NK cell-derived extracellular granzyme B drives epithelial ulceration during HSV-2 genital infection.

Genital herpes is characterized by recurrent episodes of epithelial blistering. The mechanisms causing this pathology are ill defined. Using a mouse model of vaginal herpes simplex virus 2 (HSV-2) infection, we show that interleukin-18 (IL-18) acts upon natural killer (NK) cells to promote accumulation of the serine protease granzyme B in the vagina, coinciding with vaginal epithelial ulceration. Genetic loss of granzyme B or therapeutic inhibition by a specific protease inhibitor reduces disease and restores epithelial integrity without altering viral control. Distinct effects of granzyme B and perforin deficiency on pathology indicates that granzyme B acts independent of its classic cytotoxic role. IL-18 and granzyme B are markedly elevated in human herpetic ulcers compared with non-herpetic ulcers, suggesting engagement of these pathways in HSV-infected patients. Our study reveals a role for granzyme B in destructing mucosal epithelium during HSV-2 infection, identifying a therapeutic target to augment treatment of genital herpes.

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.

Synthetic Mucin Gels with Self-Healing Properties Augment Lubricity and Inhibit HIV-1 and HSV-2 Transmission.

Mucus is a self-healing gel that lubricates the moist epithelium and provides protection against viruses by binding to viruses smaller than the gel's mesh size and removing them from the mucosal surface by active mucus turnover. As the primary nonaqueous components of mucus (≈0.2%-5%, wt/v), mucins are critical to this function because the dense arrangement of mucin glycans allows multivalence of binding. Following nature's example, bovine submaxillary mucins (BSMs) are assembled into "mucus-like" gels (5%, wt/v) by dynamic covalent crosslinking reactions. The gels exhibit transient liquefaction under high shear strain and immediate self-healing behavior. This study shows that these material properties are essential to provide lubricity. The gels efficiently reduce human immunodeficiency virus type 1 (HIV-1) and genital herpes virus type 2 (HSV-2) infectivity for various types of cells. In contrast, simple mucin solutions, which lack the structural makeup, inhibit HIV-1 significantly less and do not inhibit HSV-2. Mechanistically, the prophylaxis of HIV-1 infection by BSM gels is found to be that the gels trap HIV-1 by binding to the envelope glycoprotein gp120 and suppress cytokine production during viral exposure. Therefore, the authors believe the gels are promising for further development as personal lubricants that can limit viral transmission.

Studies on the antiviral activity of chebulinic acid against dengue and chikungunya viruses and in silico investigation of its mechanism of inhibition.

Chebulinic acid (CA), originally isolated from the flower extract of the plant Terminalia chebula, has been shown to inhibit infection of herpes simplex virus-2 (HSV-2), suggestively by inhibiting the host entry step of viral infection. Like HSV-2, the dengue virus (DENV) and chikungunya virus (CHIKV) also use receptor glycosaminoglycans (GAG) to gain host entry, therefore, the activity of CA was tested against these viruses. Co-treatment of 8 µM CA with DENV-2 caused 2 log decrease in the virus titer (4.0 log10FFU/mL) at 120 h post infection, compared to virus control (5.95 log10FFU/mL). In contrast, no inhibitory effect of CA was observed against CHIKV infection under any condition. The mechanism of action of CA was investigated in silico by employing DENV-2 and CHIKV envelope glycoproteins. During docking, CA demonstrated equivalent binding at multiple sites on DENV-2 envelope protein, including GAG binding site, which have previously been reported to play a crucial role in host attachment and fusion, indicating blocking of these sites. However, CA did not show binding to the GAG binding site on envelope protein-2 of CHIKV. The in vitro and in silico findings suggest that CA possesses the ability to inhibit DENV-2 infection at the entry stage of its infection cycle and may be developed as a potential therapeutic agent against it.

Herpesvirus DNA polymerase: Structures, functions, and mechanisms.

Herpesviruses comprise a family of DNA viruses that cause a variety of human and veterinary diseases. During productive infection, mammalian, avian, and reptilian herpesviruses replicate their genomes using a set of conserved viral proteins that include a two subunit DNA polymerase. This enzyme is both a model system for family B DNA polymerases and a target for inhibition by antiviral drugs. This chapter reviews the structure, function, and mechanisms of the polymerase of herpes simplex viruses 1 and 2 (HSV), with only occasional mention of polymerases of other herpesviruses such as human cytomegalovirus (HCMV). Antiviral polymerase inhibitors have had the most success against HSV and HCMV. Detailed structural information regarding HSV DNA polymerase is available, as is much functional information regarding the activities of the catalytic subunit (Pol), which include a DNA polymerization activity that can utilize both DNA and RNA primers, a 3'-5' exonuclease activity, and other activities in DNA synthesis and repair and in pathogenesis, including some remaining to be biochemically defined. Similarly, much is known regarding the accessory subunit, which both resembles and differs from sliding clamp processivity factors such as PCNA, and the interactions of this subunit with Pol and DNA. Both subunits contribute to replication fidelity (or lack thereof). The availability of both pharmacologic and genetic tools not only enabled the initial identification of Pol and the pol gene, but has also helped dissect their functions. Nevertheless, important questions remain for this long-studied enzyme, which is still an attractive target for new drug discovery.

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.

Phenotypic and Genotypic Testing of HSV-1 and HSV-2 Resistance to Antivirals.

Resistance testing of antivirals to herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) can be done by phenotypic and genotypic methods. The determination of a resistant phenotype is based on the calculation of inhibitory concentrations for the antiviral drug, which should be tested. The main advantage of this resistance test is a clear interpretation of laboratory findings, but the method is time-consuming and a considerable experience is required by handling infectious virus. Genotypic resistance testing is based on the detection of resistance-related mutations in viral genes encoding the thymidine kinase and DNA polymerase, which need to be amplified and sequenced. This approach has the advantage of being faster, but only frameshift mutations, stops of translation, and amino acid substitutions described in the literature can be interpreted without doubt. By contrast, numerous novel amino acid substitutions are diagnostically less conclusive.

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.

Herpes Simplex Virus 2 in Autonomic Ganglia: Evidence for Spontaneous Reactivation.

Herpes simplex virus 2 (HSV-2) can be transmitted in the presence or absence of lesions, allowing efficient spread among the general population. Recurrent HSV genital lesions are thought to arise from reactivated latent virus in sensory cell bodies of the dorsal root ganglia (DRG). However, HSV-2 has also been found latent in autonomic ganglia. Spontaneous reactivation or a low level of chronic infection could theoretically also occur in these peripheral nervous tissues, contributing to the presence of infectious virus in the periphery and to viral transmission. Use of a recently described, optimized virus with a monomeric mNeonGreen protein fused to viral capsid protein 26 (VP26) permitted detection of reactivating virus in explanted ganglia and cryosections of DRG and the sacral sympathetic ganglia (SSG) from latently infected guinea pigs. Immediate early, early, and late gene expression were quantified by droplet digital reverse transcription-PCR (ddRT-PCR), providing further evidence of viral reactivation not only in the expected DRG but also in the sympathetic SSG. These findings indicate that viral reactivation from autonomic ganglia is a feature of latent viral infection and that these reactivations likely contribute to viral pathogenesis.IMPORTANCE HSV-2 is a ubiquitous important human pathogen that causes recurrent infections for the life of its host. We hypothesized that the autonomic ganglia have important roles in viral reactivation, and this study sought to determine whether this is correct in the clinically relevant guinea pig vaginal infection model. Our findings indicate that sympathetic ganglia are sources of reactivating virus, helping explain how the virus causes lifelong recurrent disease.

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.

Histidine-Rich Glycoprotein Inhibits HIV-1 Infection in a pH-Dependent Manner.

Histidine-rich glycoprotein (HRG) is an abundant plasma protein with a multidomain structure, allowing its interaction with many ligands, including phospholipids, plasminogen, fibrinogen, IgG antibodies, and heparan sulfate. HRG has been shown to regulate different biological responses, such as angiogenesis, coagulation, and fibrinolysis. Here, we found that HRG almost completely abrogated the infection of Ghost cells, Jurkat cells, CD4+ T cells, and macrophages by HIV-1 at a low pH (range, 6.5 to 5.5) but not at a neutral pH. HRG was shown to interact with the heparan sulfate expressed by target cells, inhibiting an early postbinding step associated with HIV-1 infection. More importantly, by acting on the viral particle itself, HRG induced a deleterious effect, which reduces viral infectivity. Because cervicovaginal secretions in healthy women show low pH values, even after semen deposition, our observations suggest that HRG might represent a constitutive defense mechanism in the vaginal mucosa. Of note, low pH also enabled HRG to inhibit the infection of HEp-2 cells and Vero cells by respiratory syncytial virus (RSV) and herpes simplex virus 2 (HSV-2), respectively, suggesting that HRG might display broad antiviral activity under acidic conditions.IMPORTANCE Vaginal intercourse represents a high-risk route for HIV-1 transmission. The efficiency of male-to-female HIV-1 transmission has been estimated to be 1 in every 1,000 episodes of sexual intercourse, reflecting the high degree of protection conferred by the genital mucosa. However, the contribution of different host factors to the protection against HIV-1 at mucosal surfaces remains poorly defined. Here, we report for the first time that acidic values of pH enable the plasma protein histidine-rich glycoprotein (HRG) to strongly inhibit HIV-1 infection. Because cervicovaginal secretions usually show low pH values, our observations suggest that HRG might represent a constitutive antiviral mechanism in the vaginal mucosa. Interestingly, infection by other viruses, such as respiratory syncytial virus and herpes simplex virus 2, was also markedly inhibited by HRG at low pH values, suggesting that extracellular acidosis enables HRG to display broad antiviral activity.

A merged molecular docking, ADME-T and dynamics approaches towards the genus of Arisaema as herpes simplex virus type 1 and type 2 inhibitors.

An attempt toward screening of phytoconstituents (Arisaema genus) against herpes viruses (HSV-1 and HSV-2) was carried out using in silico approaches. Human HSV-1 and HSV-2 are accountable for cold sores genital herpes, respectively. Two drug targets, namely thymidine kinase (TK; PDB: 2ki5) serine protease (PDB: 1at3) were selected for HSV-1 and HSV-2. Initially, molecular docking tool was employed to screened apex hits phytoconstituents against herpes infections. ADME-T studies of top ranked were also further highlighted to achieve their effectiveness. Following, molecular dynamics studies were also examined to further optimize the stability of ligands. Glide scores and binding interactions of phytoconstituents were compared with Acyclovir, the main drug used in treatment of HSV, the screened top hits exhibited more glide scores and better binding for both HSV-1 and HSV-2 receptors. Additionally, ADME-T showed an ideal range for top hits while molecular dynamics results also illustrated stability of models. Ultimately, the whole efforts reveal to top three most promising hits for HSV-1 (39, 21, 19) and HSV-2 (20, 51, 19) receptors which can be explored further in wet lab experiments as promising agents against HSV infections.

Nitro-fatty acids are formed in response to virus infection and are potent inhibitors of STING palmitoylation and signaling.

The adaptor molecule stimulator of IFN genes (STING) is central to production of type I IFNs in response to infection with DNA viruses and to presence of host DNA in the cytosol. Excessive release of type I IFNs through STING-dependent mechanisms has emerged as a central driver of several interferonopathies, including systemic lupus erythematosus (SLE), Aicardi-Goutières syndrome (AGS), and stimulator of IFN genes-associated vasculopathy with onset in infancy (SAVI). The involvement of STING in these diseases points to an unmet need for the development of agents that inhibit STING signaling. Here, we report that endogenously formed nitro-fatty acids can covalently modify STING by nitro-alkylation. These nitro-alkylations inhibit STING palmitoylation, STING signaling, and subsequently, the release of type I IFN in both human and murine cells. Furthermore, treatment with nitro-fatty acids was sufficient to inhibit production of type I IFN in fibroblasts derived from SAVI patients with a gain-of-function mutation in STING. In conclusion, we have identified nitro-fatty acids as endogenously formed inhibitors of STING signaling and propose for these lipids to be considered in the treatment of STING-dependent inflammatory diseases.

A bimodal switch in global protein translation coupled to eIF4H relocalisation during advancing cell-cell transmission of herpes simplex virus.

We used the bioorthogonal protein precursor, homopropargylglycine (HPG) and chemical ligation to fluorescent capture agents, to define spatiotemporal regulation of global translation during herpes simplex virus (HSV) cell-to-cell spread at single cell resolution. Translational activity was spatially stratified during advancing infection, with distal uninfected cells showing normal levels of translation, surrounding zones at the earliest stages of infection with profound global shutoff. These cells further surround previously infected cells with restored translation close to levels in uninfected cells, reflecting a very early biphasic switch in translational control. While this process was dependent on the virion host shutoff (vhs) function, in certain cell types we also observed temporally altered efficiency of shutoff whereby during early transmission, naïve cells initially exhibited resistance to shutoff but as infection advanced, naïve target cells succumbed to more extensive translational suppression. This may reflect spatiotemporal variation in the balance of oscillating suppression-recovery phases. Our results also strongly indicate that a single particle of HSV-2, can promote pronounced global shutoff. We also demonstrate that the vhs interacting factor, eIF4H, an RNA helicase accessory factor, switches from cytoplasmic to nuclear localisation precisely correlating with the initial shutdown of translation. However translational recovery occurs despite sustained eIF4H nuclear accumulation, indicating a qualitative change in the translational apparatus before and after suppression. Modelling simulations of high multiplicity infection reveal limitations in assessing translational activity due to sampling frequency in population studies and how analysis at the single cell level overcomes such limitations. The work reveals new insight and a revised model of translational manipulation during advancing infection which has important implications both mechanistically and with regards to the physiological role of translational control during virus propagation. The work also demonstrates the potential of bioorthogonal chemistry for single cell analysis of cellular metabolic processes during advancing infections in other virus systems.

Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4+ T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4.

HSV-2 infection-induced CXCR3 ligands are important for the recruitment of virus-specific CD8+ T cells, but their impact on CD4+ T cell trafficking remains to be further determined. Given that recruitment of CD4+ T cells to infection areas may be one of the mechanisms that account for HSV-2 infection-mediated enhancement of HIV-1 sexual transmission, here we investigated the functionality of HSV-2 infection-induced CXCR3 ligands CXCL9, CXCL10, and CXCL11 in vivo and in vitro, and determined the viral components responsive for such induction and the underlying mechanisms. We first found that the expression of CXCR3 ligands CXCL9, CXCL10, and CXCL11 was increased in mice following vaginal challenge with HSV-2, while CXCL9 played a predominant role in the recruitment of CD4+ T cells to the vaginal foci of infected mice. HSV-2 infection also induced the production of CXCL9, CXCL10, and CXCL11 in human cervical epithelial cells. Of note, although HSV-2 induced the expression of all the three CXCR3 ligands, the induced CXCL9 appeared to play a predominant role in promoting CD4+ T cell migration, reflecting that the concentrations of CXCL10 and CXCL11 required for CD4+ T cell migration are higher than that of CXCL9. We further revealed that, ICP4, an immediate-early protein of HSV-2, is crucial in promoting CXCR3 ligand expression through the activation of p38 MAPK pathway. Mechanistically, ICP4 binds to corresponding promoters of CXCR3 ligands via interacting with the TATA binding protein (TBP), resulting in the transcriptional activation of the corresponding promoters. Taken together, our study highlights HSV-2 ICP4 as a vital viral protein in promoting CXCR3 ligand expression and CXCL9 as the key induced chemokine in mediating CD4+ T cell migration. Findings in this study have shed light on HSV-2 induced leukocyte recruitment which may be important for understanding HSV-2 infection-enhanced HIV-1 sexual transmission and the development of intervention strategies.