Bioprospecting the American Alligator Peptidome for antiviral peptides against Venezuelan equine encephalitis virus.

The innate immune protection provided by cationic antimicrobial peptides (CAMPs) has been shown to extend to antiviral activity, with putative mechanisms of action including direct interaction with host cells or pathogen membranes. The lack of therapeutics available for the treatment of viruses such as Venezuelan equine encephalitis virus (VEEV) underscores the urgency of novel strategies for antiviral discovery. American alligator plasma has been shown to exhibit strong in vitro antibacterial activity, and functionalized hydrogel particles have been successfully employed for the identification of specific CAMPs from alligator plasma. Here, a novel bait strategy in which particles were encapsulated in membranes from either healthy or VEEV-infected cells was implemented to identify peptides preferentially targeting infected cells for subsequent evaluation of antiviral activity. Statistical analysis of peptide identification results was used to select five candidate peptides for testing, of which one exhibited a dose-dependent inhibition of VEEV and also significantly inhibited infectious titers. Results suggest our bioprospecting strategy provides a versatile platform that may be adapted for antiviral peptide identification from complex biological samples.

Resveratrol Inhibits Venezuelan Equine Encephalitis Virus Infection by Interfering with the AKT/GSK Pathway.

The host proteins Protein Kinase B (AKT) and glycogen synthase kinase-3 (GSK-3) are associated with multiple neurodegenerative disorders. They are also important for the replication of Venezuelan equine encephalitis virus (VEEV), thereby making the AKT/GSK-3 pathway an attractive target for developing anti-VEEV therapeutics. Resveratrol, a natural phytochemical, has been shown to substantially inhibit the AKT pathway. Therefore, we attempted to explore whether it exerts any antiviral activity against VEEV. In this study, we utilized green fluorescent protein (GFP)- and luciferase-encoding recombinant VEEV to determine the cytotoxicity and antiviral efficacy via luciferase reporter assays, flow cytometry, and immunofluorescent assays. Our results indicate that resveratrol treatment is capable of inhibiting VEEV replication, resulting in increased viability of Vero and U87MG cells as well as reduced virion production and viral RNA contents within host cells for at least 48 h with a single treatment. Furthermore, the suppression of apoptotic signaling adaptors, caspase-3, caspase-7, and annexin V may also be implicated in resveratrol-mediated antiviral activity. We found that decreased phosphorylation of the AKT/GSK-3 pathway, mediated by resveratrol, can be triggered during the early stages of VEEV infection, suggesting that resveratrol disrupts the viral replication cycle and consequently promotes cell survival. Finally, molecular docking and dynamics simulation studies revealed that resveratrol can directly bind to VEEV glycoproteins, which may interfere with virus attachment and entry. In conclusion, our results suggest that resveratrol exerts inhibitory activity against VEEV infection and upon further modification could be a useful compound to study in neuroprotective research and veterinary sciences.

Protein Kinase C subtype δ interacts with Venezuelan equine encephalitis virus capsid protein and regulates viral RNA binding through modulation of capsid phosphorylation.

Protein phosphorylation plays an important role during the life cycle of many viruses. Venezuelan equine encephalitis virus (VEEV) capsid protein has recently been shown to be phosphorylated at four residues. Here those studies are extended to determine the kinase responsible for phosphorylation and the importance of capsid phosphorylation during the viral life cycle. Phosphorylation site prediction software suggests that Protein Kinase C (PKC) is responsible for phosphorylation of VEEV capsid. VEEV capsid co-immunoprecipitated with PKCδ, but not other PKC isoforms and siRNA knockdown of PKCδ caused a decrease in viral replication. Furthermore, knockdown of PKCδ by siRNA decreased capsid phosphorylation. A virus with capsid phosphorylation sites mutated to alanine (VEEV CPD) displayed a lower genomic copy to pfu ratio than the parental virus; suggesting more efficient viral assembly and more infectious particles being released. RNA:capsid binding was significantly increased in the mutant virus, confirming these results. Finally, VEEV CPD is attenuated in a mouse model of infection, with mice showing increased survival and decreased clinical signs as compared to mice infected with the parental virus. Collectively our data support a model in which PKCδ mediated capsid phosphorylation regulates viral RNA binding and assembly, significantly impacting viral pathogenesis.

EGR1 upregulation following Venezuelan equine encephalitis virus infection is regulated by ERK and PERK pathways contributing to cell death.

Venezuelan equine encephalitis virus (VEEV) is a neurotropic virus that causes significant disease in both humans and equines. Here we characterized the impact of VEEV on signaling pathways regulating cell death in human primary astrocytes. VEEV productively infected primary astrocytes and caused an upregulation of early growth response 1 (EGR1) gene expression at 9 and 18 h post infection. EGR1 induction was dependent on extracellular signal-regulated kinase1/2 (ERK1/2) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), but not on p38 mitogen activated protein kinase (MAPK) or phosphoinositide 3-kinase (PI3K) signaling. Knockdown of EGR1 significantly reduced VEEV-induced apoptosis and impacted viral replication. Knockdown of ERK1/2 or PERK significantly reduced EGR1 gene expression, dramatically reduced viral replication, and increased cell survival as well as rescued cells from VEEV-induced apoptosis. These data indicate that EGR1 activation and subsequent cell death are regulated through ERK and PERK pathways in VEEV infected primary astrocytes.

Magnetic Nanotrap Particles Preserve the Stability of Venezuelan Equine Encephalitis Virus in Blood for Laboratory Detection.

Most of the modern techniques used for identification of viral-induced disease are based on identification of viral antigens and/or nucleic acids in patient's blood. Diagnosis in the field or in remote locations can be challenging and alternatively samples are shipped to diagnostic labs for testing. Shipments must occur under controlled temperature conditions to prevent loss of sample integrity. We have tested the ability of magnetic Nanotrap® (NT) particles to improve stability and detection of Venezuelan equine encephalitis virus (VEEV), viral capsid protein, and viral genomic RNA in whole human blood at elevated temperature and prolonged storage conditions. NT particles have previously been shown to capture and enrich multiple pathogens including respiratory syncytial virus, influenza virus, coronavirus, and Rift Valley fever virus. Our study indicates that samples incubated with NT particles had detectable levels of infectious VEEV in blood equal to or greater than samples without NT treatment across all temperatures. Viral RNA detection was increased in the presence of NT particles at later time points (72 h) and higher temperature (40°C) conditions. Likewise, detection of VEEV capsid protein was enhanced in the presence of NT particles up to 72 h at 40°C. Finally, we intranasally infected C3H mice with TC-83, the live attenuated vaccine strain of VEEV, and demonstrated that NT particles could substantially increase the detection of VEEV capsid in infected blood incubated up to 72 h at 40°C. Samples without NT particles had undetectable capsid protein levels. Taken together, our data demonstrate the ability of NT particles to preserve and enable detection of VEEV in human and mouse blood samples over time and at elevated temperatures.

Author Correction: Novel inhibitors targeting Venezuelan equine encephalitis virus capsid protein identified using In Silico Structure-Based-Drug-Design.

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Repurposed FDA-Approved drug sorafenib reduces replication of Venezuelan equine encephalitis virus and other alphaviruses.

The New World alphaviruses -Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, and WEEV respectively) - cause a febrile disease that is often lethal in equines and children and leads to long-term neurological sequelae in survivors. Endemic to the Americas, epizootic outbreaks of the three viruses occur sporadically in the continental United States. All three viruses aerosolize readily, replicate to high titers in cell culture, and have low infectious doses. Additionally, there are no FDA-approved vaccines or therapeutics for human use. To address the therapeutic gap, a high throughput assay utilizing a luciferase reporter virus, TC83-luc, was performed to screen a library of commercially available, FDA-approved drugs for antiviral activity. From a group of twenty compounds found to significantly decrease luminescence, the carcinoma therapeutic sorafenib inhibited replication of VEEV-TC83 and TrD in vitro. Additionally, sorafenib inhibited replication of EEEV and two Old World alphaviruses, Sindbis virus and chikungunya virus, at 8 and 16 h post-infection. Sorafenib caused no toxicity in Vero cells, and coupled with a low EC50 value, yielded a selectivity index of >19. Mechanism of actions studies suggest that sorafenib inhibited viral translation through dephosphorylation of several key proteins, including eIF4E and p70S6K, leading to a reduction in viral protein production and overall viral replication.

Venezuelan Equine Encephalitis Virus Capsid Implicated in Infection-Induced Cell Cycle Delay in vitro.

Venezuelan equine encephalitis virus (VEEV) is a positive sense, single-stranded RNA virus and member of the New World alphaviruses. It causes a biphasic febrile illness that can be accompanied by central nervous system involvement and moderate morbidity in humans and severe mortality in equines. The virus has a history of weaponization, lacks FDA-approved therapeutics and vaccines in humans, and is considered a select agent. Like other RNA viruses, VEEV replicates in the cytoplasm of infected cells and eventually induces apoptosis. The capsid protein, which contains a nuclear localization and a nuclear export sequence, induces a shutdown of host transcription and nucleocytoplasmic trafficking. Here we show that infection with VEEV causes a dysregulation of cell cycling and a delay in the G0/G1 phase in Vero cells and U87MG astrocytes. Cells infected with VEEV encoding a capsid NLS mutant or treated with the capsid-importin α interaction inhibitor G281-1485 were partially rescued from this cell cycle dysregulation. Pathway analysis of previously published RNA-sequencing data from VEEV infected U87MG astrocytes identified alterations of canonical pathways involving cell cycle, checkpoint regulation, and proliferation. Multiple cyclins including cyclin D1, cyclin A2 and cyclin E2 and other regulators of the cell cycle were downregulated in infected cells in a capsid NLS dependent manner. Loss of Rb phosphorylation, which is a substrate for cyclin/cdk complexes was also observed. These data demonstrate the importance of capsid nuclear localization and/or importin α binding for inducing cell cycle arrest and transcriptional downregulation of key cell cycle regulators.

Novel inhibitors targeting Venezuelan equine encephalitis virus capsid protein identified using In Silico Structure-Based-Drug-Design.

Therapeutics are currently unavailable for Venezuelan equine encephalitis virus (VEEV), which elicits flu-like symptoms and encephalitis in humans, with an estimated 14% of cases resulting in neurological disease. Here we identify anti-VEEV agents using in silico structure-based-drug-design (SBDD) for the first time, characterising inhibitors that block recognition of VEEV capsid protein (C) by the host importin (IMP) α/ß1 nuclear transport proteins. From an initial screen of 1.5 million compounds, followed by in silico refinement and screening for biological activity in vitro, we identified 21 hit compounds which inhibited IMPα/ß1:C binding with IC50s as low as 5 µM. Four compounds were found to inhibit nuclear import of C in transfected cells, with one able to reduce VEEV replication at µM concentration, concomitant with reduced C nuclear accumulation in infected cells. Further, this compound was inactive against a mutant VEEV that lacks high affinity IMPα/ß1:C interaction, supporting the mode of its antiviral action to be through inhibiting C nuclear localization. This successful application of SBDD paves the way for lead optimization for VEEV antivirals, and is an exciting prospect to identify inhibitors for the many other viral pathogens of significance that require IMPα/ß1 in their infectious cycle.