Machine Learning Analysis of RNA-Seq Data Identifies Key Gene Signatures and Pathways in Mpox Virus-Induced Gastrointestinal Complications Using Colon Organoid Models.

Mpox, caused by the Mpox virus (MPXV), emerged globally in 2022 with the Clade IIb strain, presenting a critical public health challenge. While MPXV is primarily characterized by fever and rash, gastrointestinal (GI) complications, such as diarrhea and proctitis, have also been observed. This study is a reanalysis of GSE219036 without own data and focuses on the impact of MPXV infection on the colon, using human-induced pluripotent stem cell-derived colon organoids as a model. We applied a tailored statistical framework for RNA-seq data, Generalized Linear Models with Quasi-Likelihood F-tests and Relaxed Magnitude-Altitude Scoring (GLMQL-RMAS), to identify differentially expressed genes (DEGs) across MPXV clades: MPXV I (Zr-599 Congo Basin), MPXV IIa (Liberia), and MPXV IIb (2022 MPXV). Through a novel methodology called Cross-RMAS, we ranked genes by integrating statistical significance and biological relevance across all clades. Machine learning analysis using the genes identified by Cross-RMAS, demonstrated 100% accuracy in differentiating between the different MPXV strains and mock samples. Furthermore, our findings reveal that MPXV Clade I induces the most extensive alterations in gene expression, with significant upregulation of stress response genes, such as HSPA6 and FOS, and downregulation of genes involved in cytoskeletal organization and vesicular trafficking, such as PSAP and CFL1. In contrast, Clade IIb shows the least impact on gene expression. Through Gene Ontology (GO) analysis, we identified pathways involved in protein folding, immune response, and epithelial integrity that are disrupted in infected cells, suggesting mechanisms by which MPXV may contribute to GI symptoms.

Durability of Viral Neutralization in Asymptomatic Coronavirus Disease 2019 for at Least 60 Days.

A cohort consisting of asymptomatic healthcare workers donated temporal serum samples after infection with severe acute respiratory syndrome coronavirus 2. Analysis shows that all asymptomatic healthcare workers had neutralizing antibodies, that these antibodies persist for ≥60 days, and that anti-spike receptor-binding domain immunoglobulin G levels were correspondingly durable over the same time period.

Zika Virus-Like Particle (VLP) vaccine displaying Envelope (E) protein CD loop antigen elicits protective and specific immune response in a murine model.

Zika virus (ZIKV) is a mosquito-borne flavivirus associated with Congenital Zika Syndrome (CZS), reflecting a wide range of congenital abnormalities in fetuses and infants infected with ZIKV before birth. ZIKV infections have also been associated with the neurological autoimmune disorder known as Guillian-Barré syndrome (GBS). To date, no vaccines or antiviral strategies are licensed for ZIKV. We used rational design to develop a novel ZIKV vaccine candidate using a Woodchuck Hepatitis core Antigen (WHcAg) Virus-Like Particle (VLP) scaffold for displaying selected antigens from the ZIKV Envelope (E) protein. A Zika-VLP vaccine candidate containing the CD Loop sub-structural domain from ZIKV E protein Domain III (WHcAg CD Loop) elicited a strong immune response in a murine model. Analysis of serum immunoglobulins demonstrated induction of both Th1- and Th2- mediated immune response. No cross-reacting antibodies were detected between Zika, dengue and yellow fever virus, demonstrating a high level of specificity for the ZIKV CD Loop antigen. Immunization with the WHcAg CD Loop vaccine candidate demonstrated immunoprotection in a murine model of ZIKV infection, stimulating protective antibodies associated with antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities. The WHcAg CD Loop candidate may represent a safer vaccine for preventing antibody dependent enhancement (ADE).

Zika-virus-infected human full-term placental explants display pro-inflammatory responses and undergo apoptosis.

Zika virus (ZIKV) is a flavivirus that has been highly correlated with the development of neurological disorders and other malformations in newborns and stillborn fetuses after congenital infection. This association is supported by the presence of ZIKV in the fetal brain and amniotic fluid, and findings suggest that infection of the placental barrier is a critical step for fetal ZIKV infection in utero. Therefore, relevant models to investigate the interaction between ZIKV and placental tissues are essential for understanding the pathogenesis of Zika syndrome. In this report, we demonstrate that explant tissue from full-term human placentas sustains a productive ZIKV infection, though the results depend on the strain. Viral infection was found to be associated with pro-inflammatory cytokine expression and apoptosis of the infected tissue, and these findings confirm that placental explants are targets of ZIKV replication. We propose that human placental explants are useful as a model for studying ZIKV infection ex vivo.

S-nitrosylation of peroxiredoxin 1 contributes to viability of lung epithelial cells during Bacillus anthracis infection.

BACKGROUND: Using Bacillus anthracis as a model gram-positive bacterium, we investigated the effects of host protein S-nitrosylation during bacterial infection. B. anthracis possesses a bacterial nitric oxide synthase (bNOS) that is important for its virulence and survival. However, the role of S-nitrosylation of host cell proteins during B. anthracis infection has not been determined. METHODS: Nitrosoproteomic analysis of human small airway epithelial cells (HSAECs) infected with toxigenic B. anthracis Sterne was performed, identifying peroxiredoxin 1 (Prx1) as one predominant target. Peroxidase activity of Prx during infection was measured using 2-Cys-Peroxiredoxin activity assay. Chaperone activity of S-nitrosylated Prx1 was measured by insulin aggregation assay, and analysis of formation of multimeric species using Native PAGE. Griess assay and DAF-2DA fluorescence assay were used to measure NO production. Cell viability was measured using the Alamar Blue assay and the ATPlite assay (Perkin Elmer). RESULTS: S-nitrosylation of Prx1 in Sterne-infected HSAECs leads to a decrease in its peroxidase activity while enhancing its chaperone function. Treatment with bNOS inhibitor, or infection with bNOS deletion strain, reduces S-nitrosylation of Prx1 and decreases host cell survival. Consistent with this, siRNA knockdown of Prx1 lowers bNOS-dependent protection of HSAEC viability. CONCLUSIONS: Anthrax infection results in S-nitrosylation of multiple host proteins, including Prx1. The nitrosylation-dependent decrease in peroxidase activity of Prx1 and increase in its chaperone activity is one factor contributing to enhancing infected cell viability. GENERAL SIGNIFICANCE: These results provide a new venue of mechanistic investigation for inhalational anthrax that could lead to novel and potentially effective countermeasures.

Human T-lymphotropic virus type 1-infected cells secrete exosomes that contain Tax protein.

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. The HTLV-1 transactivator protein Tax controls many critical cellular pathways, including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Extracellular vesicles called exosomes play critical roles during pathogenic viral infections as delivery vehicles for host and viral components, including proteins, mRNA, and microRNA. We hypothesized that exosomes derived from HTLV-1-infected cells contain unique host and viral proteins that may contribute to HTLV-1-induced pathogenesis. We found exosomes derived from infected cells to contain Tax protein and proinflammatory mediators as well as viral mRNA transcripts, including Tax, HBZ, and Env. Furthermore, we observed that exosomes released from HTLV-1-infected Tax-expressing cells contributed to enhanced survival of exosome-recipient cells when treated with Fas antibody. This survival was cFLIP-dependent, with Tax showing induction of NF-κB in exosome-recipient cells. Finally, IL-2-dependent CTLL-2 cells that received Tax-containing exosomes were protected from apoptosis through activation of AKT. Similar experiments with primary cultures showed protection and survival of peripheral blood mononuclear cells even in the absence of phytohemagglutinin/IL-2. Surviving cells contained more phosphorylated Rb, consistent with the role of Tax in regulation of the cell cycle. Collectively, these results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells.

Novel neuroprotective GSK-3ß inhibitor restricts Tat-mediated HIV-1 replication.

The implementation of new antiretroviral therapies targeting transcription of early viral proteins in postintegrated HIV-1 can aid in overcoming current therapy limitations. Using high-throughput screening assays, we have previously described a novel Tat-dependent HIV-1 transcriptional inhibitor named 6-bromoindirubin-3'-oxime (6BIO). The screening of 6BIO derivatives yielded unique compounds that show potent inhibition of HIV-1 transcription. We have identified a second-generation derivative called 18BIOder as an inhibitor of HIV-1 Tat-dependent transcription in TZM-bl cells and a potent inhibitor of GSK-3ß kinase in vitro. Structurally, 18BIOder is half the molecular weight and structure of its parental compound, 6BIO. More importantly, we also have found a different GSK-3ß complex present only in HIV-1-infected cells. 18BIOder preferentially inhibits this novel kinase complex from infected cells at nanomolar concentrations. Finally, we observed that neuronal cultures treated with Tat protein are protected from Tat-mediated cytotoxicity when treated with 18BIOder. Overall, our data suggest that HIV-1 Tat-dependent transcription is sensitive to small-molecule inhibition of GSK-3ß.

Exosomes derived from HIV-1-infected cells contain trans-activation response element RNA.

Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 10(4)-10(6) copies/ml TAR RNA in exosomes derived from infected culture supernatants and 10(3) copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS.

Machine learning-based analysis of Ebola virus' impact on gene expression in nonhuman primates.

Introduction: This study introduces the Supervised Magnitude-Altitude Scoring (SMAS) methodology, a novel machine learning-based approach for analyzing gene expression data from non-human primates (NHPs) infected with Ebola virus (EBOV). By focusing on host-pathogen interactions, this research aims to enhance the understanding and identification of critical biomarkers for Ebola infection. Methods: We utilized a comprehensive dataset of NanoString gene expression profiles from Ebola-infected NHPs. The SMAS system combines gene selection based on both statistical significance and expression changes. Employing linear classifiers such as logistic regression, the method facilitates precise differentiation between RT-qPCR positive and negative NHP samples. Results: The application of SMAS led to the identification of IFI6 and IFI27 as key biomarkers, which demonstrated perfect predictive performance with 100% accuracy and optimal Area Under the Curve (AUC) metrics in classifying various stages of Ebola infection. Additionally, genes including MX1, OAS1, and ISG15 were significantly upregulated, underscoring their vital roles in the immune response to EBOV. Discussion: Gene Ontology (GO) analysis further elucidated the involvement of these genes in critical biological processes and immune response pathways, reinforcing their significance in Ebola pathogenesis. Our findings highlight the efficacy of the SMAS methodology in revealing complex genetic interactions and response mechanisms, which are essential for advancing the development of diagnostic tools and therapeutic strategies. Conclusion: This study provides valuable insights into EBOV pathogenesis, demonstrating the potential of SMAS to enhance the precision of diagnostics and interventions for Ebola and other viral infections.

Inhibitor of the non-structural protein 2 protease shows promising efficacy in mouse models of chikungunya.

Chikungunya virus (CHIKV) is responsible for the most endemic alphavirus infections called Chikungunya. The endemicity of Chikungunya has increased over the past two decades, and it is a pathogen with pandemic potential. There is currently no approved direct-acting antiviral to treat the disease. As part of our antiviral drug discovery program focused on alphaviruses and the non-structural protein 2 protease, we discovered that J12 and J13 can inhibit CHIKV nsP2 protease and block the replication of CHIKV in cell cultures. Both compounds are metabolically stable to human liver microsomal and S9 enzymes. J13 has excellent oral bioavailability in pharmacokinetics studies in mice and ameliorated Chikungunya symptoms in preliminary efficacy studies in mice. J13 exhibited an excellent safety profile in in vitro safety pharmacology and off-target screening assays, making J13 and its analogs good candidates for drug development against Chikungunya.

Gravity-perfused airway-on-a-chip optimized for quantitative BSL-3 studies of SARS-CoV-2 infection: barrier permeability, cytokine production, immunohistochemistry, and viral load assays.

Human microphysiological systems, such as organs on chips, are an emerging technology for modeling human physiology in a preclinical setting to understand the mechanism of action of drugs, to evaluate the efficacy of treatment options for human disease and impairment, and to assess drug toxicity. By using human cells co-cultured in three-dimensional constructs, organ chips can provide greater fidelity to the human cellular condition than their two-dimensional predecessors. However, with the rise of SARS-CoV-2 and the global COVID-19 pandemic, it became clear that many microphysiological systems were not compatible with or optimized for studies of infectious disease and operation in a Biosafety Level 3 (BSL-3) environment. Given that one of the early sites of SARS-CoV-2 infection is the airway, we created a human airway organ chip that could operate in a BSL-3 space with high throughput and minimal manipulation, while retaining the necessary physical and physiological components to recapitulate tissue response to infectious agents and the immune response to infection.

Antiviral activity of the host defense peptide piscidin 1: investigating a membrane-mediated mode of action.

Outbreaks of viral diseases are on the rise, fueling the search for antiviral therapeutics that act on a broad range of viruses while remaining safe to human host cells. In this research, we leverage the finding that the plasma membranes of host cells and the lipid bilayers surrounding enveloped viruses differ in lipid composition. We feature Piscidin 1 (P1), a cationic host defense peptide (HDP) that has antimicrobial effects and membrane activity associated with its N-terminal region where a cluster of aromatic residues and copper-binding motif reside. While few HDPs have demonstrated antiviral activity, P1 acts in the micromolar range against several enveloped viruses that vary in envelope lipid composition. Notably, it inhibits HIV-1, a virus that has an envelope enriched in cholesterol, a lipid associated with higher membrane order and stability. Here, we first document through plaque assays that P1 boasts strong activity against SARS-CoV-2, which has an envelope low in cholesterol. Second, we extend previous studies done with homogeneous bilayers and devise cholesterol-containing zwitterionic membranes that contain the liquid disordered (Ld; low in cholesterol) and ordered (Lo, rich in cholesterol) phases. Using dye leakage assays and cryo-electron microscopy on vesicles, we show that P1 has dramatic permeabilizing capability on the Lo/Ld, an effect matched by a strong ability to aggregate, fuse, and thin the membranes. Differential scanning calorimetry and NMR experiments demonstrate that P1 mixes the lipid content of vesicles and alters the stability of the Lo. Structural studies by NMR indicate that P1 interacts with the Lo/Ld by folding into an α-helix that lies parallel to the membrane surface. Altogether, these results show that P1 is more disruptive to phase-separated than homogenous cholesterol-containing bilayers, suggesting an ability to target domain boundaries. Overall, this multi-faceted research highlights how a peptide that interacts strongly with membranes through an aromatic-rich N-terminal motif disrupt viral envelope mimics. This represents an important step towards the development of novel peptides with broad-spectrum antiviral activity.

Induction of DNA damage signaling upon Rift Valley fever virus infection results in cell cycle arrest and increased viral replication.

Rift Valley fever virus (RVFV) is a highly pathogenic arthropod-borne virus infecting a wide range of vertebrate hosts. Of particular interest is the nonstructural NSs protein, which forms large filamentous fibril bundles in the nucleus. Past studies have shown NSs to be a multifaceted protein important for virulence through modulation of the interferon response as well acting as a general inhibitor of transcription. Here we investigated the regulation of the DNA damage signaling cascades by RVFV infection and found virally inducted phosphorylation of the classical DNA damage signaling proteins, ataxia-telangiectasia mutated (ATM) (Ser-1981), Chk.2 (Thr-68), H2A.X (Ser-139), and p53 (Ser-15). In contrast, ataxia-telangiectasia mutated and Rad3-related kinase (ATR) (Ser-428) phosphorylation was decreased following RVFV infection. Importantly, both the attenuated vaccine strain MP12 and the fully virulent strain ZH548 showed strong parallels in their up-regulation of the ATM arm of the DNA damage response and in the down-regulation of the ATR pathway. The increase in DNA damage signaling proteins did not result from gross DNA damage as no increase in DNA damage was observed following infection. Rather the DNA damage signaling was found to be dependent on the viral protein NSs, as an NSs mutant virus was not found to induce the equivalent signaling pathways. RVFV MP12-infected cells also displayed an S phase arrest that was found to be dependent on NSs expression. Use of ATM and Chk.2 inhibitors resulted in a marked decrease in S phase arrest as well as viral production. These results indicate that RVFV NSs induces DNA damage signaling pathways that are beneficial for viral replication.

Curcumin inhibits Rift Valley fever virus replication in human cells.

Rift Valley fever virus (RVFV) is an arbovirus that is classified as a select agent, an emerging infectious virus, and an agricultural pathogen. Understanding RVFV-host interactions is imperative to the design of novel therapeutics. Here, we report that an infection by the MP-12 strain of RVFV induces phosphorylation of the p65 component of the NFκB cascade. We demonstrate that phosphorylation of p65 (serine 536) involves phosphorylation of IκBα and occurs through the classical NFκB cascade. A unique, low molecular weight complex of the IKK-ß subunit can be observed in MP-12-infected cells, which we have labeled IKK-ß2. The IKK-ß2 complex retains kinase activity and phosphorylates an IκBα substrate. Inhibition of the IKK complex using inhibitors impairs viral replication, thus alluding to the requirement of an active IKK complex to the viral life cycle. Curcumin strongly down-regulates levels of extracellular infectious virus. Our data demonstrated that curcumin binds to and inhibits kinase activity of the IKK-ß2 complex in infected cells. Curcumin partially exerts its inhibitory influence on RVFV replication by interfering with IKK-ß2-mediated phosphorylation of the viral protein NSs and by altering the cell cycle of treated cells. Curcumin also demonstrated efficacy against ZH501, the fully virulent version of RVFV. Curcumin treatment down-regulated viral replication in the liver of infected animals. Our data point to the possibility that RVFV infection may result in the generation of novel versions of host components (such as IKK-ß2) that, by virtue of altered protein interaction and function, qualify as unique therapeutic targets.

Bacillus anthracis-derived nitric oxide induces protein S-nitrosylation contributing to macrophage death.

Bacillus anthracis, a causative agent of anthrax, is able to germinate and survive within macrophages. A recent study suggested that B. anthracis-derived nitric oxide (bNO) is a key aspect of bacterial defense that protects bacterial DNA from oxidative burst in the macrophages. However, the virulent effect of bNO in host cells has not been investigated. Here, we report that bNO contributes macrophage killing by S-nitrosylation of bioenergetic-relating proteins within mitochondria. Toxigenic Sterne induces expression of the bnos gene and produces bNO during early stage of infection. Nitroso-proteomic analysis coupled with a biotin-switch technique demonstrated that toxigenic infection induces protein S-nitrosylation in B. anthracis-susceptible RAW264.7. For each target enzyme tested (complex I, complex III and complex IV), infection by B. anthracis Sterne caused enzyme inhibition. Nω-nitro-L-arginine methyl ester, a NO synthase inhibitor, reduced S-nitrosylation and partially restored cell viability evaluated by intracellular ATP levels in macrophages. Our data suggest that bNO leads to energy depletion driven by impaired mitochondrial bioenergetic machinery that ultimately contributes to macrophage death. This novel mechanism of anthrax pathogenesis may offer specific approach to the development of therapeutics.

Interactions of Equine Viruses with the Host Kinase Machinery and Implications for One Health and Human Disease.

Zoonotic pathogens that are vector-transmitted have and continue to contribute to several emerging infections globally. In recent years, spillover events of such zoonotic pathogens have increased in frequency as a result of direct contact with livestock, wildlife, and urbanization, forcing animals from their natural habitats. Equines serve as reservoir hosts for vector-transmitted zoonotic viruses that are also capable of infecting humans and causing disease. From a One Health perspective, equine viruses, therefore, pose major concerns for periodic outbreaks globally. Several equine viruses have spread out of their indigenous regions, such as West Nile virus (WNV) and equine encephalitis viruses (EEVs), making them of paramount concern to public health. Viruses have evolved many mechanisms to support the establishment of productive infection and to avoid host defense mechanisms, including promoting or decreasing inflammatory responses and regulating host machinery for protein synthesis. Viral interactions with the host enzymatic machinery, specifically kinases, can support the viral infectious process and downplay innate immune mechanisms, cumulatively leading to a more severe course of the disease. In this review, we will focus on how select equine viruses interact with host kinases to support viral multiplication.

Exogenous Klotho Extends Survival in COVID-19 Model Mice.

A striking feature of COVID-19 disease is the broad spectrum of risk factors associated with case severity, as well as the diversity of clinical manifestations. While no central agent has been able to explain the pathogenesis of SARS-CoV-2 infection, the factors that most robustly correlate with severity are risk factors linked to aging. Low serum levels of Klotho, an anti-aging protein, strongly correlate with the pathogenesis of the same risk factors and manifestations of conditions similar to those expressed in severe COVID-19 cases. The current manuscript presents original research on the effects of the exogenous application of Klotho, an anti-aging protein, in COVID-19 model mice. Klotho supplementation resulted in a statistically significant survival benefit in parametric and non-parametric models. Further research is required to elucidate the mechanistic role Klotho plays in COVID-19 pathogenesis as well as the possible modulation SARS-CoV-2 may have on the biological aging process.

Brilacidin as a Broad-Spectrum Inhibitor of Enveloped, Acutely Infectious Viruses.

Alphaviruses, belonging to the Togaviridae family, and bunyaviruses, belonging to the Paramyxoviridae family, are globally distributed and lack FDA-approved vaccines and therapeutics. The alphaviruses Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV) are known to cause severe encephalitis, whereas Sindbis virus (SINV) causes arthralgia potentially persisting for years after initial infection. The bunyavirus Rift Valley Fever virus (RVFV) can lead to blindness, liver failure, and hemorrhagic fever. Brilacidin, a small molecule that was designed de novo based on naturally occurring host defensins, was investigated for its antiviral activity against these viruses in human small airway epithelial cells (HSAECs) and African green monkey kidney cells (Veros). This testing was further expanded into a non-enveloped Echovirus, a Picornavirus, to further demonstrate brilacidin's effect on early steps of the viral infectious cycle that leads to inhibition of viral load. Brilacidin demonstrated antiviral activity against alphaviruses VEEV TC-83, VEEV TrD, SINV, EEEV, and bunyavirus RVFV. The inhibitory potential of brilacidin against the viruses tested in this study was dependent on the dosing strategy which necessitated compound addition pre- and post-infection, with addition only at the post-infection stage not eliciting a robust inhibitory response. The inhibitory activity of brilacidin was only modest in the context of the non-enveloped Picornavirus Echovirus, suggesting brilacidin may be less potent against non-enveloped viruses.

Venezuelan equine encephalitis virus E1 protein interacts with PDIA6 and PDI inhibition reduces alphavirus production.

Venezuelan equine encephalitis virus (VEEV) is an alphavirus transmitted by mosquitos that can cause a febrile illness and induce severe neurological complications in humans and equine populations. Currently there are no FDA approved vaccines or antiviral treatments to combat VEEV. Proteomic techniques were utilized to create an interactome of the E1 fusion glycoprotein of VEEV. VEEV E1 interacted with a number of cellular chaperone proteins including protein disulfide isomerase family A member 6 (PDIA6). PDI inhibition through LOC14 and/or nitazoxanide treatment effectively decreased production of VEEV and other alphaviruses in vitro, including eastern equine encephalitis virus, Sindbis virus, and chikungunya virus. Decreased oxidoreductive capabilities of PDIs through LOC14 or nitazoxanide treatment impacted both early and late events in viral replication, including the production of non-infectious virions and decreased VEEV E1 disulfide bond formation. Results from this study identified PDIs as critical regulators of alphavirus replication and potential therapeutic targets.

Inhibitors of the Ubiquitin-Mediated Signaling Pathway Exhibit Broad-Spectrum Antiviral Activities against New World Alphaviruses.

New World alphaviruses including Venezuelan Equine Encephalitis Virus (VEEV) and Eastern Equine Encephalitis Virus (EEEV) are mosquito-transmitted viruses that cause disease in humans and equines. There are currently no FDA-approved therapeutics or vaccines to treat or prevent exposure-associated encephalitic disease. The ubiquitin proteasome system (UPS)-associated signaling events are known to play an important role in the establishment of a productive infection for several acutely infectious viruses. The critical engagement of the UPS-associated signaling mechanisms by many viruses as host-pathogen interaction hubs led us to hypothesize that small molecule inhibitors that interfere with these signaling pathways will exert broad-spectrum inhibitory activity against alphaviruses. We queried eight inhibitors of the UPS signaling pathway for antiviral outcomes against VEEV. Three of the tested inhibitors, namely NSC697923 (NSC), bardoxolone methyl (BARM) and omaveloxolone (OMA) demonstrated broad-spectrum antiviral activity against VEEV and EEEV. Dose dependency and time of addition studies suggest that BARM and OMA exhibit intracellular and post-entry viral inhibition. Cumulatively, our studies indicate that inhibitors of the UPS-associated signaling pathways exert broad-spectrum antiviral outcomes in the context of VEEV and EEEV infection, supporting their translational application as therapeutic candidates to treat alphavirus infections.