Hsp90 Inhibitors Inhibit the Entry of Herpes Simplex Virus 1 Into Neuron Cells by Regulating Cofilin-Mediated F-Actin Reorganization

Herpes simplex virus 1 (HSV-1) is a common neurotropic virus, the herpes simplex encephalitis (HSE) caused by which is considered to be the most common sporadic but fatal encephalitis. Traditional antiviral drugs against HSV-1 are limited to nucleoside analogs targeting viral factors. Inhibition of heat shock protein 90 (Hsp90) has potent anti-HSV-1 activities via numerous mechanisms, but the effects of Hsp90 inhibitors on HSV-1 infection in neuronal cells, especially in the phase of virus entry, are still unknown. In this study, we aimed to investigate the effects of the Hsp90 inhibitors on HSV-1 infection of neuronal cells. Interestingly, we found that Hsp90 inhibitors promoted viral adsorption but inhibited subsequent penetration in neuronal cell lines and primary neurons, which jointly confers the antiviral activity of the Hsp90 inhibitors. Mechanically, Hsp90 inhibitors mainly impaired the interaction between Hsp90 and cofilin, resulting in reduced cofilin membrane distribution, which led to F-actin polymerization to promote viral attachment. However, excessive polymerization of F-actin inhibited subsequent viral penetration. Consequently, unidirectional F-actin polymerization limits the entry of HSV-1 virions into neuron cells. Our research extended the molecular mechanism of Hsp90 in HSV-1 infection in neuron cells and provided a theoretical basis for developing antiviral drugs targeting Hsp90.

A novel H129-based anterograde monosynaptic tracer exhibits features of strong labeling intensity, high tracing efficiency, and reduced retrograde labeling

Background Viral tracers are important tools for mapping brain connectomes. The feature of predominant anterograde transneuronal transmission offers herpes simplex virus-1 (HSV-1) strain H129 (HSV1-H129) as a promising candidate to be developed as anterograde viral tracers. In our earlier studies, we developed H129-derived anterograde polysynaptic tracers and TK deficient (H129-dTK) monosynaptic tracers. However, their broad application is limited by some intrinsic drawbacks of the H129-dTK tracers, such as low labeling intensity due to TK deficiency and potential retrograde labeling caused by axon terminal invasion. The glycoprotein K (gK) of HSV-1 plays important roles in virus entry, egress, and virus-induced cell fusion. Its deficiency severely disables virus egress and spread, while only slightly limits viral genome replication and expression of viral proteins. Therefore, we created a novel H129-derived anterograde monosynaptic tracer (H129-dgK) by targeting gK, which overcomes the limitations of H129-dTK. Methods Using our established platform and pipeline for developing viral tracers, we generated a novel tracer by deleting the gK gene from the H129-G4. The gK-deleted virus (H129-dgK-G4) was reconstituted and propagated in the Vero cell expressing wildtype H129 gK (gKwt) or the mutant gK (gKmut, A40V, C82S, M223I, L224V, V309M), respectively. Then the obtained viral tracers of gKmut pseudotyped and gKwt coated H129-dgK-G4 were tested in vitro and in vivo to characterize their tracing properties. Results H129-dgK-G4 expresses high levels of fluorescent proteins, eliminating the requirement of immunostaining for imaging detection. Compared to the TK deficient monosynaptic tracer H129-dTK-G4, H129-dgK-G4 labeled neurons with 1.76-fold stronger fluorescence intensity, and visualized 2.00-fold more postsynaptic neurons in the downstream brain regions. gKmut pseudotyping leads to a 77% decrease in retrograde labeling by reducing axon terminal invasion, and thus dramatically improves the anterograde-specific tracing of H129-dgK-G4. In addition, assisted by the AAV helper trans-complementarily expressing gKwt, H129-dgK-G4 allows for mapping monosynaptic connections and quantifying the circuit connectivity difference in the Alzheimer’s disease and control mouse brains. Conclusions gKmut pseudotyped H129-dgK-G4, a novel anterograde monosynaptic tracer, overcomes the limitations of H129-dTK tracers, and demonstrates desirable features of strong labeling intensity, high tracing efficiency, and improved anterograde specificity.

Antiviral CD19 + CD27 + Memory B Cells Are Associated with Protection from Recurrent Asymptomatic Ocular Herpes Infection

Reactivation of herpes simplex virus 1 (HSV-1) from latently infected neurons of the trigeminal ganglia (TG) leads to blinding recurrent herpetic disease in symptomatic (SYMP) individuals. Although the role of T cells in herpes immunity seen in asymptomatic (ASYMP) individuals is heavily explored, the role of B cells is less investigated. In the present study, we evaluated whether B cells are associated with protective immunity against recurrent ocular herpes. The frequencies of circulating HSV-specific memory B cells and of memory follicular helper T cells (CD4 + T fh cells), that help B cells produce antibodies, were compared between HSV-1 infected SYMP and ASYMP individuals. The levels of IgG/IgA and neutralizing antibodies were compared in SYMP and ASYMP individuals. We found that: ( i ) the ASYMP individuals had increased frequencies of HSV-specific CD19 + CD27 + memory B cells; and ( ii ) high frequencies of HSV-specific switched IgG + CD19 + CD27 + memory B cells detected in ASYMP individuals were directly proportional to high frequencies of CD45R0 + CXCR5 + CD4 + memory T fh cells. However, no differences were detected in the level of HSV-specific IgG/IgA antibodies in SYMP and ASYMP individuals. Using the UV-B-induced HSV-1 reactivation mouse model, we found increased frequencies of HSV-specific antibody-secreting plasma HSV-1 gD + CD138 + B cells within the TG and circulation of ASYMP mice compared to SYMP mice. In contrast, no significant differences in the frequencies of B cells were found in the cornea, spleen, and bone-marrow. Our findings suggest that circulating antibody-producing HSV-specific memory B cells recruited locally to the TG may contribute to protection from symptomatic recurrent ocular herpes. IMPORTANCE Reactivation of herpes simplex virus 1 (HSV-1) from latently infected neurons of the trigeminal ganglia (TG) leads to blinding recurrent herpetic disease in symptomatic (SYMP) individuals. Although the role of T cells in herpes immunity against blinding recurrent herpetic disease is heavily explored, the role of B cells is less investigated. In the present study, we found that in both asymptomatic (ASYMP) individuals and ASYMP mice there was increased frequencies of HSV-specific memory B cells that were directly proportional to high frequencies of memory T fh cells. Moreover, following UV-B induce reactivation, we found increased frequencies of HSV-specific antibody-secreting plasma B cells within the TG and circulation of ASYMP mice, compared to SYMP mice. Our findings suggest that circulating antibody-producing HSV-specific memory B cells recruited locally to the TG may contribute to protection from recurrent ocular herpes.

Herpes simplex virus 1 expressing GFP-tagged virion host shutoff (vhs) protein uncouples the activities of degradation and nuclear retention of the infected cell transcriptome

Virion host shutoff (vhs) protein is an endoribonuclease encoded by herpes simplex virus 1 (HSV1). Vhs causes a number of changes to the infected cell environment that favour translation of late (L) virus proteins: cellular mRNAs are degraded, immediate-early (IE) and early (E) viral transcripts are sequestered in the nucleus with polyA binding protein (PABPC1), and dsRNA is degraded to help dampen the PKR-dependent stress response. To further our understanding of the cell biology of vhs, we constructed a virus expressing vhs tagged at its C-terminus with GFP. When first expressed, vhs-GFP localised to juxtanuclear clusters, and later it colocalised and interacted with its binding partner VP16, and was packaged into virions. Despite vhs-GFP maintaining activity when expressed in isolation, it failed to degrade mRNA or relocalise PABPC1 during infection, while viral transcript levels were similar to those seen for a vhs knockout virus. PKR phosphorylation was also enhanced in vhs-GFP infected cells, in line with a failure to degrade dsRNA. Nonetheless, mRNA FISH revealed that as in Wt but not Dvhs infection, IE and E, but not L transcripts were retained in the nucleus of vhs-GFP infected cells at late times. Moreover, a representative cellular transcript which is ordinarily highly susceptible to vhs degradation, was also retained in the nucleus. These results reveal that the vhs-induced nuclear retention of the infected cell transcriptome is dependent on vhs expression but not on its endoribonuclease activity, uncoupling these two functions of vhs.

Blue Laser Light Counteracts HSV-1 in the SH-SY5Y Neuronal Cell Model of Infection

Herpes simplex virus 1 (HSV-1) is wide-spread virus that triggers painful and recurrent infections, as herpes labialis, causing blister lesions on the lip. HSV-1 infection can be a lifelong condition starting from childhood due to the latency of the virus hidden in the trigeminal ganglia. Despite the use of antiviral treatments, there is not a resolutive cure for herpes. In our study, we tested blue light against HSV-1 in a neuronal cellular model, aimed at mimicking the neuronal tropism of HSV-1. Two laser protocols employing continuous wave and pulse modalities were delivered to infected cell cultures and to the virus alone. A significant reduction of viral replication was observed when the beam was directly applied to the virus, along with an increase in cell survival. Our findings, considering the limitation of the still-unknown mechanisms by which the blue light acts on the virus, suggested a potential use of photobiomodulation therapy for clinical applications against herpes labialis in pediatric patients.

Efficacy and Tolerance of Thymus Vulgaris Extract in Patients with Covid-19

The coronavirus disease 19 (COVID-19) has been a pandemic since February 2020. So far, no effective treatment has been found. WHO has recommended research on medicinal plants as an alternative treatment course. Several studies conducted on Thymus vulgaris have established its antioxidant, antiviral and immunomodulatory properties that induce the elimination of viruses such as Herpes simplex Virus 1 and 2. Following this, we initiated a study entitled Efficacy and tolerance of Thymus vulgaris extract in patients with coronavirus 2019. Material and method: Thymus vulgaris powder was used in this study. A consent letter and a questionnaire about the patients’ symptoms were prepared to be used by a research investigator. According to the statistical calculations of this cohort study, 161 patients testing positive for COVID-19 PCR were consecutively recruited, of which 75 patients were not exposed to Thymus vulgaris and 86 patients were exposed. Information from the questionnaire was gathered from the patients before the initiation of conventional treatment (vitamin C 1000 1 tablet/day, Zinc 20 mg 1tablet/day, Azithromycin 500 1cp day and amoxicillin/clavulamic acid 1g/125 1 tablet per 12 hours for six days in both cohorts) and by combining Thymus vulgaris (1 teaspoon, i.e. 5g, in 100 ml of hot water to be taken every 8 hours) by the patients in the exposed cohort. After three days of this treatment, the evaluative part of the questionnaire was completed to assess the impact of taking or not taking Thymus vulgaris on early symptoms and tolerance; on the 10th day after the start of treatment, the PCR control test was carried out. Thereafter, the various statistical analyses were performed. Results: Statistical evaluation after three days of treatment shows that taking Thymus vulgaris has a statistically significant positive effect on cough (p <0.01), dyspnoea (p <0.001), dizziness (p <0.029), fatigue (p <0.001), anorexia (p <0.001), chest pain (p <0.001), fever (p <0.024), agueusia (p <0.029) and anosmia (p <0.001). There was a significant decrease in neutrophils (p <0.01); in addition, the lymphocyte count increased significantly (p <0.001) as did the serum calcium level (p <0.03). Blood urea level decreased significantly (p <0.01). Significant negative results of the COVID-19 PCR were obtained at Day 10 in the exposed group (p <0.001). In addition, there was no significant change in other biological parameters such as creatinine, blood glucose, aspartate amino transferase. Conclusion: Results of this study show that the use of the powder of Thymus vulgaris, a medicinal plant, with antioxidant, immunomodulatory and antiviral properties, was very effective on coronavirus-induced symptoms and virus elimination. Moreover, there was good tolerance after taking Thymus vulgaris.

Development of a Multiplex Droplet Digital PCR Assay for Detection of Enterovirus, Parechovirus, Herpes Simplex Virus 1 and 2 Simultaneously for Diagnosis of Viral CNS Infections

Background: Enterovirus (EV), parechovirus (HPeV), herpes simplex virus 1 and 2 (HSV1/2) are common viruses leading to viral central nervous system (CNS) infections which are increasingly predominant but exhibit deficiency in definite pathogen diagnosis with gold-standard quantitative PCR method. Previous studies have shown that droplet digital PCR (ddPCR) has great potential in pathogen detection and quantification especially in low concentration samples.Methods: Targeting four common viruses of EV, HPeV, HSV1, and HSV2 in cerebrospinal fluid (CSF), we developed a multiplex ddPCR assay using probe ratio-based multiplexing strategy, analyzed the performance, and evaluated it in 97 CSF samples collected from patients with suspected viral CNS infections on a two-channel ddPCR detection system.Results: The four viruses were clearly distinguished by their corresponding fluorescence amplitude. The limits of detection for EV, HPeV, HSV1, and HSV2 were 5, 10, 5, and 10 copies per reaction, respectively. The dynamic range was at least four orders of magnitude spanned from 2000 to 2 copies per reaction. The results of 97 tested clinical CSF specimens were identical to those deduced from qPCR/qRT-PCR assays using commercial kits.Conclusion: The multiplex ddPCR assay was demonstrated to be an accurate and robust method which could detect EV, HPeV, HSV1, and HSV2 simultaneously. It provides a useful tool for clinical diagnosis and disease monitoring of viral CNS infections.

The US3 kinase of Herpes Simplex virus phosphorylates the RNA sensor RIG-I to suppress innate immunity

Recent studies demonstrated that the signaling activity of the cytosolic pathogen sensor retinoic acid-inducible gene-I (RIG-I) is modulated by a variety of post-translational modifications (PTMs) to fine-tune the antiviral type I interferon (IFN) response. Whereas K63-linked ubiquitination of the RIG-I caspase activation and recruitment domains (CARDs) catalyzed by TRIM25 or other E3 ligases activates RIG-I, phosphorylation of RIG-I at S8 and T170 represses RIG-I signal transduction by preventing the TRIM25-RIG-I interaction and subsequent RIG-I ubiquitination. While strategies to suppress RIG-I signaling by interfering with its K63-polyubiquitin-dependent activation have been identified for several viruses, evasion mechanisms that directly promote RIG-I phosphorylation to escape antiviral immunity are unknown. Here, we show that the serine/threonine (Ser/Thr) kinase US3 of herpes simplex virus 1 (HSV-1) binds to RIG-I and phosphorylates RIG-I specifically at S8. US3-mediated phosphorylation suppressed TRIM25-mediated RIG-I ubiquitination, RIG-I-MAVS binding, and type I IFN induction. We constructed a mutant HSV-1 encoding a catalytically-inactive US3 protein (K220A) and found that, in contrast to the parental virus, the US3 mutant HSV-1 is unable to phosphorylate RIG-I at S8 and elicited higher levels of type I IFNs, IFN-stimulated genes (ISGs), and proinflammatory cytokines in a RIG-I-dependent manner. Finally, we show that this RIG-I evasion mechanism is conserved among the alphaherpesvirus US3 kinase family. Collectively, our study reveals a novel immune evasion mechanism of herpesviruses in which their US3 kinases phosphorylate the sensor RIG-I to keep it in the signaling-repressed state. IMPORTANCE Herpes simplex virus 1 (HSV-1) establishes life-long latency in the majority of the human population worldwide. HSV-1 occasionally reactivates to produce infectious virus and to facilitate dissemination. While often remaining subclinical, both primary infection and reactivation occasionally cause debilitating eye diseases, which can lead to blindness, as well as life-threatening encephalitis and newborn infections. To identify new therapeutic targets for HSV-1-induced diseases, it is important to understand the HSV-1-host interactions that may influence infection outcome and disease. Our work uncovered direct phosphorylation of the pathogen sensor RIG-I by alphaherpesvirus-encoded kinases as a novel viral immune escape strategy and also underscores the importance of RNA sensors in surveilling DNA virus infection.