Exposure of negative-sense viral RNA in the cytoplasm initiates innate immunity to West Nile virus.

For many RNA viruses, immunity is triggered when RIG-I-like receptors (RLRs) detect viral RNA. However, only a minority of infected cells undergo innate immune activation. By examining these "first responder" cells during West Nile virus infection, we found that specific accumulation of anti- genomic negative-sense viral RNA (-vRNA) underlies innate immune activation and that RIG-I preferentially interacts with -vRNA. However, flaviviruses sequester -vRNA into membrane-bound replication compartments away from cytosolic sensors. We found that single-stranded -vRNA accumulates outside of replication compartments in "first responder" cells, rendering it accessible to RLRs. Exposure of this -vRNA occurs at late timepoints of infection, is linked to viral assembly, and depends on the expression of viral structural proteins. These findings reveal that while most infected cells replicate high levels of vRNA, release of -vRNA from replication compartments during assembly occurs at low frequency and is critical for initiation of innate immunity during flavivirus infection.

Oligodendrocyte-derived IL-33 functions as a microglial survival factor during neuroinvasive flavivirus infection.

In order to recover from infection, organisms must balance robust immune responses to pathogens with the tolerance of immune-mediated pathology. This balance is particularly critical within the central nervous system, whose complex architecture, essential function, and limited capacity for self-renewal render it susceptible to both pathogen- and immune-mediated pathology. Here, we identify the alarmin IL-33 and its receptor ST2 as critical for host survival to neuroinvasive flavivirus infection. We identify oligodendrocytes as the critical source of IL-33, and microglia as the key cellular responders. Notably, we find that the IL-33/ST2 axis does not impact viral control or adaptive immune responses; rather, it is required to promote the activation and survival of microglia. In the absence of intact IL-33/ST2 signaling in the brain, neuroinvasive flavivirus infection triggered aberrant recruitment of monocyte-derived peripheral immune cells, increased neuronal stress, and neuronal cell death, effects that compromised organismal survival. These findings identify IL-33 as a critical mediator of CNS tolerance to pathogen-initiated immunity and inflammation.

Oligodendrocyte-derived IL-33 functions as a microglial survival factor during neuroinvasive flavivirus infection.

In order to recover from infection, organisms must balance robust immune responses to pathogens with the tolerance of immune-mediated pathology. This balance is particularly critical within the central nervous system, whose complex architecture, essential function, and limited capacity for self-renewal render it susceptible to both pathogen- and immune-mediated pathology. Here, we identify the alarmin IL-33 and its receptor ST2 as critical for host survival to neuroinvasive flavivirus infection. We identify oligodendrocytes as the critical source of IL-33, and microglia as the key cellular responders. Notably, we find that the IL-33/ST2 axis does not impact viral control or adaptive immune responses; rather, it is required to promote the activation and survival of microglia. In the absence of intact IL-33/ST2 signaling in the brain, neuroinvasive flavivirus infection triggered aberrant recruitment of monocyte-derived peripheral immune cells, increased neuronal stress, and neuronal cell death, effects that compromised organismal survival. These findings identify IL-33 as a critical mediator of CNS tolerance to pathogen-initiated immunity and inflammation.

Pathophysiology of reinfection by exogenous HSV-1 is driven by heparanase dysfunction.

Limited knowledge exists on exogenous DNA virus reinfections. Herpes simplex virus-1 (HSV-1), a prototype DNA virus, causes multiple human diseases including vision-threatening eye infections. While reinfection with an exogenous HSV-1 strain is considered plausible, little is known about the underlying mechanisms governing its pathophysiology in a host. Heparanase (HPSE), a host endoglycosidase, when up-regulated by HSV-1 infection dictates local inflammatory response by destabilizing tissue architecture. Here, we demonstrate that HSV-1 reinfection in mice causes notable pathophysiology in wild-type controls compared to the animals lacking HPSE. The endoglycosidase promotes infected cell survival and supports a pro-disease environment. In contrast, lack of HPSE strengthens intrinsic immunity by promoting cytokine expression, inducing necroptosis of infected cells, and decreasing leukocyte infiltration into the cornea. Collectively, we report that immunity from a recent prior infection fails to abolish disease manifestation during HSV-1 reinfection unless HPSE is rendered inactive.

ADAR1 mutation causes ZBP1-dependent immunopathology.

The RNA-editing enzyme ADAR1 is essential for the suppression of innate immune activation and pathology caused by aberrant recognition of self-RNA, a role it carries out by disrupting the duplex structure of endogenous double-stranded RNA species1,2. A point mutation in the sequence encoding the Z-DNA-binding domain (ZBD) of ADAR1 is associated with severe autoinflammatory disease3-5. ZBP1 is the only other ZBD-containing mammalian protein6, and its activation can trigger both cell death and transcriptional responses through the kinases RIPK1 and RIPK3, and the protease caspase 8 (refs. 7-9). Here we show that the pathology caused by alteration of the ZBD of ADAR1 is driven by activation of ZBP1. We found that ablation of ZBP1 fully rescued the overt pathology caused by ADAR1 alteration, without fully reversing the underlying inflammatory program caused by this alteration. Whereas loss of RIPK3 partially phenocopied the protective effects of ZBP1 ablation, combined deletion of caspase 8 and RIPK3, or of caspase 8 and MLKL, unexpectedly exacerbated the pathogenic effects of ADAR1 alteration. These findings indicate that ADAR1 is a negative regulator of sterile ZBP1 activation, and that ZBP1-dependent signalling underlies the autoinflammatory pathology caused by alteration of ADAR1.

Safety, efficacy and delivery of multiple nucleoside analogs via drug encapsulated carbon (DECON) based sustained drug release platform.

Acyclovir and similar nucleoside analogs form an essential frontline treatment for various herpesvirus infections of the eye. However, these drugs have low ocular retention when delivered topically and need to be administered several times every day. We have previously demonstrated that acyclovir loaded into activated carbon can be used to significantly decrease dosage frequency in a murine model of ocular infection. In this study, we demonstrate that other nucleoside analogs such as ganciclovir, penciclovir and famciclovir have excellent loading and release profile similar to acyclovir. Similarly we also demonstrate that nucleoside analog loaded carbons termed DECON are effective at very low concentrations in treating active viral infection of human corneal epithelial cells. In this study, using a variety of techniques to evaluate corneal dryness, nerve sensitivity, intraocular pressure, corneal thickness, and somatic inflammation, we report that DECON is well tolerated after administration three times daily over the course of four weeks.

Intrinsic Antiviral Activity of Optineurin Prevents Hyperproliferation of a Primary Herpes Simplex Virus Type 2 Infection.

Very little knowledge exists on virus-specific host cell intrinsic mechanisms that prevent hyperproliferation of primary HSV type 2 (HSV-2) genital infections. In this study, we provide evidence that the Nemo-related protein, optineurin (OPTN), plays a key role in restricting HSV-2 infection both in vitro and in vivo. Contrary to previous reports regarding the proviral role of OPTN during Sendai virus infection, we demonstrate that lack of OPTN in cells causes enhanced virus production. OPTN deficiency negatively affects the host autophagy response and results in a marked reduction of CCL5 induction. OPTN knockout (OPTN-/-) mice display exacerbated genital disease and dysregulated T cell frequencies in infected tissues and lymph nodes. A human transcriptomic profile dataset provides further credence that a strong positive correlation exists between CCL5 upregulation and OPTN expression during HSV-2 genital infection. Our findings underscore a previously unknown OPTN/CCL5 nexus that restricts hyperproliferative spread of primary HSV-2 infection, which may constitute an intrinsic host defense mechanism against herpesviruses in general.

mTORC2 confers neuroprotection and potentiates immunity during virus infection.

Herpes simplex virus type-1 (HSV-1) causes ocular and orofacial infections. In rare cases, HSV-1 can cause encephalitis, which leads to permanent brain injuries, memory loss or even death. Host factors protect humans from viral infections by activating the immune response. However, factors that confer neuroprotection during viral encephalitis are poorly understood. Here we show that mammalian target of rapamycin complex 2 (mTORC2) is essential for the survival of experimental animals after ocular HSV-1 infection in vivo. We find the loss of mTORC2 causes systemic HSV-1 infection due to defective innate and adaptive immune responses, and increased ocular and neuronal cell death that turns lethal for the infected mice. Furthermore, we find that mTORC2 mediated cell survival channels through the inactivation of the proapoptotic factor FoxO3a. Our results demonstrate how mTORC2 potentiates host defenses against viral infections and implicate mTORC2 as a necessary factor for survival of the infected host.

OPTN is a host intrinsic restriction factor against neuroinvasive HSV-1 infection.

Fast-replicating neurotropic herpesviruses exemplified by herpes simplex virus-1 (HSV-1) naturally infect the central nervous system (CNS). However, most individuals intrinsically suppress the virus during a primary infection and preclude it from significantly damaging the CNS. Optineurin (OPTN) is a conserved autophagy receptor with little understanding of its role in neurotropic viral infections. We show that OPTN selectively targets HSV-1 tegument protein, VP16, and the fusion glycoprotein, gB, to degradation by autophagy. OPTN-deficient mice challenged with HSV-1 show significant cognitive decline and susceptibility to lethal CNS infection. OPTN deficiency unveils severe consequences for recruitment of adaptive immunity and suppression of neuronal necroptosis. Ocular HSV-1 infection is lethal without OPTN and is rescued using a necroptosis inhibitor. These results place OPTN at the crux of neuronal survival from potentially lethal CNS viral infections.

Heparan Sulfate Binding Cationic Peptides Restrict SARS-CoV-2 Entry.

A novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. While the world is striving for a treatment modality against SARS-CoV-2, our understanding about the virus entry mechanisms may help to design entry inhibitors, which may help to limit the virus spreading. Owing to the importance of cellular ACE2 and heparan sulfate in SARS-CoV-2 entry, we aimed to evaluate the efficacy of cationic G1 and G2 peptides in virus entry inhibition. In silico binding affinity studies revealed possible binding sites of G1 and G2 peptides on HS and ACE2, which are required for the spike-HS and spike-ACE2 interactions. Prophylactic treatment of G1 and G2 peptide was also proved to decrease the cell surface HS, an essential virus entry receptor. With these two mechanisms we confirm the possible use of cationic peptides to inhibit the entry of SARS-CoV-2.

Entry receptor bias in evolutionarily distant HSV-1 clinical strains drives divergent ocular and nervous system pathologies.

PURPOSE: Herpes simplex virus-1 (HSV-1) infection leads to varying pathologies including the development of ocular lesions, stromal keratitis and encephalitis. While the role for host immunity in disease progression is well understood, the contribution of genetic variances in generating preferential viral entry receptor usage and resulting immunopathogenesis in humans are not known. METHODS: Ocular cultures were obtained from patients presenting distinct pathologies of herpes simplex keratitis (HSK). Next-generation sequencing and subsequent analysis characterized genetic variances among the strains and estimated evolutionary divergence. Murine model of ocular infection was used to assess phenotypic contributions of strain variances on damage to the ocular surface and propagation of innate immunity. Flow cytometry of eye tissue identified differential recruitment of immune cell populations, cytokine array probed for programming of local immune response in the draining lymph node and histology was used to assess inflammation of the trigeminal ganglion (TG). Ex-vivo corneal cultures and in-vitro studies elucidated the role of genetic variances in altering host-pathogen interactions, leading to divergent host responses. RESULTS: Phylogenetic analysis of the clinical isolates suggests evolutionary divergence among currently circulating HSV-1 strains. Mutations causing alterations in functional host interactions were identified, particularly in viral entry glycoproteins which generated a receptor bias to herpesvirus entry mediator, an immune modulator involved in immunopathogenic diseases like HSK, leading to exacerbated ocular surface pathologies and heightened viral burden in the TG and brainstem. CONCLUSIONS: Our data suggests receptor bias resulting from genetic variances in clinical strains may dictate disease severity and treatment outcome.

Disruption of innate defense responses by endoglycosidase HPSE promotes cell survival.

The drive to withstand environmental stresses and defend against invasion is a universal trait extant in all forms of life. While numerous canonical signaling cascades have been characterized in detail, it remains unclear how these pathways interface to generate coordinated responses to diverse stimuli. To dissect these connections, we followed heparanase (HPSE), a protein best known for its endoglycosidic activity at the extracellular matrix but recently recognized to drive various forms of late-stage disease through unknown mechanisms. Using herpes simplex virus-1 (HSV-1) infection as a model cellular perturbation, we demonstrate that HPSE acts beyond its established enzymatic role to restrict multiple forms of cell-intrinsic defense and facilitate host cell reprogramming by the invading pathogen. We reveal that cells devoid of HPSE are innately resistant to infection and counteract viral takeover through multiple amplified defense mechanisms. With a unique grasp of the fundamental processes of transcriptional regulation and cell death, HPSE represents a potent cellular intersection with broad therapeutic potential.

Standalone or combinatorial phenylbutyrate therapy shows excellent antiviral activity and mimics CREB3 silencing.

Herpesviruses are ubiquitous human pathogens that tightly regulate many cellular pathways including the unfolded protein response to endoplasmic reticulum (ER) stress. Pharmacological modulation of this pathway results in the inhibition of viral replication. In this study, we tested 4-phenylbutyrate (PBA), a chemical chaperone-based potent alleviator of ER stress, for its effects on herpes simplex virus (HSV) type 1 infection. Through in vitro studies, we observed that application of PBA to HSV-infected cells results in the down-regulation of a proviral, ER-localized host protein CREB3 and a resultant inhibition of viral protein synthesis. PBA treatment caused viral inhibition in cultured human corneas and human skin grafts as well as murine models of ocular and genital HSV infection. Thus, we propose that this drug can provide an alternative to current antivirals to treat both ocular HSV-1 and genital HSV-2 infections and may be a strong candidate for human trials.

Bacterial Pigment Prodigiosin Demonstrates a Unique Antiherpesvirus Activity That Is Mediated through Inhibition of Prosurvival Signal Transducers.

Herpes simplex virus (HSV) is among the most prevalent viral infections worldwide and remains incurable. While nucleoside analogs are used to relieve symptoms of infection, they suffer from having serious adverse effects and are unable to abolish the virus from the host. Here, we demonstrate a unique antiviral effect of prodigiosin (PG), a natural secondary metabolite produced by Serratia marcescens, on HSV infection. We show that PG naturally exerts antiviral activity against HSV-1 and HSV-2 infections. PG treatment resulted in robust inhibition of viral replication in vitro and ex vivo in cultured porcine corneas. Additionally, PG protected against HSV-1 infection and disease progression in a murine model of ocular infection. In our quest to determine the molecular mechanisms of its antiviral activity, we show that PG specifically inhibits NF-κB and Akt signaling pathways and promotes accelerated cell death in HSV-infected cells. Our findings reveal novel antiviral properties of PG, suggesting its high potential as an alternative treatment for herpetic diseases. They also provide new information on antiviral effects of HSV-bacterial metabolite interactions.IMPORTANCE In this article, we provide a new role for a commonly found bacterial pigment in controlling herpes simplex virus infection, for which diverse and multimodal antiviral agents are needed to prevent drug resistance. Serratia marcescens is a red pigment (prodigiosin)-producing Gram-negative bacillus that is naturally found in soil and water. It is associated with many kinds of human infections, including wound and eye infections, and meningitis. Taking cues from previous studies on prodigiosin, including possible proapoptotic anticancer properties, we investigated how it might affect HSV infection. Interestingly, we found that it is a potent virucidal compound that disrupts host signaling pathways needed for HSV growth and survival. The mode of antiviral action suggests potentially broad activity against enveloped viruses. Our results also indicate that interactions with commensal bacteria may inhibit HSV infection, underscoring the importance of studying these microbial metabolites and their implications for viral pathogenesis and treatment.

Prior inhibition of AKT phosphorylation by BX795 can define a safer strategy to prevent herpes simplex virus-1 infection of the eye.

PURPOSE: To evaluate the prophylactic antiviral efficacy, corneal tolerance and toxicity of topically dosed BX795, a non-nucleoside small-molecule inhibitor of herpes simplex virus type-1 (HSV-1). METHODS: Prophylactic treatment with BX795 was performed both in-vitro on human corneal epithelial cells and in-vivo on mice prior to HSV-1 challenge. Viral burden was evaluated using a standard plaque assay. In a separate experiment, mice were treated topically 3-times daily for 4-weeks with BX795 to evaluate corneal tolerance and toxicity. Phenol-red thread measurements, fluorescein staining and optical coherence tomography (OCT) were used to evaluate tear production, dryness and corneal structural changes. Corneal sensitivity and intraocular pressure were measured using esthesiometery and tonometery respectively. RESULTS: Both in-vitro and in-vivo results showed a robust suppression of HSV-1 infection when treated prophylactically with BX795. The fluorescein stain and phenol-red results for the BX795-treated eyes did not show signs of corneal surface dryness when compared to trifluridine (TFT), an FDA-approved topical antiviral. The OCT measurements showed no signs of structural changes to the cornea suggesting that BX795 treatment was well tolerated without any apparent signs of toxicity or inflammation. The corneal sensitivity of BX795-treated eyes was not significantly different from TFT-treated eyes. No significant increase in the intraocular pressure of BX795-treated mice was observed. CONCLUSIONS: Prophylactic treatment with BX795 protects corneal cells from HSV-1 infection. The antiviral is well-tolerated on murine corneas without any detectable toxicity.

Drug-encapsulated carbon (DECON): A novel platform for enhanced drug delivery.

Current drug-delivery systems are designed primarily for parenteral applications and are either lipid or polymer drug conjugates. In our quest to inhibit herpes simplex virus infection via the compounds found in commonly used cosmetic products, we found that activated carbon particles inhibit infection and, in addition, substantially improve topical delivery and, hence, the efficacy of a common antiviral drug, acyclovir (ACV). Our in vitro studies demonstrate that highly porous carbon structures trapped virions, blocked infection and substantially improved efficacy when ACV was loaded onto them. Also, using murine models of corneal and genital herpes infections, we show that the topical use of drug-encapsulated carbon (DECON) reduced dosing frequency, shortened treatment duration, and exhibited higher therapeutic efficacy than currently approved topical or systemic antivirals alone. DECON is a nontoxic, cost-effective and nonimmunogenic alternative to current topical drug-delivery systems that is uniquely triggered for drug release by virus trapping.

An off-target effect of BX795 blocks herpes simplex virus type 1 infection of the eye.

Herpes simplex virus type 1 (HSV-1) causes recurrent mucocutaneous lesions in the eye that may advance to corneal blindness. Nucleoside analogs exemplified by acyclovir (ACV) form the primary class of antiherpetic drugs, but this class suffers limitations due to the emergence of viral resistance and other side effects. While studying the molecular basis of ocular HSV-1 infection, we observed that BX795, a commonly used inhibitor of TANK-binding kinase 1 (TBK1), strongly suppressed infection by multiple strains of HSV-1 in transformed and primary human cells, cultured human and animal corneas, and a murine model of ocular infection. Our investigations revealed that the antiviral activity of BX795 relies on targeting Akt phosphorylation in infected cells, leading to the blockage of viral protein synthesis. This small-molecule inhibitor, which was also effective against an ACV-resistant HSV-1 strain, shows promise as an alternative to existing drugs and as an effective topical therapy for ocular herpes infection. Collectively, our results obtained using multiple infection models and virus strains establish BX795 as a promising lead compound for broad-spectrum antiviral applications in humans.

Herpes Simplex Virus 1 Induces Phosphorylation and Reorganization of Lamin A/C through the γ134.5 Protein That Facilitates Nuclear Egress.

Herpes simplex virus 1 (HSV-1) remodels nuclear membranes during virus egress. Although the UL31 and UL34 proteins control nucleocapsid transit in infected cells, the molecular interactions required for their function are unclear. Here we report that the γ134.5 gene product of HSV-1 facilitates nucleocapsid release to the cytoplasm through bridging the UL31/UL34 complex, cellular p32, and protein kinase C. Unlike wild-type virus, an HSV mutant devoid of γ134.5 or its amino terminus is crippled for viral growth and release. This is attributable to a defect in virus nuclear egress. In infected cells, wild-type virus recruits protein kinase C to the nuclear membrane and triggers its activation, whereas the γ134.5 mutants fail to exert such an effect. Accordingly, the γ134.5 mutants are unable to induce phosphorylation and reorganization of lamin A/C. When expressed in host cells γ134.5 targets p32 and protein kinase C. Meanwhile, it communicates with the UL31/UL34 complex through UL31. Deletion of the amino terminus from γ134.5 disrupts its activity. These results suggest that disintegration of the nuclear lamina mediated by γ134.5 promotes HSV replication. IMPORTANCE: HSV nuclear egress is a key step that determines the outcome of viral infection. While the nuclear egress complex mediates capsid transit across the nuclear membrane, the regulatory components are not clearly defined in virus-infected cells. We report that the γ134.5 gene product, a virulence factor of HSV-1, facilitates nuclear egress cooperatively with cellular p32, protein kinase C, and the nuclear egress complex. This work highlights a viral mechanism that may contribute to the pathogenesis of HSV infection.

Inactivation of RNA Viruses by Gamma Irradiation: A Study on Mitigating Factors.

Effective inactivation of biosafety level 4 (BSL-4) pathogens is vital in order to study these agents safely. Gamma irradiation is a commonly used method for the inactivation of BSL-4 viruses, which among other advantages, facilitates the study of inactivated yet morphologically intact virions. The reported values for susceptibility of viruses to inactivation by gamma irradiation are sometimes inconsistent, likely due to differences in experimental protocols. We analyzed the effects of common sample attributes on the inactivation of a recombinant vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein and green fluorescent protein. Using this surrogate virus, we found that sample volume and protein content of the sample modulated viral inactivation by gamma irradiation but that air volume within the sample container and the addition of external disinfectant surrounding the sample did not. These data identify several factors which alter viral susceptibility to inactivation and highlight the usefulness of lower biosafety level surrogate viruses for such studies. Our results underscore the need to validate inactivation protocols of BSL-4 pathogens using "worst-case scenario" procedures to ensure complete sample inactivation.