Gene editing for latent herpes simplex virus infection reduces viral load and shedding in vivo.

Anti-HSV therapies are only suppressive because they do not eliminate latent HSV present in ganglionic neurons, the source of recurrent disease. We have developed a potentially curative approach against HSV infection, based on gene editing using HSV-specific meganucleases delivered by adeno-associated virus (AAV) vectors. Gene editing performed with two anti-HSV-1 meganucleases delivered by a combination of AAV9, AAV-Dj/8, and AAV-Rh10 can eliminate 90% or more of latent HSV DNA in mouse models of orofacial infection, and up to 97% of latent HSV DNA in mouse models of genital infection. Using a pharmacological approach to reactivate latent HSV-1, we demonstrate that ganglionic viral load reduction leads to a significant decrease of viral shedding in treated female mice. While therapy is well tolerated, in some instances, we observe hepatotoxicity at high doses and subtle histological evidence of neuronal injury without observable neurological signs or deficits. Simplification of the regimen through use of a single serotype (AAV9) delivering single meganuclease targeting a duplicated region of the HSV genome, dose reduction, and use of a neuron-specific promoter each results in improved tolerability while retaining efficacy. These results reinforce the curative potential of gene editing for HSV disease.

Glycoengineered keratinocyte library reveals essential functions of specific glycans for all stages of HSV-1 infection.

Viral and host glycans represent an understudied aspect of host-pathogen interactions, despite potential implications for treatment of viral infections. This is due to lack of easily accessible tools for analyzing glycan function in a meaningful context. Here we generate a glycoengineered keratinocyte library delineating human glycosylation pathways to uncover roles of specific glycans at different stages of herpes simplex virus type 1 (HSV-1) infectious cycle. We show the importance of cellular glycosaminoglycans and glycosphingolipids for HSV-1 attachment, N-glycans for entry and spread, and O-glycans for propagation. While altered virion surface structures have minimal effects on the early interactions with wild type cells, mutation of specific O-glycosylation sites affects glycoprotein surface expression and function. In conclusion, the data demonstrates the importance of specific glycans in a clinically relevant human model of HSV-1 infection and highlights the utility of genetic engineering to elucidate the roles of specific viral and cellular carbohydrate structures.

IRF7 and UNC93B1 variants in an infant with recurrent herpes simplex virus infection.

Neonatal herpes simplex virus (HSV) infection is a devastating disease with substantial morbidity and mortality. The genetic basis of susceptibility to HSV in neonates remains undefined. We evaluated a male infant with neonatal skin/eye/mouth (SEM) HSV-1 disease, who had complete recovery after acyclovir but developed HSV-1 encephalitis at 1 year of age. An immune workup showed an anergic PBMC cytokine response to TLR3 stimulation but no other TLRs. Exome sequencing identified rare missense variants in IFN-regulatory factor 7 (IRF7) and UNC-93 homolog B1 (UNC93B1). PBMC single-cell RNA-Seq done during childhood revealed decreased expression of several innate immune genes and a repressed TLR3 pathway signature at baseline in several immune cell populations, including CD14 monocytes. Functional studies in fibroblasts and human leukemia monocytic THP1 cells showed that both variants individually suppressed TLR3-driven IRF3 transcriptional activity and the type I IFN response in vitro. Furthermore, fibroblasts expressing the IRF7 and UNC93B1 variants had higher intracellular viral titers with blunting of the type I IFN response upon HSV-1 challenge. This study reports an infant with recurrent HSV-1 disease complicated by encephalitis associated with deleterious variants in the IRF7 and UNC93B1 genes. Our results suggest that TLR3 pathway mutations may predispose neonates to recurrent, severe HSV.

No Association of IFNL4 Genotype With Opportunistic Infections and Cancers Among Men With Human Immunodeficiency Virus 1 Infection.

BACKGROUND: IFNL4 genetic variants that are strongly associated with clearance of hepatitis C virus have been linked to risk of certain opportunistic infections (OIs) and cancers, including Kaposi sarcoma, cytomegalovirus infection, and herpes simplex virus infection. As the interferon (IFN) λ family plays a role in response to viral, bacterial, and fungal infections, IFNL4 genotype might affect risk for a wide range of OIs/cancers. METHODS: We examined associations between genotype for the functional IFNL4 rs368234815 polymorphism and incidence of 16 OIs/cancers among 2310 men with human immunodeficiency virus (2038 white; 272 black) enrolled in the Multicenter AIDS Cohort Study during 1984-1990. Our primary analyses used Cox proportional hazards models adjusted for self-reported racial ancestry to estimate hazard ratios with 95% confidence intervals, comparing participants with the genotypes that generate IFN-λ4 and those with the genotype that abrogates IFN-λ4. We censored follow-up at the introduction of highly effective antiretroviral therapies. RESULTS: We found no statistically significant association between IFNL4 genotype and the incidence of Kaposi sarcoma (hazard ratio, 0.92 [95% confidence interval, .76-1.11]), cytomegalovirus infection (0.94 [.71-1.24]), herpes simplex virus infection (1.37 [.68-2.93]), or any other OI/cancer. We observed consistent results using additive genetic models and after controlling for CD4 cell count through time-dependent adjustment or restriction to participants with a low CD4 cell count. CONCLUSIONS: The absence of associations between IFNL4 genotype and these OIs/cancers provides evidence that this gene does not affect the risk of disease from opportunistic pathogens.

Swarms of chemically modified antiviral siRNA targeting herpes simplex virus infection in human corneal epithelial cells.

Herpes simplex virus type 1 (HSV-1) is a common virus of mankind and HSV-1 infections are a significant cause of blindness. The current antiviral treatment of herpes infection relies on acyclovir and related compounds. However, acyclovir resistance emerges especially in the long term prophylactic treatment that is required for prevention of recurrent herpes keratitis. Earlier we have established antiviral siRNA swarms, targeting sequences of essential genes of HSV, as effective means of silencing the replication of HSV in vitro or in vivo. In this study, we show the antiviral efficacy of 2´-fluoro modified antiviral siRNA swarms against HSV-1 in human corneal epithelial cells (HCE). We studied HCE for innate immunity responses to HSV-1, to immunostimulatory cytotoxic double stranded RNA, and to the antiviral siRNA swarms, with or without a viral challenge. The panel of studied innate responses included interferon beta, lambda 1, interferon stimulated gene 54, human myxovirus resistance protein A, human myxovirus resistance protein B, toll-like receptor 3 and interferon kappa. Our results demonstrated that HCE cells are a suitable model to study antiviral RNAi efficacy and safety in vitro. In HCE cells, the antiviral siRNA swarms targeting the HSV UL29 gene and harboring 2´-fluoro modifications, were well tolerated, induced only modest innate immunity responses, and were highly antiviral with more than 99% inhibition of viral release. The antiviral effect of the 2'-fluoro modified swarm was more apparent than that of the unmodified antiviral siRNA swarm. Our results encourage further research in vitro and in vivo on antiviral siRNA swarm therapy of corneal HSV infection, especially with modified siRNA swarms.

The In Vitro Replication, Spread, and Oncolytic Potential of Finnish Circulating Strains of Herpes Simplex Virus Type 1.

Herpes simplex virus type 1 (HSV-1) is the only FDA- and EMA- approved oncolytic virus, and accordingly, many potential oncolytic HSVs (oHSV) are in clinical development. The utilized oHSV parental strains are, however, mostly based on laboratory reference strains, which may possess a compromised cytolytic capacity in contrast to circulating strains of HSV-1. Here, we assess the phenotype of thirty-six circulating HSV-1 strains from Finland to uncover their potential as oHSV backbones. First, we determined their capacity for cell-to-cell versus extracellular spread, to find strains with replication profiles favorable for each application. Second, to unfold the differences, we studied the genetic diversity of two relevant viral glycoproteins (gB/UL27, gI/US7). Third, we examined the oncolytic potential of the strains in cells representing glioma, lymphoma, and colorectal adenocarcinoma. Our results suggest that the phenotype of a circulating isolate, including the oncolytic potential, is highly related to the host cell type. Nevertheless, we identified isolates with increased oncolytic potential in comparison with the reference viruses across many or all of the studied cancer cell types. Our research emphasizes the need for careful selection of the backbone virus in early vector design, and it highlights the potential of clinical isolates as backbones in oHSV development.

PML Body Component Sp100A Restricts Wild-Type Herpes Simplex Virus 1 Infection.

Sp100 (speckled protein 100 kDa) is a constituent component of nuclear structure PML (promyelocytic leukemia) bodies, playing important roles in mediating intrinsic and innate immunity. The Sp100 gene encodes four isoforms with distinct roles in the transcriptional regulation of both cellular and viral genes. Since Sp100 is a primary intranuclear target of infected-cell protein 0 (ICP0), an immediate early E3 ligase encoded by herpes simplex virus 1 (HSV-1), previous investigations attempting to analyze the functions of individual Sp100 variants during HSV-1 infection mostly avoided using a wild-type virus. Therefore, the role of Sp100 under natural infection by HSV-1 remains to be clarified. Here, we reappraised the antiviral capacity of four Sp100 isoforms during infection by a nonmutated HSV-1, examined the molecular behavior of the Sp100 protein in detail, and revealed the following intriguing observations. First, Sp100 isoform A (Sp100A) inhibited wild-type HSV-1 propagation in HEp-2, Sp100-/-, and PML-/- cells. Second, endogenous Sp100 is located in both the nucleus and the cytoplasm. During HSV-1 infection, the nuclear Sp100 level decreased drastically upon the detection of ICP0 in the same subcellular compartment, but cytosolic Sp100 remained stable. Third, transfected Sp100A showed subcellular localizations similar to those of endogenous Sp100 and matched the protein size of endogenous cytosolic Sp100. Fourth, HSV-1 infection induced increased secretion of endogenous Sp100 and ectopically expressed Sp100A, which copurified with extracellular vesicles (EVs) but not infectious virions. Fifth, the Sp100A level in secreting cells positively correlated with its level in EVs, and EV-associated Sp100A restricted HSV-1 in recipient cells. IMPORTANCE Previous studies show that the PML body component Sp100 protein is immediately targeted by ICP0 of HSV-1 in the nucleus during productive infection. Therefore, extensive studies investigating the interplay of Sp100 isoforms with HSV-1 were conducted using a mutant virus lacking ICP0 or in the absence of infection. The role of Sp100 variants during natural HSV-1 infection remains blurry. Here, we report that Sp100A potently and independently inhibited wild-type HSV-1 and that during HSV-1 infection, cytosolic Sp100 remained stable and was increasingly secreted into the extracellular space, in association with EVs. Furthermore, the Sp100A level in secreting cells positively correlated with its level in EVs and the anti-HSV-1 potency of these EVs in recipient cells. In summary, this study implies an active antiviral role of Sp100A during wild-type HSV-1 infection and reveals a novel mechanism of Sp100A to restrict HSV-1 through extracellular communications.

Host MOV10 is induced to restrict herpes simplex virus 1 lytic infection by promoting type I interferon response.

Moloney leukemia virus 10 protein (MOV10) is an interferon (IFN)-inducible RNA helicase implicated in antiviral activity against RNA viruses, yet its role in herpesvirus infection has not been investigated. After corneal inoculation of mice with herpes simplex virus 1 (HSV-1), we observed strong upregulation of both MOV10 mRNA and protein in acutely infected mouse trigeminal ganglia. MOV10 suppressed HSV-1 replication in both neuronal and non-neuronal cells, and this suppression required the N-terminus, but not C-terminal helicase domain of MOV10. MOV10 repressed expression of the viral gene ICP0 in transfected cells, but suppressed HSV-1 replication independently of ICP0. MOV10 increased expression of type I IFN in HSV-1 infected cells with little effect on IFN downstream signaling. Treating the cells with IFN-α or an inhibitor of the IFN receptor eliminated MOV10 suppression of HSV-1 replication. MOV10 enhanced IFN production stimulated by cytoplasmic RNA rather than DNA. IKKε co-immunoprecipitated with MOV10 and was required for MOV10 restriction of HSV-1 replication. Mass spectrometry identified ICP27 as a viral protein interacting with MOV10. Co-immunoprecipitation results suggested that this interaction depended on the RGG box of ICP27 and both termini of MOV10. Overexpressed ICP27, but not its RGG-Box deletion mutant, rendered MOV10 unable to regulate HSV-1 replication and type I IFN production. In summary, MOV10 is induced to restrict HSV-1 lytic infection by promoting the type I IFN response through an IKKε-mediated RNA sensing pathway, and its activity is potentially antagonized by ICP27 in an RGG box dependent manner.

Targeting NECTIN-1 Based on CRISPR/Cas9 System Attenuated the Herpes Simplex Virus Infection in Human Corneal Epithelial Cells In Vitro.

PURPOSE: Viral keratitis caused by herpes simplex virus 1 (HSV-1) is a lifelong recurring disease and an unignored cause of blindness worldwide. Current antiviral therapy cannot eliminate the transcriptionally silent HSV-1 in latently infected patients. With the explosive applications of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9 gene-editing system in recent years, we aim to develop a CRISPR/Cas9 system targeting down the major HSV receptor, NECTIN-1 on human corneal epithelial cells (HCECs), to provide a novel strategy for herpes simplex keratitis (HSK) treatment. METHODS: The selected single guide RNAs (sgRNAs) targeting human nectin cell adhesion molecule 1 (NECTIN-1), together with Cas-9, were assembled into lentivirus. HCECs were infected with Lenti-Cas9-gRNAs to establish NECTIN-1 knockdown cells. Following HSV-green fluorescent protein (GFP) infection, cell survival and virus infection were determined by fluorescence microscopy and flow cytometry. Relative HSV DNA amount was also compared through quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Lentivirus packaged with the CRISPR/Cas9 system and the two selected sgRNAs both successfully edited down the protein levels of NECTIN-1 of HCECs. After HSV-GFP infection, the infection rate of HCECs in knockdown groups dramatically decreased, especially in the NECTIN-1 knockdown group 1. In addition, the relative HSV DNA amount of both knockdown groups was only 30% when compared with the control group. CONCLUSIONS: We successfully knocked down the NECTIN-1 expression in vitro by the CRISPR/Cas9 system, which alleviated the HSV infection in HCECs. TRANSLATIONAL RELEVANCE: This study offered a promising target for the cure of HSK.

Suicide gene therapy by canine mesenchymal stem cell transduced with thymidine kinase in a u-87 glioblastoma murine model: Secretory profile and antitumor activity.

The role played by certain domestic species such as dogs as a translational model in comparative oncology shows great interest to develop new therapeutic strategies in brain tumors. Gliomas are a therapeutic challenge that represents the most common form of malignant primary brain tumors in humans and the second most common form in dogs. Gene-directed enzyme/prodrug therapy using adipose mesenchymal stem cells (Ad-MSCs) expressing the herpes simplex virus thymidine kinase (TK) has proven to be a promising alternative in glioblastoma therapy, through its capacity to migrate and home to the tumor and delivering local cytotoxicity avoiding other systemic administration. In this study, we demonstrate the possibility for canine Ad-MSCs (cAd-MSCs) to be genetically engineered efficiently with a lentiviral vector to express TK (TK-cAd-MSCs) and in combination with ganciclovir (GCV) prodrug demonstrated its potential antitumor efficacy in vitro and in vivo in a mice model with the human glioblastoma cell line U87. TK-cAd-MSCs maintained cell proliferation, karyotype stability, and MSCs phenotype. Genetic modification significantly affects its secretory profile, both the analyzed soluble factors and exosomes. TK-cAd-MSCs showed a high secretory profile of some active antitumor immune response cytokines and a threefold increase in the amount of secreted exosomes, with changes in their protein cargo. We also found that the prodrug protein is not released directly into the culture medium by TK-cAd-MSCs. We believe that our work provides new perspectives for glioblastoma gene therapy in dogs and a better understanding of this therapy in view of its possible implantation in humans.

STING-Mediated Interferon Induction by Herpes Simplex Virus 1 Requires the Protein Tyrosine Kinase Syk.

The nature and the intensity of innate immune response to virus infection determine the course of pathogenesis in the host. Among the many pathogen-associated molecular pattern recognition receptors, STING, an endoplasmic reticulum (ER)-associated protein, plays a pivotal role in triggering responses to microbial or cellular cytoplasmic DNA. Herpes simplex virus 1 (HSV-1), a common human pathogen, activates STING signaling, and the resultant induction of type I interferon causes inhibition of virus replication. In this context, we have observed that phosphorylation of Tyr245 of STING by epidermal growth factor receptor kinase is necessary for interferon induction. Here, we report that phosphorylation of Tyr240 by the tyrosine kinase Syk is essential for all signaling activities of STING. Our analysis showed that upon ligand-binding, STING dimerizes and interacts with membrane-bound EGFR, which autophosphorylates and provides the platform for the recruitment of cytoplasmic Syk to the signaling complex and its activation. Activated Syk phosphorylates Tyr240 of STING, followed by phosphorylation of Tyr245 by epidermal growth factor receptor (EGFR). Pharmacological or genetic ablation of Syk activity resulted in an arrest of STING in the ER compartment and a complete block of gene induction. Consequently, in the absence of Syk, HSV-1 could not induce interferon, and it replicated more robustly. IMPORTANCE The innate immune response to virus infection leads to interferon production and inhibition of viral replication. STING, an ER-bound protein, mediates such a response to cytoplasmic cellular or microbial DNA. HSV-1, a DNA virus, activates STING, and it replicates more efficiently in the absence of STING signaling. We demonstrate that phosphorylation of Tyr240 of STING by the protein tyrosine kinase Syk is essential for STING-mediated gene induction. To signal, ligand-activated STING recruits two kinases, Syk and EGFR, which phosphorylate Tyr240 and Tyr245, respectively. The dependence of STING signaling on Syk has broad significance, because STING plays a major role in many microbial, mitochondrial, and autoimmune diseases as well as in cancer development and therapy.

Herpes Simplex Virus Type 1 Infection Disturbs the Mitochondrial Network, Leading to Type I Interferon Production through the RNA Polymerase III/RIG-I Pathway.

Viruses have evolved a plethora of mechanisms to impair host innate immune responses. Herpes simplex virus type 1 (HSV-1), a double-stranded linear DNA virus, impairs the mitochondrial network and dynamics predominantly through the UL12.5 gene. We demonstrated that HSV-1 infection induced a remodeling of mitochondrial shape, resulting in a fragmentation of the mitochondria associated with a decrease in their volume and an increase in their sphericity. This damage leads to the release of mitochondrial DNA (mtDNA) to the cytosol. By generating a stable THP-1 cell line expressing the DNase I-mCherry fusion protein and a THP-1 cell line specifically depleted of mtDNA upon ethidium bromide treatment, we showed that cytosolic mtDNA contributes to type I interferon and APOBEC3A upregulation. This was confirmed by using an HSV-1 strain (KOS37 UL98-SPA) with a deletion of the UL12.5 gene that impaired its ability to induce mtDNA stress. Furthermore, by using an inhibitor of RNA polymerase III, we demonstrated that upon HSV-1 infection, cytosolic mtDNA enhanced type I interferon induction through the RNA polymerase III/RIG-I pathway. APOBEC3A was in turn induced by interferon. Deep sequencing analyses of cytosolic mtDNA mutations revealed an APOBEC3A signature predominantly in the 5'TpCpG context. These data demonstrate that upon HSV-1 infection, the mitochondrial network is disrupted, leading to the release of mtDNA and ultimately to its catabolism through APOBEC3-induced mutations. IMPORTANCE Herpes simplex virus 1 (HSV-1) impairs the mitochondrial network through the viral protein UL12.5. This leads to the fusion of mitochondria and simultaneous release of mitochondrial DNA (mtDNA) in a mouse model. We have shown that released mtDNA is recognized as a danger signal, capable of stimulating signaling pathways and inducing the production of proinflammatory cytokines. The expression of the human cytidine deaminase APOBEC3A is highly upregulated by interferon responses. This enzyme catalyzes the deamination of cytidine to uridine in single-stranded DNA substrates, resulting in the catabolism of edited DNA. Using human cell lines deprived of mtDNA and viral strains deficient in UL12, we demonstrated the implication of mtDNA in the production of interferon and APOBEC3A expression during viral infection. We have shown that HSV-1 induces mitochondrial network fragmentation in a human model and confirmed the implication of RNA polymerase III/RIG-I signaling in the capture of cytosolic mtDNA.

Heparanase-Induced Activation of AKT Stabilizes ß-Catenin and Modulates Wnt/ß-Catenin Signaling during Herpes Simplex Virus 1 Infection.

Under pathological conditions like herpes simplex virus 1 (HSV-1) infection, host-pathogen interactions lead to major reconstruction of the host protein network, which contributes to the dysregulation of signaling pathways and disease onset. Of note is the upregulation of a multifunctional host protein, heparanase (HPSE), following infection, which serves as a mediator in HSV-1 replication. In this study, we identify a novel function of HPSE and highlight it as a key regulator of ß-catenin signal transduction. The regulatory role of HPSE on the activation, nuclear translocation, and signal transduction of ß-catenin disrupts cellular homeostasis and establishes a pathogenic environment that promotes viral replication. Under normal physiological conditions, ß-catenin is bound to a group of proteins, referred to as the destruction complex, and targeted for ubiquitination and, ultimately, degradation. We show that virus-induced upregulation of HPSE leads to the activation of Akt and subsequent stabilization and activation of ß-catenin through (i) the release of ß-catenin from the destruction complex, and (ii) direct phosphorylation of ß-catenin at Ser552. This study also provides an in-depth characterization of the proviral role of ß-catenin signaling during HSV-1 replication using physiologically relevant cell lines and in vivo models of ocular infection. Furthermore, pharmacological inhibitors of this pathway generated a robust antiviral state against multiple laboratory and clinical strains of HSV-1. Collectively, our findings assign a novel regulatory role to HPSE as a driver of ß-catenin signaling in HSV-1 infection. IMPORTANCE Heparanase (HPSE) and ß-catenin have independently been implicated in regulating key pathophysiological processes, including neovascularization, angiogenesis, and inflammation; however, the relationship between the two proteins has remained elusive thus far. For that reason, characterizing this relationship is crucial and can lead to the development of novel therapeutics. For HSV-1 specifically, current antivirals are not able to abolish the virus from the host, leaving patients susceptible to episodes of viral reactivation. Identifying a host-based intervention can provide a better alternative with enhanced efficacy and sustained relief.

In Situ Cancer Vaccination and Immunovirotherapy Using Oncolytic HSV.

Herpes simplex virus (HSV) can be genetically altered to acquire oncolytic properties so that oncolytic HSV (oHSV) preferentially replicates in and kills cancer cells, while sparing normal cells, and inducing anti-tumor immune responses. Over the last three decades, a better understanding of HSV genes and functions, and improved genetic-engineering techniques led to the development of oHSV as a novel immunovirotherapy. The concept of in situ cancer vaccination (ISCV) was first introduced when oHSV was found to induce a specific systemic anti-tumor immune response with an abscopal effect on non-injected tumors, in the process of directly killing tumor cells. Thus, the use of oHSV for tumor vaccination in situ is antigen-agnostic. The research and development of oHSVs have moved rapidly, with the field of oncolytic viruses invigorated by the FDA/EMA approval of oHSV talimogene laherparepvec in 2015 for the treatment of advanced melanoma. Immunovirotherapy can be enhanced by arming oHSV with immunomodulatory transgenes and/or using them in combination with other chemotherapeutic and immunotherapeutic agents. This review offers an overview of the development of oHSV as an agent for ISCV against solid tumors, describing the multitude of different oHSVs and their efficacy in immunocompetent mouse models and in clinical trials.

PML-NB-dependent type I interferon memory results in a restricted form of HSV latency.

Herpes simplex virus (HSV) establishes latent infection in long-lived neurons. During initial infection, neurons are exposed to multiple inflammatory cytokines but the effects of immune signaling on the nature of HSV latency are unknown. We show that initial infection of primary murine neurons in the presence of type I interferon (IFN) results in a form of latency that is restricted for reactivation. We also find that the subnuclear condensates, promyelocytic leukemia nuclear bodies (PML-NBs), are absent from primary sympathetic and sensory neurons but form with type I IFN treatment and persist even when IFN signaling resolves. HSV-1 genomes colocalize with PML-NBs throughout a latent infection of neurons only when type I IFN is present during initial infection. Depletion of PML prior to or following infection does not impact the establishment latency; however, it does rescue the ability of HSV to reactivate from IFN-treated neurons. This study demonstrates that viral genomes possess a memory of the IFN response during de novo infection, which results in differential subnuclear positioning and ultimately restricts the ability of genomes to reactivate.

Parallel G-quadruplexes recruit the HSV-1 transcription factor ICP4 to promote viral transcription in herpes virus-infected human cells.

G-quadruplexes (G4s) are four-stranded nucleic acid structures abundant at gene promoters. They can adopt several distinctive conformations. G4s have been shown to form in the herpes simplex virus-1 (HSV-1) genome during its viral cycle. Here by cross-linking/pull-down assay we identified ICP4, the major HSV-1 transcription factor, as the protein that most efficiently interacts with viral G4s during infection. ICP4 specific and direct binding and unfolding of parallel G4s, including those present in HSV-1 immediate early gene promoters, induced transcription in vitro and in infected cells. This mechanism was also exploited by ICP4 to promote its own transcription. Proximity ligation assay allowed visualization of G4-protein interaction at the single selected G4 in cells. G4 ligands inhibited ICP4 binding to G4s. Our results indicate the existence of a well-defined G4-viral protein network that regulates the productive HSV-1 cycle. They also point to G4s as elements that recruit transcription factors to activate transcription in cells.

Mutational pressure by host APOBEC3s more strongly affects genes expressed early in the lytic phase of herpes simplex virus-1 (HSV-1) and human polyomavirus (HPyV) infection.

Herpes-Simplex Virus 1 (HSV-1) infects most humans when they are young, sometimes with fatal consequences. Gene expression occurs in a temporal order upon lytic HSV-1 infection: immediate early (IE) genes are expressed, then early (E) genes, followed by late (L) genes. During this infection cycle, the HSV-1 genome has the potential for exposure to APOBEC3 (A3) proteins, a family of cytidine deaminases that cause C>U mutations on single-stranded DNA (ssDNA), often resulting in a C>T transition. We developed a computational model for the mutational pressure of A3 on the lytic cycle of HSV-1 to determine which viral kinetic gene class is most vulnerable to A3 mutations. Using in silico stochastic methods, we simulated the infectious cycle under varying intensities of A3 mutational pressure. We found that the IE and E genes are more vulnerable to A3 than L genes. We validated this model by analyzing the A3 evolutionary footprints in 25 HSV-1 isolates. We find that IE and E genes have evolved to underrepresent A3 hotspot motifs more so than L genes, consistent with greater selection pressure on IE and E genes. We extend this model to two-step infections, such as those of polyomavirus, and find that the same pattern holds for over 25 human Polyomavirus (HPyVs) genomes. Genes expressed earlier during infection are more vulnerable to mutations than those expressed later.

Novel XTENylated AWRK6 analog with hypoglycemic activity, and anti-HSV-2 potential in combination with double shRNA.

AIMS: To design and evaluate a novel AWRK6 peptide-based long-acting GLP-1 receptor agonist (GLP-1RA) conjugated a recombinant polyethylene glycol mimetic (XTEN protein) with significant therapeutic potential on type 2 diabetes mellitus (T2DM) alone as well as Herpes simplex virus type 2 (HSV-2) infection in combination with double shRNA. MAIN METHODS: First, four AWRK6 analogs (termed XA-1 to XA-4) were designed and produced by solid phase synthesis strategy. Further surface plasmon resonance (SPR) measurement and in vitro cAMP accumulation assay were performed to detect the GLP-1R binding affinities and GLP-1R activation, respectively. The in vivo efficacy evaluation including pharmacokinetic test, oral glucose tolerance test (OGTT), hypoglycemic duration test and chronic pharmacodynamics study in rodent animals were all carefully performed. KEY FINDINGS: Four XA peptides were synthesized with purity >99%. High binding affinity as well as activation potency of XA-4 for GLP-1R were demonstrated by SPR and cell-based luciferase reporter assay, respectively. Additionally, XA-4 exhibited the long-lasting antidiabetic effects in the multiple OGTTs, hypoglycemic duration test and chronic study in mice. Furthermore, combined treatment of XA-4 and double shRNA (D-shRNA) achieved potent antiviral effects in HSV-2 infected HEK293 cells. SIGNIFICANCE: XA-4 exhibited promising pharmaceutical potential to be a therapeutic drug for treating T2D, and also held potential to against the HSV-2 infection, which is really an accidental discovery. The strategy of recombinant XTENylation can also be applied to other peptides or small molecules for the development of long-acting therapeutic drugs.

Species-Specific Deamidation of RIG-I Reveals Collaborative Action between Viral and Cellular Deamidases in HSV-1 Lytic Replication.

Retinoic acid-inducible gene I (RIG-I) is a sensor that recognizes cytosolic double-stranded RNA derived from microbes to induce host immune response. Viruses, such as herpesviruses, deploy diverse mechanisms to derail RIG-I-dependent innate immune defense. In this study, we discovered that mouse RIG-I is intrinsically resistant to deamidation and evasion by herpes simplex virus 1 (HSV-1). Comparative studies involving human and mouse RIG-I indicate that N495 of human RIG-I dictates species-specific deamidation by HSV-1 UL37. Remarkably, deamidation of the other site, N549, hinges on that of N495, and it is catalyzed by cellular phosphoribosylpyrophosphate amidotransferase (PPAT). Specifically, deamidation of N495 enables RIG-I to interact with PPAT, leading to subsequent deamidation of N549. Collaboration between UL37 and PPAT is required for HSV-1 to evade RIG-I-mediated antiviral immune response. This work identifies an immune regulatory role of PPAT in innate host defense and establishes a sequential deamidation event catalyzed by distinct deamidases in immune evasion.IMPORTANCE Herpesviruses are ubiquitous pathogens in human and establish lifelong persistence despite host immunity. The ability to evade host immune response is pivotal for viral persistence and pathogenesis. In this study, we investigated the evasion, mediated by deamidation, of species-specific RIG-I by herpes simplex virus 1 (HSV-1). Our findings uncovered a collaborative and sequential action between viral deamidase UL37 and a cellular glutamine amidotransferase, phosphoribosylpyrophosphate amidotransferase (PPAT), to inactivate RIG-I and mute antiviral gene expression. PPAT catalyzes the rate-limiting step of the de novo purine synthesis pathway. This work describes a new function of cellular metabolic enzymes in host defense and viral immune evasion.

LAMP2 deficiency attenuates the neurodegeneration markers induced by HSV-1 infection.

Mounting evidence suggests a major role of infectious agents in the pathogenesis of sporadic Alzheimer's disease (AD). Among them, herpes simplex virus type 1 (HSV-1) infection has emerged as a major factor in the etiology of AD. HSV-1 is able to induce some of the main alterations of the disease such as hyperphosphorylation of tau protein and accumulation of amyloid-ß peptide. Functional genomic analysis of a cell model of HSV-1 infection and oxidative stress developed in our laboratory revealed lysosomal system to be the main pathway altered, and the lysosome-associated membrane protein 2 (LAMP2) gene one of the most strongly modulated genes. The aim of this work is to study LAMP2 as an AD candidate gene and to investigate its role in the neurodegeneration induced by HSV-1 using a LAMP2 knockdown cell model. LAMP2 deficiency led to a significant reduction of viral DNA replication and formation of infectious particles. In addition, tau hyperphosphorylation and inhibition of Aß secretion induced by the virus were attenuated by the absence of LAMP2. Finally, genetic association studies revealed LAMP2 genetic variants to be associated with AD risk. In summary, our data indicate that LAMP2 could be a suitable candidate to mediate the AD-like phenotype caused by HSV-1.