focal herpetic viral changes in the epidermis, as patients with HIV possibly tend to amount a predominantly eosinophilic immune response in inflammatory skin conditions.

Cell-to-Cell Spread Blocking Activity Is Extremely Limited in the Sera of Herpes Simplex Virus 1 (HSV-1)- and HSV-2-Infected Subjects.

Herpes simplex virus 1 (HSV-1) and HSV-2 can evade serum antibody-mediated neutralization through cell-to-cell transmission mechanisms, which represent one of the central steps in disease reactivation. To address the role of humoral immunity in controlling HSV-1 and HSV-2 replication, we analyzed serum samples from 44 HSV-1 and HSV-2 seropositive subjects by evaluating (i) their efficiency in binding both the purified viral particles and recombinant gD and gB viral glycoproteins, (ii) their neutralizing activity, and (iii) their capacity to inhibit the cell-to-cell virus passage in vitro All of the sera were capable of binding gD, gB, and whole virions, and all sera significantly neutralized cell-free virus. However, neither whole sera nor purified serum IgG fraction was able to inhibit significantly cell-to-cell virus spreading in in vitro post-virus-entry infectious assays. Conversely, when spiked with an already described anti-gD human monoclonal neutralizing antibody capable of inhibiting HSV-1 and -2 cell-to-cell transmission, each serum boosted both its neutralizing and post-virus-entry inhibitory activity, with no interference exerted by serum antibody subpopulations.IMPORTANCE Despite its importance in the physiopathology of HSV-1 and -2 infections, the cell-to-cell spreading mechanism is still poorly understood. The data shown here suggest that infection-elicited neutralizing antibodies capable of inhibiting cell-to-cell virus spread can be underrepresented in most infected subjects. These observations can be of great help in better understanding the role of humoral immunity in controlling virus reactivation and in the perspective of developing novel therapeutic strategies, studying novel correlates of protection, and designing effective vaccines.

Increased Frequency of Virus Shedding by Herpes Simplex Virus 2-Infected Guinea Pigs in the Absence of CD4+ T Lymphocytes.

Reactivation of herpes simplex virus 2 (HSV-2) results in infection of epithelial cells at the neuro-epithelial junction and shedding of virus at the epithelial surface. Virus shedding can occur in either the presence or absence of clinical disease and is usually of short duration, although the shedding frequency varies among individuals. The basis for host control of virus shedding is not well understood, although adaptive immune mechanisms are thought to play a central role. To determine the importance of CD4+ T cells in control of HSV-2 shedding, this subset of immune cells was depleted from HSV-2-infected guinea pigs by injection of an anti-CD4 monoclonal antibody (MAb). Guinea pigs were treated with the depleting MAb after establishment of a latent infection, and vaginal swabs were taken daily to monitor shedding by quantitative PCR. The cumulative number of HSV-2 shedding days and the mean number of days virus was shed were significantly increased in CD4-depleted compared to control-treated animals. However, there was no difference in the incidence of recurrent disease between the two treatment groups. Serum antibody levels and the number of HSV-specific antibody-secreting cells in secondary lymphoid tissues were unaffected by depletion of CD4+ T cells; however, the frequency of functional HSV-specific, CD8+ gamma interferon-secreting cells was significantly decreased. Together, these results demonstrate an important role for CD4+ T lymphocytes in control of virus shedding that may be mediated in part by maintenance of HSV-specific CD8+ T cell populations. These results have important implications for development of therapeutic vaccines designed to control HSV-2 shedding.IMPORTANCE Sexual transmission of HSV-2 results from viral shedding following reactivation from latency. The immune cell populations and mechanisms that control HSV-2 shedding are not well understood. This study examined the role of CD4+ T cells in control of virus shedding using a guinea pig model of genital HSV-2 infection that recapitulates the shedding of virus experienced by humans. We found that the frequency of virus-shedding episodes, but not the incidence of clinical disease, was increased by depletion of CD4+ T cells. The HSV-specific antibody response was not diminished, but frequency of functional HSV-reactive CD8+ T cells was significantly diminished by CD4 depletion. These results confirm the role of cell-mediated immunity and highlight the importance of CD4+ T cells in controlling HSV shedding, suggesting that therapeutic vaccines designed to reduce transmission by controlling HSV shedding should include specific enhancement of HSV-specific CD4+ T cell responses.

Herpes simplex viruses type 1 and 2 photoinactivated in the presence of methylene blue transform human and mouse cells in vitro.

Three strains of herpes simplex virus, K17syn- and HSZPsyn+ of type 1 (HSV-1) and USsyn- of type 2 (HSV-2), were photoinactivated in the presence of methylene blue and used to infect 3 cell lines, normal human lung tissue cells (MRC-5), mouse epithelial cells (NIH3T3), and human lung carcinoma cells (A549). The virus titer and phenotype of cells were evaluated to compare the characteristics of normal and carcinoma cells infected with non-syncytial (non-syn) and syncytial (syn) strains of herpes simplex viruses. We found that the cells of both normal cell lines infected with photoinactivated K17syn- and USsyn- but not HSZPsyn+ acquired transformed phenotype accompanied by the presence of virus. Surprisingly, the infection with photoinactivated viruses K17syn- and USsyn- but not HSZPsyn+ resulted in the suppression of the transformed phenotype of A549 cells. Using nested PCR, herpesviral DNA was identified in newly transformed cells and cells that lost the transformed phenotype. The effect of putative herpesvirus-related growth factors (HRGF) produced by cells infected with photoinactivated viruses was quantified and compared. Since methylene blue is currently used in phototherapy of herpetic lesions, these results raise the question of whether such therapy is risky to human health.

Comparison of the antiviral potential among soluble forms of herpes simplex virus type-2 glycoprotein D receptors, herpes virus entry mediator A, nectin-1 and nectin-2, in transgenic mice.

Herpesvirus entry mediator A (HVEM), nectin-1 and nectin-2 are cellular receptors of glycoprotein D (gD) of herpes simplex virus type-2 (HSV-2). It has been shown that soluble forms of HSV gD receptors have the antiviral potential in cultured cells and transgenic mice. Here, to compare antiviral potential of soluble forms of HVEM, nectin-1 and nectin-2 against HSV-2 infections in vivo, transgenic mice expressing fusion proteins consisting of the entire ectodomain of HVEM, nectin-1 or nectin-2 and the Fc portion of human IgG (HVEMIg, nectin-1Ig and nectin-2Ig, respectively) were intraperitoneally infected with HSV-2. In the infection with 3 MLD50 (50 % mouse lethal dose), effective resistance was not observed in transgenic mice expressing nectin-2Ig. In a transgenic mouse line with high expression of nectin-1Ig, significant protection from the infection with 30 and 300 MLD50 was observed (survival rate of 100 and 71 %, respectively). On the other hand, transgenic mice expressing HVEMIg showed a complete resistance to the lethal infection even with 300 MLD50 (survival rate of 100 %). These results demonstrated that HVEMIg could exert effective antiviral activities against HSV-2 infections in vivo as compared with other soluble forms of HSV gD receptors.

Herpes simplex virus particles interact with chemokines and enhance cell migration.

Herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2, respectively) are among the most prevalent human pathogens, causing a variety of diseases. HSV modulation of the chemokine network remains poorly understood. We have previously identified secreted glycoprotein G (SgG) as the first viral chemokine-binding protein that enhances chemokine function as a novel viral immunomodulatory mechanism. However, gG is also present at the viral envelope and its role in the virus particle remains unknown. Here we have addressed the chemokine-binding capacity of HSV particles and the functionality of such interaction in vitro. We adapted surface plasmon resonance assays and demonstrated the ability of HSV particles to bind a specific set of human chemokines with high affinity. Moreover, we identified gG as the envelope glycoprotein mediating such interaction, as shown by the lack of binding to a HSV-1 gG mutant. In contrast to HSV-1, HSV-2 gG is cleaved and the chemokine-binding domain is secreted (SgG2). However, we found that HSV-2 particles retain the ability to bind chemokines, potentially through SgG2 associated to the viral envelope or non-processed precursor protein. Moreover, we found that HSV particles increase cell migration independently of chemokine binding to envelope gG. This work provides insights into HSV manipulation of the host immune system.

B Virus (Macacine Herpesvirus 1) Divergence: Variations in Glycoprotein D from Clinical and Laboratory Isolates Diversify Virus Entry Strategies.

UNLABELLED: B virus (Macacine herpesvirus 1) can cause deadly zoonotic disease in humans. Molecular mechanisms of B virus cell entry are poorly understood for both macaques and humans. Here we investigated the abilities of clinical B virus isolates to use entry receptors of herpes simplex viruses (HSV). We showed that resistant B78H1 cells became susceptible to B virus clinical strains upon expression of either human nectin-2 or nectin-1. Antibody against glycoprotein D (gD) protected these nectin-bearing cells from B virus infection, and a gD-negative recombinant B virus failed to enter these cells, indicating that the nectin-mediated B virus entry depends on gD. We observed that the infectivity of B virus isolates with a single amino acid substitution (D122N) in the IgV-core of the gD ectodomain was impaired on nectin-1-bearing cells. Computational homology-based modeling of the B virus gD-nectin-1 complex revealed conformational differences between the structures of the gD-122N and gD-122D variants that affected the gD-nectin-1 protein-protein interface and binding affinity. Unlike HSV, B virus clinical strains were unable to use herpesvirus entry mediator (HVEM) as a receptor, regardless of conservation of the gD amino acid residues essential for HSV-1 entry via HVEM. Based on the model of the B virus gD-HVEM interface, we predict that residues R7, R11, and G15 are largely responsible for the inability of B virus to utilize HVEM for entry. The ability of B virus to enter cells of a human host by using a combination of receptors distinct from those for HSV-1 or HSV-2 suggests a possible mechanism of enhanced neuropathogenicity associated with zoonotic infections. IMPORTANCE: B virus causes brainstem destruction in infected humans in the absence of timely diagnosis and intervention. Nectins are cell adhesion molecules that are widely expressed in human tissues, including neurons and neuronal synapses. Here we report that human nectin-2 is a target receptor for B virus entry, in addition to the reported receptor human nectin-1. Similar to a B virus lab strain, B virus clinical strains can effectively use both nectin-1 and nectin-2 as cellular receptors for entry into human cells, but unlike HSV-1 and HSV-2, none of the clinical strains uses an HVEM-mediated entry pathway. Ultimately, these differences between B virus and HSV-1 and -2 may provide insight into the neuropathogenicity of B virus during zoonotic infections.

[Efficacy of polyprenyl phosphates in the experimental genital herpes model].

An experimental model of the primary genital herpes (herpes simplex type 2, HSV-2) in the female guinea pigs was suggested to study the infectious process activity of polyprenyl phosphates (PPP) and PPP+acyclovir (AC) complex treatment. The morphofunctional features of the guinea pig ovaries were studied in the control and experimental groups (the latter were inoculated with PPP and/or AC as a primary infection treatment) at the stage of the recurrent genital herpes aggravation. It was shown that in the case of combined PPP +AC use significant changes in the disease symptoms were observed, as well as a decrease in the infectious process activity and duration, and positive remote effect on the ovarian morphophysiology.

A spiroketal-enol ether derivative from Tanacetum vulgare selectively inhibits HSV-1 and HSV-2 glycoprotein accumulation in Vero cells.

The inhibitory effects of Tanacetum vulgare rhizome extracts on HSV-1 and HSV-2 in vitro replication were assessed. Unlike extracts obtained from the aerial parts, adsorption inhibition and virucidal activities seemed not to be relevant for the observed antiviral action of tansy rhizome extracts. Instead, the most significant effects were the inhibition of virus penetration and a novel mechanism consisting of the specific arrest of viral gene expression and consequently the decrease of viral protein accumulation within infected cells. Through a bioactivity-guided fractionation protocol we isolated and identified the spiroketal-enol ether derivative (E)-2-(2,4-hexadiynyliden)-1,6-dioxaspiro[4.5]dec-3-ene as the active compound responsible for this inhibitory effect.

Houttuynia cordata targets the beginning stage of herpes simplex virus infection.

Herpes simplex virus (HSV), a common latent virus in humans, causes certain severe diseases. Extensive use of acyclovir (ACV) results in the development of drug-resistant HSV strains, hence, there is an urgent need to develop new drugs to treat HSV infection. Houttuynia cordata (H. cordata), a natural herbal medicine, has been reported to exhibit anti-HSV effects which is partly NF-κB-dependent. However, the molecular mechanisms by which H. cordata inhibits HSV infection are not elucidated thoroughly. Here, we report that H. cordata water extracts (HCWEs) inhibit the infection of HSV-1, HSV-2, and acyclovir-resistant HSV-1 mainly via blocking viral binding and penetration in the beginning of infection. HCWEs also suppress HSV replication. Furthermore, HCWEs attenuate the first-wave of NF-κB activation, which is essential for viral gene expressions. Further analysis of six compounds in HCWEs revealed that quercetin and isoquercitrin inhibit NF-κB activation and additionally, quercetin also has an inhibitory effect on viral entry. These results indicate that HCWEs can inhibit HSV infection through multiple mechanisms and could be a potential lead for development of new drugs for treating HSV.

Herpes simplex virus 2 (HSV-2) infected cell proteins are among the most dominant antigens of a live-attenuated HSV-2 vaccine.

Virion glycoproteins such as glycoprotein D (gD) are believed to be the dominant antigens of herpes simplex virus 2 (HSV-2). We have observed that mice immunized with a live HSV-2 ICP0- mutant virus, HSV-2 0ΔNLS, are 10 to 100 times better protected against genital herpes than mice immunized with a HSV-2 gD subunit vaccine (PLoS ONE 6:e17748). In light of these results, we sought to determine which viral proteins were the dominant antibody-generators (antigens) of the live HSV-2 0ΔNLS vaccine. Western blot analyses indicated the live HSV-2 0ΔNLS vaccine elicited an IgG antibody response against 9 or more viral proteins. Many antibodies were directed against infected-cell proteins of >100 kDa in size, and only 10 ± 5% of antibodies were directed against gD. Immunoprecipitation (IP) of total HSV-2 antigen with 0ΔNLS antiserum pulled down 19 viral proteins. Mass spectrometry suggested 44% of immunoprecipitated viral peptides were derived from two HSV-2 infected cells proteins, RR-1 and ICP8, whereas only 14% of immunoprecipitated peptides were derived from HSV-2's thirteen glycoproteins. Collectively, the results suggest the immune response to the live HSV-2 0ΔNLS vaccine includes antibodies specific for infected cell proteins, capsid proteins, tegument proteins, and glycoproteins. This increased breadth of antibody-generating proteins may contribute to the live HSV-2 vaccine's capacity to elicit superior protection against genital herpes relative to a gD subunit vaccine.

Enhancing the bystander killing effect of an oncolytic HSV by arming it with a secretable apoptosis activator.

Although oncolytic viruses have shown great promise as cancer therapeutics, results from a recent phase III clinical trial indicate that their potency may need further improvement for a clear clinical benefit. Here, we report a novel strategy to increase the bystander effect of virotherapy by arming an oncolytic virus with a secreted form of a Her2 single chain antibody linked to a self-multimerizing Fas ligand extracellular domain (Her2-COL-sFasL). The rationale is that, due to its much smaller size, this apoptosis activator can overcome obstacles such as the dense collagen in the tumor tissues to spread more freely than the viral particles. When measured in vitro, Her2-COL-sFasL was found to efficiently induce caspase cleavage, resulting in an 80% reduction in cell viability. Once incorporated into the genome of an oncolytic type 2 herpes simplex virus, FusOn-H3, Her2-COL-sFasL potentiates the therapeutic efficacy of the virus in an aggressive syngeneic mammary tumor model. Our data suggest that arming an oncolytic virus with a secretable and self-multimerizing apoptosis inducer is a feasible strategy to improve the potency of virotherapy.

Secreted herpes simplex virus-2 glycoprotein G modifies NGF-TrkA signaling to attract free nerve endings to the site of infection.

Herpes simplex virus type 1 (HSV-1) and HSV-2 are highly prevalent viruses that cause a variety of diseases, from cold sores to encephalitis. Both viruses establish latency in peripheral neurons but the molecular mechanisms facilitating the infection of neurons are not fully understood. Using surface plasmon resonance and crosslinking assays, we show that glycoprotein G (gG) from HSV-2, known to modulate immune mediators (chemokines), also interacts with neurotrophic factors, with high affinity. In our experimental model, HSV-2 secreted gG (SgG2) increases nerve growth factor (NGF)-dependent axonal growth of sympathetic neurons ex vivo, and modifies tropomyosin related kinase (Trk)A-mediated signaling. SgG2 alters TrkA recruitment to lipid rafts and decreases TrkA internalization. We could show, with microfluidic devices, that SgG2 reduced NGF-induced TrkA retrograde transport. In vivo, both HSV-2 infection and SgG2 expression in mouse hindpaw epidermis enhance axonal growth modifying the termination zone of the NGF-dependent peptidergic free nerve endings. This constitutes, to our knowledge, the discovery of the first viral protein that modulates neurotrophins, an activity that may facilitate HSV-2 infection of neurons. This dual function of the chemokine-binding protein SgG2 uncovers a novel strategy developed by HSV-2 to modulate factors from both the immune and nervous systems.

Fungal metabolite myriocin promotes human herpes simplex virus-2 infection.

AIMS: Myriocin is a fungal metabolite with antiviral activity, including influenza, hepatitis B, and hepatitis C viruses. We investigated whether myriocin has activity against human HSV-2, one of the most prevalent pathogens of sexually transmitted disease. MAIN METHODS: Cell culture systems were used to evaluate myriocin effect on HSV-2 infection. Plaque forming assay and immunoblotting studies were used to determine virus production and viral protein expression, respectively. KEY FINDINGS: Myriocin showed no cytotoxic effect at up to 5 µM. Myriocin treatment did not inhibit HSV-2 infection. Instead, the treatment resulted in accelerated replication of HSV-2 and increased titers of infectious virion. The effect was detected at concentrations as low as 3 nM and plateaued at approximately 30 nM. Myriocin at 30 nM increased HSV-2 production by approximately 1.7 logs. Myriocin also promoted HSV-1 infection but required higher concentrations. A time course study revealed that myriocin promoted HSV-2 infection by acceleration of virus replication. Unlike trichostatin A that promotes HSV-2 infection and histone modifications, myriocin treatment did not alter histone modifications. Myriocin is a well characterized inhibitor of sphingolipid biosynthesis pathway. Structurally different inhibitors of the pathway showed no effect on HSV-2 infection. Exogenous sphingolipids did not reverse the effect of myriocin on HSV-2 infection either. SIGNIFICANCE: We found that myriocin promotes HSV-2 replication at nanomolar concentrations with yet unknown mechanisms. Further studies may uncover novel mechanisms regulating HSV replication and targets of myriocin action. This may have potential application in enhancing efficacy of oncolytic HSV for cancer therapy and other diseases.