Inhibition of Venezuelan Equine Encephalitis Virus Using Small Interfering RNAs.

Acutely infectious new world alphaviruses such as Venezuelan Equine Encephalitis Virus (VEEV) pose important challenges to the human population due to a lack of effective therapeutic intervention strategies. Small interfering RNAs that can selectively target the viral genome (vsiRNAs) has been observed to offer survival advantages in several in vitro and in vivo models of acute virus infections, including alphaviruses such as Chikungunya virus and filoviruses such as Ebola virus. In this study, novel vsiRNAs that targeted conserved regions in the nonstructural and structural genes of the VEEV genome were designed and evaluated for antiviral activity in mammalian cells in the context of VEEV infection. The data demonstrate that vsiRNAs were able to effectively decrease the infectious virus titer at earlier time points post infection in the context of the attenuated TC-83 strain and the virulent Trinidad Donkey strain, while the inhibition was overcome at later time points. Depletion of Argonaute 2 protein (Ago2), the catalytic component of the RISC complex, negated the inhibitory effect of the vsiRNAs, underscoring the involvement of the siRNA pathway in the inhibition process. Depletion of the RNAi pathway proteins Dicer, MOV10, TRBP2 and Matrin 3 decreased viral load in infected cells, alluding to an impact of the RNAi pathway in the establishment of a productive infection. Additional studies focused on rational combinations of effective vsiRNAs and delivery strategies to confer better in vivo bioavailability and distribution to key target tissues such as the brain can provide effective solutions to treat encephalitic diseases resulting from alphavirus infections.

Inhibitors of Venezuelan Equine Encephalitis Virus Identified Based on Host Interaction Partners of Viral Non-Structural Protein 3.

Venezuelan equine encephalitis virus (VEEV) is a new world alphavirus and a category B select agent. Currently, no FDA-approved vaccines or therapeutics are available to treat VEEV exposure and resultant disease manifestations. The C-terminus of the VEEV non-structural protein 3 (nsP3) facilitates cell-specific and virus-specific host factor binding preferences among alphaviruses, thereby providing targets of interest when designing novel antiviral therapeutics. In this study, we utilized an overexpression construct encoding HA-tagged nsP3 to identify host proteins that interact with VEEV nsP3 by mass spectrometry. Bioinformatic analyses of the putative interactors identified 42 small molecules with the potential to inhibit the host interaction targets, and thus potentially inhibit VEEV. Three inhibitors, tomatidine, citalopram HBr, and Z-VEID-FMK, reduced replication of both the TC-83 strain and the Trinidad donkey (TrD) strain of VEEV by at least 10-fold in astrocytoma, astroglial, and microglial cells. Further, these inhibitors reduced replication of the related New World (NW) alphavirus Eastern equine encephalitis virus (EEEV) in multiple cell types, thus demonstrating broad-spectrum antiviral activity. Time-course assays revealed all three inhibitors reduced both infectious particle production and positive-sense RNA levels post-infection. Further evaluation of the putative host targets for the three inhibitors revealed an interaction of VEEV nsP3 with TFAP2A, but not eIF2S2. Mechanistic studies utilizing siRNA knockdowns demonstrated that eIF2S2, but not TFAP2A, supports both efficient TC-83 replication and genomic RNA synthesis, but not subgenomic RNA translation. Overall, this work reveals the composition of the VEEV nsP3 proteome and the potential to identify host-based, broad spectrum therapeutic approaches to treat new world alphavirus infections.

The transcriptional landscape of Venezuelan equine encephalitis virus (TC-83) infection.

Venezuelan Equine Encephalitis Virus (VEEV) is a major biothreat agent that naturally causes outbreaks in humans and horses particularly in tropical areas of the western hemisphere, for which no antiviral therapy is currently available. The host response to VEEV and the cellular factors this alphavirus hijacks to support its effective replication or evade cellular immune responses are largely uncharacterized. We have previously demonstrated tremendous cell-to-cell heterogeneity in viral RNA (vRNA) and cellular transcript levels during flaviviral infection using a novel virus-inclusive single-cell RNA-Seq approach. Here, we used this unbiased, genome-wide approach to simultaneously profile the host transcriptome and vRNA in thousands of single cells during infection of human astrocytes with the live-attenuated vaccine strain of VEEV (TC-83). Host transcription was profoundly suppressed, yet "superproducer cells" with extremely high vRNA abundance emerged during the first viral life cycle and demonstrated an altered transcriptome relative to both uninfected cells and cells with high vRNA abundance harvested at later time points. Additionally, cells with increased structural-to-nonstructural transcript ratio exhibited upregulation of intracellular membrane trafficking genes at later time points. Loss- and gain-of-function experiments confirmed pro- and antiviral activities in both vaccine and virulent VEEV infections among the products of transcripts that positively or negatively correlated with vRNA abundance, respectively. Lastly, comparison with single cell transcriptomic data from other viruses highlighted common and unique pathways perturbed by infection across evolutionary scales. This study provides a high-resolution characterization of the VEEV (TC-83)-host interplay, identifies candidate targets for antivirals, and establishes a comparative single-cell approach to study the evolution of virus-host interactions.

Synthetic Host Defense Peptides Inhibit Venezuelan Equine Encephalitis Virus Replication and the Associated Inflammatory Response.

Venezuelan equine encephalitis virus (VEEV), a New World alphavirus of the Togaviridae family of viruses causes periodic outbreaks of disease in humans and equines. Disease following VEEV infection manifests as a febrile illness with flu-like symptoms, which can progress to encephalitis and cause permanent neurological sequelae in a small number of cases. VEEV is classified as a category B select agent due to ease of aerosolization and high retention of infectivity in the aerosol form. Currently, there are no FDA-approved vaccines or therapeutics available to combat VEEV infection. VEEV infection in vivo is characterized by extensive systemic inflammation that can exacerbate infection by potentially increasing the susceptibility of off-site cells to infection and dissemination of the virus. Hence, a therapeutic targeting both the infection and associated inflammation represents an unmet need. We have previously demonstrated that host defense peptides (HDPs), short peptides that are key components of the innate immune response, exhibit antiviral activity against a multitude of viruses including VEEV. In this study, we designed synthetic peptides derived from indolicidin, a naturally occurring HDP, and tested their efficacy against VEEV. Two candidate synthetic peptides inhibited VEEV replication by approximately 1000-fold and decreased the expression of inflammatory mediators such as IL1α, IL1ß, IFNγ, and TNFα at both the gene and protein expression levels. Furthermore, an increase in expression levels of genes involved in chemotaxis of leukocytes and anti-inflammatory genes such as IL1RN was also observed. Overall, we conclude that our synthetic peptides inhibit VEEV replication and the inflammatory burden associated with VEEV infection.

Venezuelan Equine Encephalitis Virus nsP3 Phosphorylation Can Be Mediated by IKKß Kinase Activity and Abrogation of Phosphorylation Inhibits Negative-Strand Synthesis.

Venezuelan equine encephalitis virus (VEEV), a mosquito transmitted alphavirus of the Togaviridae family, can cause a highly inflammatory and encephalitic disease upon infection. Although a category B select agent, no FDA-approved vaccines or therapeutics against VEEV currently exist. We previously demonstrated NF-κB activation and macromolecular reorganization of the IKK complex upon VEEV infection in vitro, with IKKß inhibition reducing viral replication. Mass spectrometry and confocal microscopy revealed an interaction between IKKß and VEEV non-structural protein 3 (nsP3). Here, using western blotting, a cell-free kinase activity assay, and mass spectrometry, we demonstrate that IKKß kinase activity can directly phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5. Alanine substitution mutations at sites 204/5, 142, or 134/5 reduced VEEV replication by >30-100,000-fold corresponding to a severe decrease in negative-strand synthesis. Serial passaging rescued viral replication and negative-strand synthesis, and sequencing of revertant viruses revealed reversion to the wild-type TC-83 phosphorylation capable amino acid sequences at nsP3 sites 204/5, 142, and 135. Generation of phosphomimetic mutants using aspartic acid substitutions at site 204/5 resulted in rescue of both viral replication and negative-strand RNA production, whereas phosphomimetic mutant 134/5 rescued viral replication but failed to restore negative-strand RNA levels, and phosphomimetic mutant 142 did not rescue VEEV replication. Together, these data demonstrate that IKKß can phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5, and suggest that phosphorylation is essential for negative-strand RNA synthesis at site 204/5, but may be important for infectious particle production at site 134/5.

Efficacy of FDA-Approved Anti-Inflammatory Drugs Against Venezuelan Equine Encephalitis Virus Infection.

Venezuelan equine encephalitis virus (VEEV) is a category B select agent pathogen that can be aerosolized. Infections in murine models and humans can advance to an encephalitic phenotype which may result in long-term neurological complications or death. No specific FDA-approved treatments or vaccines are available for the treatment or prevention of VEEV infection. Neurotropic viral infections have two damaging components: neuronal death caused by viral replication, and damage from the subsequent inflammatory response. Reducing the level of inflammation may lessen neurological tissue damage that often arises following VEEV infection. In this study, three commercially available anti-inflammatory drugs, Celecoxib, Rolipram, and Tofacitinib, were evaluated for antiviral activity in an astrocyte and a microglial model of VEEV infection. The inhibitors were tested against the vaccine strain VEEV TC-83, as well as the wild-type VEEV Trinidad donkey strain. Celecoxib, Tofacitinib, and Rolipram significantly decreased viral titers both after pre-treatment and post-treatment of infected cells. VEEV Trinidad Donkey (TrD) titers were reduced 6.45-fold in cells treated with 50 µM of Celecoxib, 2.45-fold when treated with 50 µM of Tofacitinib, and 1.81-fold when treated with 50 µM of Rolipram. Celecoxib was also shown to decrease inflammatory gene expression in the context of TC-83 infection. Overall, Celecoxib demonstrated potency as a countermeasure strategy that slowed VEEV infection and infection-induced inflammation in an in vitro model.

Human cathelicidin peptide LL-37 as a therapeutic antiviral targeting Venezuelan equine encephalitis virus infections.

Venezuelan equine encephalitis virus (VEEV), a new world alphavirus belonging to the Togaviridae family, causes periodic disease outbreaks in humans and equines with high associated mortality and morbidity. VEEV is highly infectious via the aerosol route and so has been developed as a biological weapon (Hawley and Eitzen, 2001). Despite its current classification as a category B select agent, there are no FDA approved vaccines or therapeutics to counter VEEV infections. Here we utilize a naturally occurring host defense peptide, LL-37, as a therapeutic strategy to inhibit VEEV multiplication in infected cells. LL-37 has previously demonstrated activity against several viruses by directly interacting with viral particles and indirectly by establishing an antiviral state in the host cell. We show that LL-37 exhibited potent antiviral activity against VEEV by inhibiting viral replication. Genomic RNA copies of the TC-83 strain of VEEV and viral titers were significantly reduced compared to non-treated controls. LL-37 also inhibited the virulent Trinidad Donkey (TrD) strain of VEEV. Entry assays revealed a robust reduction of viral RNA copies at the early stages of TC-83 infection. Pre-incubation of cells with LL-37 and TC-83 resulted in a strong inhibitory response, indicating that LL-37 impacts early stages of the infectious process. Confocal and electron microscopy images confirmed the aggregation of viral particles, which potentially accounts for entry prevention and hence reduced viral infection. LL-37 treatment also modulated type I interferon (IFN) expression in infected cells. LL-37 treatment dramatically increased IFNß1 expression in treated cells in a time-dependent manner. Our results establish LL-37 as a relevant and novel potential therapeutic strategy for the treatment of VEEV infections.

Host translation shutoff mediated by non-structural protein 2 is a critical factor in the antiviral state resistance of Venezuelan equine encephalitis virus.

Most previous studies of interferon-alpha/beta (IFN-α/ß) response antagonism by alphaviruses have focused upon interruption of IFN-α/ß induction and/or receptor signaling cascades. Infection of mice with Venezuelan equine encephalitis alphavirus (VEEV) or Sindbis virus (SINV) induces serum IFN-α/ß, that elicits a systemic antiviral state in uninfected cells successfully controlling SINV but not VEEV replication. Furthermore, VEEV replication is more resistant than that of SINV to a pre-existing antiviral state in vitro. While host macromolecular shutoff is proposed as a major antagonist of IFN-α/ß induction, the underlying mechanisms of alphavirus resistance to a pre-existing antiviral state are not fully defined, nor is the mechanism for the greater resistance of VEEV. Here, we have separated viral transcription and translation shutoff with multiple alphaviruses, identified the viral proteins that induce each activity, and demonstrated that VEEV nonstructural protein 2-induced translation shutoff is likely a critical factor in enhanced antiviral state resistance of this alphavirus.