Herpes Simplex Virus Type 1 Replication Research Articles

A novel GFP-based strategy to quantitate cellular spatial associations in HSV-1 viral pathogenesis.

Periodic reactivation of herpes simplex virus type 1 (HSV-1) triggers immune responses that result in corneal scarring (CS), known as herpes stromal keratitis (HSK). Despite considerable research, fully understanding HSK and eliminating it remains challenging due to a lack of comprehensive analysis of HSV-1-infected immune cells in both corneas and trigeminal ganglia (TG). We engineered a recombinant HSV-1 expressing green fluorescent protein (GFP) in the virulent McKrae virus strain that does not require corneal scarification for efficient virus replication (GFP-McKrae). Next-generation sequencing (NGS) analysis, along with in vitro and in vivo assays, showed that GFP-McKrae virus was similar to WT-McKrae virus. Furthermore, corneal cells infected with GFP-McKrae were quantitatively analyzed using image mass cytometry (IMC). The single-cell reconstruction data generated cellular maps of corneas based on the expression of 25 immune cell markers in GFP-McKrae-infected mice. Corneas from mock control mice showed the presence of T cells and macrophages, whereas corneas from GFP-McKrae-infected mice on days 3 and 5 post-infection (PI) exhibited increased immune cells. Notably, on day 3 PI, increased GFP expression was observed in closely situated clusters of DCs, macrophages, and epithelial cells. By day 5 PI, macrophages and T cells became prominent. Finally, immunostaining methods detected HSV-1 or GFP and gD proteins in latently infected TG. This study presents a valuable strategy for identifying cellular spatial associations in viral pathogenesis and holds promise for future therapeutic applications.IMPORTANCEThe goal of this study was to establish quantitative approaches to analyze immune cell markers in HSV-1-infected intact corneas and trigeminal ganglia from primary and latently infected mice. This allowed us to define spatial and temporal interactions between specific immune cells and their potential roles in virus replication and latency. To accomplish this important goal, we took advantage of the utility of GFP-McKrae virus as a valuable research tool while also highlighting its potential to uncover previously unrecognized cell types that play pivotal roles in HSV-1 replication and latency. Such insights will pave the way for developing targeted therapeutic approaches to tackle HSV-1 infections more effectively.

An Intrinsic Host Defense against HSV-1 Relies on the Activation of Xenophagy with the Active Clearance of Autophagic Receptors.

Autophagy engulfs cellular components in double-membrane-bound autophagosomes for clearance and recycling after fusion with lysosomes. Thus, autophagy is a key process for maintaining proteostasis and a powerful cell-intrinsic host defense mechanism, protecting cells against pathogens by targeting them through a specific form of selective autophagy known as xenophagy. In this context, ubiquitination acts as a signal of recognition of the cargoes for autophagic receptors, which direct them towards autophagosomes for subsequent breakdown. Nevertheless, autophagy can carry out a dual role since numerous viruses including members of the Orthoherpesviridae family can either inhibit or exploit autophagy for its own benefit and to replicate within host cells. There is growing evidence that Herpes simplex virus type 1 (HSV-1), a highly prevalent human pathogen that infects epidermal keratinocytes and sensitive neurons, is capable of negatively modulating autophagy. Since the effects of HSV-1 infection on autophagic receptors have been poorly explored, this study aims to understand the consequences of HSV-1 productive infection on the levels of the major autophagic receptors involved in xenophagy, key proteins in the recruitment of intracellular pathogens into autophagosomes. We found that productive HSV-1 infection in human neuroglioma cells and keratinocytes causes a reduction in the total levels of Ub conjugates and decreases protein levels of autophagic receptors, including SQSTM1/p62, OPTN1, NBR1, and NDP52, a phenotype that is also accompanied by reduced levels of LC3-I and LC3-II, which interact directly with autophagic receptors. Mechanistically, we show these phenotypes are the result of xenophagy activation in the early stages of productive HSV-1 infection to limit virus replication, thereby reducing progeny HSV-1 yield. Additionally, we found that the removal of the tegument HSV-1 protein US11, a recognized viral factor that counteracts autophagy in host cells, enhances the clearance of autophagic receptors, with a significant reduction in the progeny HSV-1 yield. Moreover, the removal of US11 increases the ubiquitination of SQSTM1/p62, indicating that US11 slows down the autophagy turnover of autophagy receptors. Overall, our findings suggest that xenophagy is a potent host defense against HSV-1 replication and reveals the role of the autophagic receptors in the delivery of HSV-1 to clearance via xenophagy.

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.

CRISPR/Cas9-mediated genome editing of the thymidine kinase gene in a clinical HSV-1 isolate identifies F289S as novel acyclovir-resistant mutation

Herpes simplex virus type 1 (HSV-1) is a neurotropic alphaherpesvirus that establishes a lifelong infection in sensory neurons of infected individuals, accompanied with intermittent reactivation of latent virus causing (a)symptomatic virus shedding. Whereas acyclovir (ACV) is a safe and highly effective antiviral to treat HSV-1 infections, long-term usage can lead to emergence of ACV resistant (ACVR) HSV-1 and subsequently ACV refractory disease. Here, we isolated an HSV-1 strain from a patient with reactivated herpetic eye disease that did not respond to ACV treatment. The isolate carried a novel non-synonymous F289S mutation in the viral UL23 gene encoding the thymidine kinase (TK) protein. Because ACV needs conversion by viral TK and subsequently cellular kinases to inhibit HSV-1 replication, the UL23 gene is commonly mutated in ACVR HSV-1 strains. The potential role of the F289S mutation causing ACVR was investigated using CRISPR/Cas9-mediated HSV-1 genome editing. Reverting the F289S mutation in the original clinical isolate to the wild-type sequence S289F resulted in an ACV-sensitive (ACVS) phenotype, and introduction of the F289S substitution in an ACVS HSV-1 reference strain led to an ACVR phenotype. In summary, we identified a new HSV-1 TK mutation in the eye of a patient with ACV refractory herpetic eye disease, which was identified as the causative ACVR mutation with the aid of CRISPR/Cas9-mediated genome engineering technology. Direct editing of clinical HSV-1 isolates by CRISPR/Cas9 is a powerful strategy to assess whether single residue substitutions are causative to a clinical ACVR phenotype.

α7 nicotinic receptor activation mitigates herpes simplex virus type 1 infection in microglia cells

Herpes simplex virus type 1 (HSV-1), a neurotropic DNA virus, establishes latency in neural tissues, with reactivation causing severe consequences like encephalitis. Emerging evidence links HSV-1 infection to chronic neuroinflammation and neurodegenerative diseases. Microglia, the central nervous system's (CNS) immune sentinels, express diverse receptors, including α7 nicotinic acetylcholine receptors (α7 nAChRs), critical for immune regulation. Recent studies suggest α7 nAChR activation protects against viral infections. Here, we show that α7 nAChR agonists, choline and PNU-282987, significantly inhibit HSV-1 replication in microglial BV2 cells. Notably, this inhibition is independent of the traditional ionotropic nAChR signaling pathway. mRNA profiling revealed that choline stimulates the expression of antiviral factors, IL-1β and Nos2, and down-regulates the apoptosis genes and type A Lamins in BV2 cells. These findings suggest a novel mechanism by which microglial α7 nAChRs restrict viral infections by regulating innate immune responses.

Herpes Simplex Virus type 1 inhibits autophagy in glial cells but requires ATG5 for the success of viral replication.

Herpes Simplex Virus type 1 (HSV-1) 1 is a neurotropic virus that has been associated with neurodegenerative disorders. The dysregulation of autophagy by HSV-1 has been proposed as a potential cause of neurodegeneration. While studies have extensively tackled the interaction between autophagy and HSV-1 in neurons, research in glial cells is currently limited. Our studies demonstrate that HSV-1 inhibits, but not completely blocks, the formation of autophagosomes in human oligodendroglioma- and astrocytoma- derived cell lines. These findings have been confirmed in murine oligodendrocyte precursor cells (OPCs). Finally, this study investigates the impact of autophagy on HSV-1 infection in glial cells. While the lack of basal autophagy in LC3B knockout glial cells does not have a significant effect on viral infection, cells without the autophagy-related protein ATG5 exhibit reduced viral production. The absence of ATG5 leads to a decrease in the transcription and replication of viral genes, as well as a delay in the initial stages of the formation of HSV-1 replication compartments. These findings indicate that while autophagy may not play a significant role in antiviral defense in glial cells, HSV-1 may be inhibiting autophagy to exploit non-canonical functions of certain components of the autophagic machinery, such as ATG5, to benefit its lifecycle.

Harmol used for the treatment of herpes simplex virus induced keratitis.

Herpes simplex virus type 1 (HSV-1) infection of the eyes results in herpes simplex keratitis (HSK), which has led to vision loss and even blindness in patients. However, the rate of drug resistance in HSV is on the rise; therefore, new antiviral agents with sufficient safety profiles must be developed. At present, we assessed the anti-HSV-1 activity of 502 natural compounds and their ability to reduce the HSV-1-induced cytopathic effect. We chose harmol for further studies because it exhibited the highest antiviral activity. We found that harmol inhibited both HSV-1F and HSV-1/153 (a clinical drug-resistant strain) replication, with an EC50 of 9.34 µM and 5.84 µM, respectively. Moreover, harmol reduced HSV-1 replication in corneal tissues and viral progeny production in tears, and also alleviated early corneal surface lesions related to HSK. For example, harmol treatment preserved corneal thickness and nerve density in HSK mice. Interestingly, harmol also showed a promising antiviral effect on HSV-1/153 induced HSK in mouse model. Furthermore, harmol combined with acyclovir (ACV) treatment showed a greater antiviral effect than either one alone in vitro. Therefore, harmol may be a promising therapeutic agent for managing HSK.

Enterocin DD14 can inhibit the infection of eukaryotic cells with enveloped viruses.

Bacteriocins are ribosomally synthesized bacterial peptides endowed with antibacterial, antiprotozoal, anticancer and antiviral activities. In the present study, we evaluated the antiviral activities of two bacteriocins, enterocin DD14 (EntDD14) and lacticaseicin 30, against herpes simplex virus type 1 (HSV-1), human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Vero, Huh7 and Vero E6 cells, respectively. In addition, the interactions of these bacteriocins with the envelope glycoprotein D of HSV-1 and the receptor binding domains of HCoV-229E and SARS-CoV-2 have been computationally evaluated using protein-protein docking and molecular dynamics simulations. HSV-1 replication in Vero cells was inhibited by EntDD14 and, to a lesser extent, by lacticaseicin 30 added to cells after virus inoculation. EntDD14 and lacticaseicin 30 had no apparent antiviral activity against HCoV-229E; however, EntDD14 was able to inhibit SARS-CoV-2 in Vero E6 cells. Further studies are needed to elucidate the antiviral mechanism of these bacteriocins.

Cytosolic DNA sensors activation of human astrocytes inhibits herpes simplex virus through IRF1 induction.

While astrocytes participate in the CNS innate immunity against herpes simplex virus type 1 (HSV-1) infection, they are the major target for the virus. Therefore, it is of importance to understand the interplay between the astrocyte-mediated immunity and HSV-1 infection. Both primary human astrocytes and the astrocyte line (U373) were used in this study. RT-qPCR and Western blot assay were used to measure IFNs, the antiviral IFN-stimulated genes (ISGs), IFN regulatory factors (IRFs) and HSV-1 DNA. IRF1 knockout or knockdown was performed with CRISPR/Cas9 and siRNA transfection techniques. Poly(dA:dT) could inhibit HSV-1 replication and induce IFN-β/IFN-λs production in human astrocytes. Poly(dA:dT) treatment of astrocytes also induced the expression of the antiviral ISGs (Viperin, ISG56 and MxA). Among IRFs members examined, poly(dA:dT) selectively unregulated IRF1 and IRF9, particularly IRF1 in human astrocytes. The inductive effects of poly(dA:dT) on IFNs and ISGs were diminished in the IRF1 knockout cells. In addition, IRF1 knockout attenuated poly(dA:dT)-mediated HSV-1 inhibition in the cells. The DNA sensors activation induces astrocyte intracellular innate immunity against HSV-1. Therefore, targeting the DNA sensors has potential for immune activation-based HSV-1 therapy.

Overexpression of NLRP12 enhances macrophage immune response and alleviates herpes simplex keratitis.

Herpes simplex keratitis (HSK) is a blinding disease caused by corneal infection of Herpes simplex virus type 1 (HSV-1). Effective clearance of HSV-1 from the infected cornea is crucial for HSK management. Macrophages play an important part in the innate immune defense against viral infections. This study investigates the immunomodulatory role of NLRP12 in macrophage immune response during HSV-1 infection. NLRP12 expression post-infection was assessed in various macrophage cell lines. Overexpression of NLRP12 was achieved by lentiviral transfection, and its effect on HSV-1 replication and immune responses were examined. Mechanistic insights into the role of NLRP12 were explored using immunofluorescence and Western Blot. For in vivo studies, ocular adoptive transfer of NLRP12-overexpressing bone marrow derived macrophages (BMDMs) was performed. HSV-1 viral loads, HSK symptoms, and macrophage-mediated immune responses were investigated. A significant decrease in NLRP12 expression post-infection was observed in various macrophage cell lines. Overexpression of NLRP12 in macrophages reduced HSV-1 replication. Mechanistically, overexpression of NLRP12 triggered early and robust pyroptosis in response to HSV-1 infection, inducing interleukin (IL)-18 production and activating downstream antiviral responses through the JAK-STAT signaling pathway. In vivo, ocular adoptive transfer of NLRP12-overexpressing BMDMs to mouse corneas alleviated HSK damage and reduced HSV-1 viral loads. NLRP12-overexpressing BMDMs improved antiviral responses in the cornea and promoted the maturation of corneal-infiltrating macrophages and dendritic cells. Additionally, NLRP12-overexpressing BMDMs amplified the adaptive immune response in the submandibular draining lymph nodes. These findings highlight the role of NLRP12 in macrophage-mediated immune response against HSV-1 infection and suggest its potential for possible immunotherapy for HSK.

Lanatoside C inhibits herpes simplex virus 1 replication by regulating NRF2 distribution within cells

BackgroundIn the past decades, extensive research has been conducted to identify new drug targets for the treatment of Herpes simplex virus type 1 (HSV-1) infections. However, the emergence of drug-resistant HSV-1 strains remains a major challenge. This necessitates the identification of new drugs with novel mechanisms of action. Lanatoside C (LanC), a cardiac glycoside (CG) approved by the US Food and Drug Administration (FDA), has demonstrated anticancer and antiviral properties. Nevertheless, its potential as an agent against HSV-1 infections and the underlying mechanism of action are currently unknown. PurposeThis study aimed to investigate the antiviral activity of LanC against HSV-1 and elucidate its molecular mechanisms. MethodsThe in vitro antiviral activity of LanC was assessed by examining the levels of viral genes, proteins, and virus titers in HSV-1-infected ARPE-19 and Vero cells. Immunofluorescence (IF) analysis was performed to determine the intracellular distribution of NRF2. Additionally, an in vivo mouse model of HSV-1 infection was developed to evaluate the antiviral activity of LanC, using indicators such as intraepidermal nerve fibers (IENFs) loss and viral gene inhibition. ResultsOur findings demonstrate that LanC significantly inhibits HSV-1 replication both in vitro and in vivo. The antiviral effect of LanC is mediated by the perinuclear translocation of NRF2. ConclusionsLanC exhibits anti-HSV-1 effects in viral infections, which are associated with the intracellular translocation of NRF2. These findings suggest that LanC has the potential to serve as a novel NRF2 modulator in the treatment of viral diseases.

Plant Cell-Engineered Gold Nanoparticles Conjugated to Quercetin Inhibit SARS-CoV-2 and HSV-1 Entry.

Recent studies have revealed considerable promise in the antiviral properties of metal nanomaterials, specifically when biologically prepared. This study demonstrates for the first time the antiviral roles of the plant cell-engineered gold nanoparticles (pAuNPs) alone and when conjugated with quercetin (pAuNPsQ). We show here that the quercetin conjugated nanoparticles (pAuNPsQ) preferentially inhibit the cell entry of two medically important viruses-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and herpes simplex virus type-1 (HSV-1) using different mechanisms. Interestingly, in the case of SARS-CoV-2, the pre-treatment of target cells with pAuNPsQ inhibited the viral entry, but the pre-treatment of the virus with pAuNPsQ did not affect viral entry into the host cell. In contrast, pAuNPsQ demonstrated effective blocking capabilities against HSV-1 entry, either during the pre-treatment of target cells or by inducing virus neutralization. In addition, pAuNPsQ also significantly affected HSV-1 replication, evidenced by the plaque-counting assay. In this study, we also tested the chemically synthesized gold nanoparticles (cAuNPs) of identical size and shape and observed comparable effects. The versatility of plant cell-based nanomaterial fabrication and its modification with bioactive compounds opens a new frontier in therapeutics, specifically in designing novel antiviral formulations.

AMPK protects endothelial cells against HSV-1 replication via inhibition of mTORC1 and ACC1.

Herpes simplex virus type 1 (HSV-1) is a widespread contagious pathogen, mostly causing mild symptoms on the mucosal entry side. However, systemic distribution, in particular upon reactivation of the virus in immunocompromised patients, may trigger an innate immune response and induce damage of organs. In these conditions, HSV-1 may infect vascular endothelial cells, but little is known about the regulation of HSV-1 replication and possible defense mechanisms in these cells. The current study addresses the question of whether the host cell protein AMP-activated protein kinase (AMPK), an important metabolic sensor, can control HSV-1 replication in endothelial cells. We show that downregulation of the catalytic subunits AMPKα1 and/or AMPKα2 increased HSV-1 replication as monitored by TCID50 titrations, while a potent AMPK agonist, MK-8722, strongly inhibited it. MK-8722 induced a persistent phosphorylation of the AMPK downstream targets acetyl-CoA carboxylase (ACC) and the rapamycin-sensitive adaptor protein of mTOR (Raptor) and, related to this, impairment of ACC1-mediated lipid synthesis and the mechanistic target of the rapamycin complex-1 (mTORC1) pathway. Since blockade of mTOR by Torin-2 as well as downregulation of ACC1 by siRNA also decreased HSV-1 replication, MK-8722 is likely to exert its anti-viral effect via mTORC1 and ACC1 inhibition. Importantly, MK-8722 was able to reduce virus replication even when added after HSV-1. Together, our data highlight the importance of endothelial cells as host cells for HSV-1 replication upon systemic infection and identify AMPK, a metabolic host cell protein, as a potential target for antiviral strategies against HSV-1 infection and its severe consequences. IMPORTANCE Herpes simplex virus type 1 (HSV-1) is a common pathogen that causes blisters or cold sores in humans. It remains latent in infected individuals and can be reactivated multiple times. In adverse conditions, for instance, in immunocompromised patients, HSV-1 can lead to serious complications such as encephalitis, meningitis, or blindness. In these situations, infection of endothelial cells lining the surface of blood vessels may contribute to the manifestation of disease. Here, we describe the role of AMP-activated protein kinase (AMPK), a potent regulator of cellular energy metabolism, in HSV-1 replication in endothelial cells. While downregulation of AMPK potentiates HSV-1 replication, pharmacological AMPK activation inhibits it by limiting the availability of required host cell macromolecules such as proteins or fatty acids. These data highlight the role of metabolic host cell proteins as antiviral targets and reveal activation of endothelial AMPK as a potential strategy to protect from severe consequences of HSV-1 infection.

Therapeutic Potential of Biochanin A in Herpes Simplex Keratitis.

Herpes simplex keratitis (HSK) is a blinding eye disease that is initiated by the herpes simplex virus type 1 (HSV-1). Resistance to acyclovir (ACV) and the side effects of corticosteroid drugs have become concerning issues, so it is crucial to develop new antivirals for treating HSK. In this study, we report that biochanin A (BCA), a naturally occurring flavonoid compound, provides multifaceted protective effects with anti-viral, anti-inflammatory, anti-oxidative stress and anti-apoptotic activities to alleviate HSK. The results show that BCA significantly inhibited HSV-1 replication in vitro and further proved that BCA principally influenced the early stage of virus infection. We reveal that BCA downregulated the expression of pro-inflammatory factors triggered by HSV-1, including TNF-α, RANTES, IL-1β and IL-6. Furthermore, BCA treatment alleviated oxidative stress and apoptotic arising from HSV-1 infection. Lastly, we induced HSK in male C57BL/6 mice and treated them with either BCA or phosphate buffer solution (PBS) eye drops. We observed the ocular surface lesions; determined the virus load in the tear fluid, corneas as well as trigeminal ganglions (TGs); and detected the levels of inflammation and apoptosis in the corneas simultaneously. These results show that BCA inhibits HSV-1 and alleviates the corneal lesion degree. Our study illustrates that BCA is a promising therapeutic approach for application in treating HSK.

Early Events after Herpes Simplex Virus-Type 1 Entry Are Necessary for the Release of Gamma-Hydroxybutyrate upon Acute Infection.

We reported that gamma-hydroxybutyrate (GHB) is released upon Herpes Simplex Virus Type-1 (HSV-1) acute infection. However, the cellular biochemical processes involved in the production of GHB in infected cells are unclear. This study aims to shed light on the biochemical pathway and the stage within the viral life cycle responsible for the release of GHB in infected cells. UV-inactivation, acyclovir (ACV), and cycloheximide (CHX) treatments were used to inhibit HSV-1 replication at various stages. Vero cells treated with UV-inactivated HSV-1 significantly decreased GHB production. However, ACV or CHX treatments did not affect GHB production. We also showed that inhibition of glycolytic enzyme enolase by sodium fluoride (NaF) significantly reduces GHB production upon infection. This finding suggests that suppression of glycolytic activity negatively affects cellular GHB production. Our data also indicated that succinic semialdehyde dehydrogenase, an enzyme involved in the shunt of the tricarboxylic acid (TCA) cycle to generate succinic acid, was decreased upon infection, suggesting that infection may trigger the accumulation of succinic semialdehyde, causing the production of GHB. Although the precise mechanism has yet to be defined, our results suggest that early events following infection modulates the release of GHB, which is generated through the metabolic pathways of glycolysis and TCA cycle.

BMS-265246, a Cyclin-Dependent Kinase Inhibitor, Inhibits the Infection of Herpes Simplex Virus Type 1.

Herpes simplex virus type 1 (HSV-1) infections are prevalent illnesses that can cause mucocutaneous ulcerative disease, keratitis, and genital herpes. In patients with compromised immune systems, the infection can lead to serious problems, such as encephalitis. Additionally, neonatal infections can cause brain problems and even death. Current first-line antiviral drugs are nucleoside analog inhibitors that target viral polymerase, and resistant strains have emerged. As a result, new drugs with distinct action modes are needed. Recent research indicates that cyclin-dependent kinases (CDKs) are prospective antiviral targets. Thus, CDK inhibitors may be effective antiviral agents against HSV-1 infection. In this study, we examined a panel of CDK inhibitors that target CDKs in the present study. BMS-265246 (BMS), a CDK 1/2 inhibitor, was found to effectively limit HSV-1 multiplication in Vero, HepG2, and Hela cells. A mechanism of action study suggested that BMS inhibits the early stages of viral replication when added early in the viral infection. The suppression of multiple steps in viral replication by BMS was revealed when HSV-1 infected cells were treated at different time periods in the viral life cycle. Our results suggest that BMS is a potent anti-HSV-1 agent and unique in that it may interfere with multiple steps in HSV-1 replication.

Herpes Simplex Virus-1 ICP27 Nuclear Export Signal Mutants Exhibit Cell Type-Dependent Deficits in Replication and ICP4 Expression.

Herpes simplex virus type-1 (HSV-1) protein ICP27 is an essential immediate early (IE) protein that promotes the expression of viral early (E) and late (L) genes via multiple mechanisms. Our understanding of this complex regulatory protein has been greatly enhanced by the characterization of HSV-1 mutants bearing engineered alterations in the ICP27 gene. However, much of this analysis has been performed in interferon-deficient Vero monkey cells. Here, we assessed the replication of a panel of ICP27 mutants in several other cell types. Our analysis shows that mutants lacking ICP27's amino (N)-terminal nuclear export signal (NES) display a striking cell type-dependent growth phenotype, i.e., they grow semi-permissively in Vero and some other cells but are tightly blocked for replication in primary human fibroblasts and multiple human cell lines. This tight growth defect correlates with a failure of these mutants to replicate viral DNA. We also report that HSV-1 NES mutants are deficient in expressing the IE protein ICP4 at early times postinfection. Analysis of viral RNA levels suggests that this phenotype is due, at least in part, to a defect in the export of ICP4 mRNA to the cytoplasm. In combination, our results (i) show that ICP27's NES is critically important for HSV-1 replication in many human cells, and (ii) suggest that ICP27 plays a heretofore unappreciated role in the expression of ICP4. IMPORTANCE HSV-1 IE proteins drive productive HSV-1 replication. The major paradigm of IE gene induction, developed over many years, involves the parallel activation of the five IE genes by the viral tegument protein VP16, which recruits the host RNA polymerase II (RNAP II) to the IE gene promoters. Here, we provide evidence that ICP27 can enhance ICP4 expression early in infection. Because ICP4 is required for transcription of viral E and L genes, this finding may be relevant to understanding how HSV-1 enters and exits the latent state in neurons.

Navoximod modulates local HSV-1 replication to reshape tumor immune microenvironment for enhanced immunotherapy via an injectable hydrogel

Oncolytic virotherapy can lead to tumor lysis and systemic anti-tumor immunity, but the therapeutic potential in humans is limited due to the impaired virus replication and the insufficient ability to overcome the immunosuppressive tumor microenvironment (TME). To solve the above problems, we identified that Indoleamine 2, 3-dioxygenase 1 (IDO1) inhibitor Navoximod promoted herpes simplex virus type 1 (HSV-1) replication and HSV-1-mediated oncolysis in tumor cells, making it a promising combination modality with HSV-1-based virotherapy. Thus, we loaded HSV-1 and Navoximod together in an injectable and biocompatible hydrogel (V-Navo@gel) for hepatocellular carcinoma (HCC) virotherapy. The hydrogel formed a local delivery reservoir to maximize the viral replication and distribution at the tumor site with a single-dose injection. Notably, V-Navo@gel improved the disease-free survival time of HCC- bearing mice and protects the mice against tumor recurrence. What’s more, V-Navo@gel also showed an effective therapeutic efficacy in the rabbit orthotopic liver cancer model. Mechanistically, we further discovered that our combination strategy entirely reprogramed the TME through single-cell RNA sequencing. All these results collectively indicated that the combination of Navoximod with HSV-1 could boost the viral replication and reshape TME for tumor eradication through the hydrogel reservoir.

Single therapeutic dose of an antiviral UL29 siRNA swarm diminishes symptoms and viral load of mice infected intranasally with HSV-1.

Herpes simplex virus type 1 (HSV-1) is a human pathogen that causes recurrent infections. Acyclovir-resistant strains exist and can cause severe complications, which are potentially untreatable with current therapies. We have developed siRNA swarms that target a 653 base pair long region of the essential HSV gene UL29. As per our previous results, the anti-UL29 siRNA swarm effectively inhibits the replication of circulating HSV strains and acyclovir-resistant HSV strains in vitro, while displaying a good safety profile. We investigated a single intranasal therapeutic dose of a siRNA swarm in mice, which were first inoculated intranasally with HSV-1 and given treatment 4h later. We utilized a luciferase-expressing HSV-1 strain, which enabled daily follow-up of infection with in vivo imaging. Our results show that a single dose of a UL29-targeted siRNA swarm can inhibit the replication of HSV-1 in orofacial tissue, which was reflected in ex vivo HSV titers and HSV DNA copy numbers as well as by a decrease in a luciferase-derived signal. Furthermore, the treatment had a tendency to protect mice from severe clinical symptoms and delay the onset of the symptoms. These results support the development of antiviral siRNA swarms as a novel treatment for HSV-1 infections.

Isoliquiritigenin inhibits virus replication and virus-mediated inflammation via NRF2 signaling

BackgroundThe transcription factor NRF2 is a master redox switch that regulates the cellular antioxidant response. However, recent advances have revealed new roles for NRF2, including the regulation of antiviral responses to various viruses, suggesting that pharmacological NRF2-activating agents may be a promising therapeutic drug for viral diseases. Isoliquiritigenin (ISL), a chalcone isolated from liquorice (Glycyrrhizae Radix) root, is reported to be a natural NRF2 agonist and has has antiviral activities against HCV (hepatitis C virus) and IAV (influenza A virus). However, the spectrum of antiviral activity and associated mechanism of ISL against other viruses are not well defined. PurposeThis study investigated the antiviral activity and underlying mechanism of ISL against vesicular stomatitis virus (VSV), influenza A virus (H1N1), encephalomyocarditis virus (EMCV), herpes simplex virus type 1 (HSV-1). MethodsWe evaluated the antiviral activity of ISL against VSV, H1N1, EMCV, and HSV-1 using flow cytometry and qRT-PCR analysis. RNA sequencing and bioinformatic analysis were performed to investigate the potential antiviral mechanism of ISL. NRF2 knockout cells were used to investigate whether NRF2 is required for the antiviral activity of ISL. The anti-apoptosis and anti-inflammatory activities of ISL were further measured by counting cell death ratio and assessing proinflammatory cytokines expression in virus-infected cells, respectively. In addition, we evaluated the antiviral effect of ISL in vivo by measuring the survival rate, body weights, histological analysis, viral load, and cytokine expression in VSV-infected mouse model. ResultsOur data demonstrated that ISL effectively suppressed VSV, H1N1, HSV-1, and EMCV replication in vitro. The antiviral activity of ISL could be partially impaired in NRF2-deficient cells. Virus-induced cell death and proinflammatory cytokines were repressed by ISL. Finally, we showed that ISL treatment protected mice against VSV infection by reducing viral titers and suppressing the expression of inflammatory cytokines in vivo. ConclusionThese findings suggest that ISL has antiviral and anti-inflammatory effects in virus infections, which are associated with its ability to activate NRF2 signaling, thus indicating that ISL has the potential to serve as an NRF2 agonist in the treatment of viral diseases.