Publications by authors named "Aarthi Narayanan"

72 Publications

Brilacidin Demonstrates Inhibition of SARS-CoV-2 in Cell Culture.

Viruses 2021 02 9;13(2). Epub 2021 Feb 9.

National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, VA 20110, USA.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the newly emergent causative agent of coronavirus disease-19 (COVID-19), has resulted in more than two million deaths worldwide since it was first detected in 2019. There is a critical global need for therapeutic intervention strategies that can be deployed to safely treat COVID-19 disease and reduce associated morbidity and mortality. Increasing evidence shows that both natural and synthetic antimicrobial peptides (AMPs), also referred to as Host Defense Proteins/Peptides (HDPs), can inhibit SARS-CoV-2, paving the way for the potential clinical use of these molecules as therapeutic options. In this manuscript, we describe the potent antiviral activity exerted by brilacidin-a de novo designed synthetic small molecule that captures the biological properties of HDPs-on SARS-CoV-2 in a human lung cell line (Calu-3) and a monkey cell line (Vero). These data suggest that SARS-CoV-2 inhibition in these cell culture models is likely to be a result of the impact of brilacidin on viral entry and its disruption of viral integrity. Brilacidin demonstrated synergistic antiviral activity when combined with remdesivir. Collectively, our data demonstrate that brilacidin exerts potent inhibition of SARS-CoV-2 against different strains of the virus in cell culture.
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http://dx.doi.org/10.3390/v13020271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916214PMC
February 2021

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

Sci Rep 2020 12 8;10(1):21491. Epub 2020 Dec 8.

National Center for Biodefence and Infectious Disease, Biomedical Research Laboratory, School of Systems Biology, George Mason University, Manassas, VA, USA.

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, IL1β, 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.
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http://dx.doi.org/10.1038/s41598-020-77990-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7722873PMC
December 2020

Role of Mitochondria in Cancer Immune Evasion and Potential Therapeutic Approaches.

Front Immunol 2020 16;11:573326. Epub 2020 Oct 16.

Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, United States.

The role of mitochondria in cancer formation and progression has been studied extensively, but much remains to be understood about this complex relationship. Mitochondria regulate many processes that are known to be altered in cancer cells, from metabolism to oxidative stress to apoptosis. Here, we review the evolving understanding of the role of mitochondria in cancer cells, and highlight key evidence supporting the role of mitochondria in cancer immune evasion and the effects of mitochondria-targeted antitumor therapy. Also considered is how knowledge of the role of mitochondria in cancer can be used to design and improve cancer therapies, particularly immunotherapy and radiation therapy. We further offer critical insights into the mechanisms by which mitochondria influence tumor immune responses, not only in cancer cells but also in immune cells. Given the central role of mitochondria in the complex interactions between cancer and the immune system, high priority should be placed on developing rational strategies to address mitochondria as potential targets in future preclinical and clinical studies. We believe that targeting mitochondria may provide additional opportunities in the development of novel antitumor therapeutics.
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http://dx.doi.org/10.3389/fimmu.2020.573326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596324PMC
October 2020

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

Viruses 2020 09 13;12(9). Epub 2020 Sep 13.

National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, VA 20110, USA.

Venezuelan equine encephalitis virus (VEEV), a mosquito transmitted alphavirus of the 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 IKKβ inhibition reducing viral replication. Mass spectrometry and confocal microscopy revealed an interaction between IKKβ and VEEV non-structural protein 3 (nsP3). Here, using western blotting, a cell-free kinase activity assay, and mass spectrometry, we demonstrate that IKKβ 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 IKKβ 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.
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http://dx.doi.org/10.3390/v12091021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7551587PMC
September 2020

Maraviroc inhibits SARS-CoV-2 multiplication and s-protein mediated cell fusion in cell culture.

bioRxiv 2020 Aug 13. Epub 2020 Aug 13.

In an effort to identify therapeutic intervention strategies for the treatment of COVID-19, we have investigated a selection of FDA-approved small molecules and biologics that are commonly used to treat other human diseases. A screen of 19 small molecules and 3 biologics was conducted in cell culture and the impact of treatment on viral titer was quantified by plaque assay. The screen identified 4 FDA-approved small molecules, Maraviroc, FTY720 (Fingolimod), Atorvastatin and Nitazoxanide that were able to inhibit SARS-CoV-2 infection. Confocal microscopy with over expressed S protein demonstrated that Maraviroc reduced the extent of S-protein mediated cell fusion as observed by fewer multinucleate cells in drug-treated cells. Mathematical modeling of drug-dependent viral multiplication dynamics revealed that prolonged drug treatment will exert an exponential decrease in viral load in a multicellular/tissue environment. Taken together, the data demonstrate that Maraviroc, Fingolimod, Atorvastatin and Nitazoxanide inhibit SARS-CoV-2 in cell culture.

Highlights: Maraviroc, FTY720, Nitazoxanide and Atorvastatin inhibit SARS-CoV-2 multiplication in cell culture.Maraviroc does not interfere with the interaction between SARS-CoV-2 spike protein and ACE2 receptor.Maraviroc exhibits only modest synergistic activities with FTY720, Nitazoxanide or Atorvastatin.Maraviroc reduces the extent of SARS-CoV-2 S-protein mediated cell fusion.Mathematical modeling reveals that Maraviroc treatment will elicit an exponential decrease in viral load in a multicellular tissue environment.
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http://dx.doi.org/10.1101/2020.08.12.246389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430595PMC
August 2020

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

Biochem Biophys Res Commun 2020 08 20;529(3):805-811. Epub 2020 Jul 20.

American Type Culture Collection (ATCC®), Manassas, VA, USA.

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).
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http://dx.doi.org/10.1016/j.bbrc.2020.05.161DOI Listing
August 2020

Rapid community-driven development of a SARS-CoV-2 tissue simulator.

bioRxiv 2020 Apr 5. Epub 2020 Apr 5.

The 2019 novel coronavirus, SARS-CoV-2, is an emerging pathogen of critical significance to international public health. Knowledge of the interplay between molecular-scale virus-receptor interactions, single-cell viral replication, intracellular-scale viral transport, and emergent tissue-scale viral propagation is limited. Moreover, little is known about immune system-virus-tissue interactions and how these can result in low-level (asymptomatic) infections in some cases and acute respiratory distress syndrome (ARDS) in others, particularly with respect to presentation in different age groups or pre-existing inflammatory risk factors like diabetes. Given the nonlinear interactions within and among each of these processes, multiscale simulation models can shed light on the emergent dynamics that lead to divergent outcomes, identify actionable "choke points" for pharmacologic interactions, screen potential therapies, and identify potential biomarkers that differentiate patient outcomes. Given the complexity of the problem and the acute need for an actionable model to guide therapy discovery and optimization, we introduce and iteratively refine a prototype of a multiscale model of SARS-CoV-2 dynamics in lung tissue. The first prototype model was built and shared internationally as open source code and an online interactive model in under 12 hours, and community domain expertise is driving rapid refinements with a two-to-four week release cycle. In a sustained community effort, this consortium is integrating data and expertise across virology, immunology, mathematical biology, quantitative systems physiology, cloud and high performance computing, and other domains to accelerate our response to this critical threat to international health.
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http://dx.doi.org/10.1101/2020.04.02.019075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7239052PMC
April 2020

Use of Nanotrap particles for the capture and enrichment of Zika, chikungunya and dengue viruses in urine.

PLoS One 2020 7;15(1):e0227058. Epub 2020 Jan 7.

National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, Manassas, VA, United States of America.

Nanotrap® (NT) particles are hydrogel microspheres developed for target analyte separation and discovery applications. NT particles consist of cross-linked N-isopropylacrylamide (NIPAm) copolymers that are functionalized with a variety of chemical affinity baits to enable broad-spectrum collection and retention of target proteins, nucleic acids, and pathogens. NT particles have been previously shown to capture and enrich arboviruses including Rift Valley fever and Venezuelan equine encephalitis viruses. Yet, there is still a need to enhance the detection ability for other re-emerging viruses such as Zika (ZIKV), chikungunya (CHIKV), and dengue (DENV) viruses. In this study, we exploited NT particles with different affinity baits, including cibacron blue, acrylic acid, and reactive red 120, to evaluate their capturing and enrichment capability for ZIKV, DENV and CHIKV in human fluids. Our results demonstrate that CN1030, a NT particle conjugated with reactive red 120, can recover between 8-16-fold greater genomic copies of ZIKV, CHIKV and DENV in virus spiked urine samples via RT-qPCR, superior to the other chemical baits. Also, we observed that CN1030 simultaneously enriched ZIKV, CHIKV and DENV in co-infection-based settings and could stabilize ZIKV, but not CHIKV infectivity in saliva spiked samples. CN1030 enriched viral detection at various viral concentrations, with significant enhancement observed at viral titers as low as 100 PFU/mL for ZIKV and 10 PFU/mL for CHIKV. The detection of ZIKV was further enhanced with NT particles by processing of larger volume urine samples. Furthermore, we developed a magnetic NT particle, CN3080, based on the same backbone of CN1030, and demonstrated that CN3080 could also capture and enrich ZIKV and CHIKV in a dose-dependent manner. Finally, in silico docking predictions support that the affinity between reactive red 120 and ZIKV or CHIKV envelope proteins appeared to be greater than acrylic acid. Overall, our data show that NT particles along with reactive red 120 can be utilized as a pre-processing technology for enhancement of detecting febrile-illness causing viruses.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0227058PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6946132PMC
April 2020

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

Viruses 2019 12 12;11(12). Epub 2019 Dec 12.

National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, VA 20110, USA.

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.
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http://dx.doi.org/10.3390/v11121151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6950191PMC
December 2019

Human Antimicrobial Peptides as Therapeutics for Viral Infections.

Viruses 2019 08 1;11(8). Epub 2019 Aug 1.

National Center for Biodefense and Infectious Disease, School of Systems Biology, George Mason University, Manassas, VA 20110, USA.

Successful in vivo infection following pathogen entry requires the evasion and subversion of multiple immunological barriers. Antimicrobial peptides (AMPs) are one of the first immune pathways upregulated during infection by multiple pathogens, in multiple organs in vivo. In humans, there are many classes of AMPs exhibiting broad antimicrobial activities, with defensins and the human cathelicidin LL-37 being the best studied examples. Whereas historically the efficacy and therapeutic potential of AMPs against bacterial infection has been the primary focus of research, recent studies have begun to elucidate the antiviral properties of AMPs as well as their role in regulation of inflammation and chemoattraction. AMPs as therapeutic tools seem especially promising against emerging infectious viral pathogens for which no approved vaccines or treatments are currently available, such as dengue virus (DENV) and Zika virus (ZIKV). In this review, we summarize recent studies elucidating the efficacy and diverse mechanisms of action of various classes of AMPs against multiple viral pathogens, as well as the potential use of human AMPs in novel antiviral therapeutic strategies.
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http://dx.doi.org/10.3390/v11080704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6722670PMC
August 2019

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

Antiviral Res 2019 04 8;164:61-69. Epub 2019 Feb 8.

National Center for Biodefense and Infectious Disease, School of Systems Biology, George Mason University, Manassas, VA, USA.

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 IFNβ1 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.
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http://dx.doi.org/10.1016/j.antiviral.2019.02.002DOI Listing
April 2019

New World alphavirus protein interactomes from a therapeutic perspective.

Antiviral Res 2019 03 26;163:125-139. Epub 2019 Jan 26.

National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, Manassas, VA, USA. Electronic address:

The New World alphaviruses, Venezuelan, eastern and western equine encephalitis viruses (VEEV, EEEV, and WEEV), are important human pathogens due to their ability to cause varying levels of morbidity and mortality in humans. There is also concern about VEEV and EEEV being used as bioweapons. Currently, a FDA-approved antiviral is lacking for New World alphaviruses. In this review, the function of each viral protein is discussed with an emphasis on how these functions can be targeted by therapeutics. Both direct acting antivirals as well as inhibitors that impact host protein interactions with viral proteins are described. Non-structural protein 3 (nsP3), capsid, and E2 proteins have garnered attention in recent years, whereas little is known regarding host protein interactions of the other viral proteins and is an important avenue for future study.
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http://dx.doi.org/10.1016/j.antiviral.2019.01.015DOI Listing
March 2019

Mitochondrial-Directed Antioxidant Reduces Microglial-Induced Inflammation in Murine In Vitro Model of TC-83 Infection.

Viruses 2018 11 2;10(11). Epub 2018 Nov 2.

National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, VA 20110, USA.

Venezuelan equine encephalitis virus (VEEV) is an arbovirus that is associated with robust inflammation that contributes to neurodegenerative phenotypes. In addition to triggering central nervous system (CNS) inflammation, VEEV will also induce mitochondrial dysfunction, resulting in increased cellular apoptosis. In this study, we utilize the TC-83 strain of VEEV to determine the role of mitochondrial oxidative stress in mediating inflammation elicited by murine brain microglial cells. Using an in vitro model, we show that murine microglia are susceptible to TC-83 infection, and that these cells undergo mitochondrial stress as the result of infection. We also indicate that bystander microglia contribute more significantly to the overall inflammatory load than directly infected microglia. Use of a mitochondrial targeted antioxidant, mitoquinone mesylate, greatly reduced the pro-inflammatory cytokines released by both direct infected and bystander microglia. Our data suggest that release of interleukin-1β, a key instigator of neuroinflammation during VEEV infection, may be the direct result of accumulating mitochondrial stress. This data improves our understanding inflammation elicited by murine microglia and will aid in the development of more accurate in vitro and in vivo murine model of VEEV-induced neuroinflammation.
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http://dx.doi.org/10.3390/v10110606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6266753PMC
November 2018

Host-based processes as therapeutic targets for Rift Valley fever virus.

Antiviral Res 2018 12 11;160:64-78. Epub 2018 Oct 11.

National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, Manassas, VA, USA. Electronic address:

Rift Valley fever virus (RVFV) is an enveloped, segmented, negative sense RNA virus that replicates within the host's cytoplasm. To facilitate its replication, RVFV must utilize host cell processes and as such, these processes may serve as potential therapeutic targets. This review summarizes key host cell processes impacted by RVFV infection. Specifically the influence of RVFV on host transcriptional regulation, post-transcriptional regulation, protein half-life and availability, host signal transduction, trafficking and secretory pathways, cytoskeletal modulation, and mitochondrial processes and oxidative stress are discussed. Therapeutics targeted towards host processes that are essential for RVFV to thrive as well as their efficacy and importance to viral pathogenesis are highlighted.
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http://dx.doi.org/10.1016/j.antiviral.2018.10.004DOI Listing
December 2018

Direct and indirect pro-inflammatory cytokine response resulting from TC-83 infection of glial cells.

Virulence 2018 ;9(1):1403-1421

a National Center for Biodefense and Infectious Diseases , George Mason University , Manassas , VA , USA.

Venezuelan equine encephalitis virus (VEEV) is a neurotropic arbovirus that is highly infectious as an aerosol and can result in an encephalitic phenotype in infected individuals. VEEV infections are known to be associated with robust inflammation that eventually contributes to neurodegenerative phenotypes. In this study, we utilize the TC-83 strain of VEEV, which is known to induce the expression of IL-6, IL-8, and other pro-inflammatory cytokines. We had previously demonstrated that TC-83 infection resulted in changes in mitochondrial function, eventually resulting in mitophagy. In this manuscript, we provide data that links upstream mitochondrial dysfunction with downstream pro-inflammatory cytokine production in the context of microglia and astrocytoma cells. We also provide data on the role of bystander cells, which significantly contribute to the overall inflammatory load. Use of a mitochondrial-targeted antioxidant, mitoquinone mesylate, greatly reduced the inflammatory cytokine load and ameliorated bystander cell inflammatory responses more significantly than a broad-spectrum anti-inflammatory compound (BAY 11-7082). Our data suggest that the inflammatory mediators, especially IL-1β, may prime naïve cells to infection and lead to increased infection rates in microglial and astrocytoma cells. Cumulatively, our data suggest that the interplay between mitochondrial dysfunction and inflammatory events elicited in a neuronal microenvironment during a TC-83 infection may contribute to the spread of infection.
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http://dx.doi.org/10.1080/21505594.2018.1509668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6141141PMC
April 2019

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

Antiviral Res 2018 09 5;157:57-67. Epub 2018 Jul 5.

National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, Manassas, VA, USA. Electronic address:

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 EC 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.
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http://dx.doi.org/10.1016/j.antiviral.2018.07.005DOI Listing
September 2018

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

Arch Virol 2018 Oct 6;163(10):2687-2699. Epub 2018 Jun 6.

Virology Research Laboratory, Department of Dermatological, Infectious, and Parasitic Diseases, São José do Rio Preto School of Medicine (FAMERP), São José do Rio Preto, São Paulo, Brazil.

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.
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http://dx.doi.org/10.1007/s00705-018-3911-xDOI Listing
October 2018

Phosphoproteomic analysis reveals Smad protein family activation following Rift Valley fever virus infection.

PLoS One 2018 6;13(2):e0191983. Epub 2018 Feb 6.

National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, Manassas, Virginia, United States of America.

Rift Valley fever virus (RVFV) infects both ruminants and humans leading to a wide variance of pathologies dependent on host background and age. Utilizing a targeted reverse phase protein array (RPPA) to define changes in signaling cascades after in vitro infection of human cells with virulent and attenuated RVFV strains, we observed high phosphorylation of Smad transcription factors. This evolutionarily conserved family is phosphorylated by and transduces the activation of TGF-β superfamily receptors. Moreover, we observed that phosphorylation of Smad proteins required active RVFV replication and loss of NSs impaired this activation, further corroborating the RPPA results. Gene promoter analysis of transcripts altered after RVFV infection identified 913 genes that contained a Smad-response element. Functional annotation of these potential Smad-regulated genes clustered in axonal guidance, hepatic fibrosis and cell signaling pathways involved in cellular adhesion/migration, calcium influx, and cytoskeletal reorganization. Furthermore, chromatin immunoprecipitation confirmed the presence of a Smad complex on the interleukin 1 receptor type 2 (IL1R2) promoter, which acts as a decoy receptor for IL-1 activation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0191983PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5800665PMC
April 2018

Interactions of Alphavirus nsP3 Protein with Host Proteins.

Front Microbiol 2017 9;8:2652. Epub 2018 Jan 9.

National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, Fairfax, VA, United States.

Alphaviruses are members of the family and are grouped into two categories: arthritogenic and encephalitic. Arthritogenic alphavirus infections, as the name implies, are associated with arthritic outcomes while encephalitic alphavirus infections can lead to encephalitic outcomes in the infected host. Of the non-structural proteins (nsPs) that the viruses code for, nsP3 is the least understood in terms of function. Alphavirus nsP3s are characterized by regions with significantly conserved domain structure along with regions of high variability. Interactions of nsP3 with several host proteins have been documented including, stress granule-related proteins, dead box proteins, heat shock proteins, and kinases. In some cases, in addition to the interaction, requirement of the interaction to support infection has been demonstrated. An understanding of the proteomic network of nsP3 and the mechanisms by which these interactions support the establishment of a productive infection would make alphavirus nsP3 an interesting target for design of effective medical countermeasures.
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http://dx.doi.org/10.3389/fmicb.2017.02652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5767282PMC
January 2018

Alterations in the host transcriptome in vitro following Rift Valley fever virus infection.

Sci Rep 2017 10 30;7(1):14385. Epub 2017 Oct 30.

National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, Manassas, VA, 20110, USA.

Rift Valley fever virus (RVFV) causes major outbreaks among livestock, characterized by "abortion storms" in which spontaneous abortion occurs in almost 100% of pregnant ruminants. Humans can also become infected with mild symptoms that can progress to more severe symptoms, such as hepatitis, encephalitis, and hemorrhagic fever. The goal of this study was to use RNA-sequencing (RNA-seq) to analyze the host transcriptome in response to RVFV infection. G2/M DNA damage checkpoint, ATM signaling, mitochondrial dysfunction, regulation of the antiviral response, and integrin-linked kinase (ILK) signaling were among the top altered canonical pathways with both the attenuated MP12 strain and the fully virulent ZH548 strain. Although several mRNA transcripts were highly upregulated, an increase at the protein level was not observed for the selected genes, which was at least partially due to the NSs dependent block in mRNA export. Inhibition of ILK signaling, which is involved in cell motility and cytoskeletal reorganization, resulted in reduced RVFV replication, indicating that this pathway is important for viral replication. Overall, this is the first global transcriptomic analysis of the human host response following RVFV infection, which could give insight into novel host responses that have not yet been explored.
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http://dx.doi.org/10.1038/s41598-017-14800-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5662566PMC
October 2017

Altered mitochondrial dynamics as a consequence of Venezuelan Equine encephalitis virus infection.

Virulence 2017 11 25;8(8):1849-1866. Epub 2017 Jan 25.

a National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University , Manassas , VA , USA.

Mitochondria are sentinel organelles that are impacted by various forms of cellular stress, including viral infections. While signaling events associated with mitochondria, including those activated by pathogen associated molecular patterns (PAMPs), are widely studied, alterations in mitochondrial distribution and changes in mitochondrial dynamics are also beginning to be associated with cellular insult. Cells of neuronal origin have been demonstrated to display remarkable alterations in several instances, including neurodegenerative disorders. Venezuelan Equine Encephalitis Virus (VEEV) is a New World alphavirus that infects neuronal cells and contributes to an encephalitic phenotype. We demonstrate that upon infection by the vaccine strain of VEEV (TC-83), astrocytoma cells experience a robust drop in mitochondrial activity, which corresponds with an increased accumulation of reactive oxygen species (ROS) in an infection-dependent manner. Infection status also corresponds with a prominent perinuclear accumulation of mitochondria. Cellular enzymatic machinery, including PINK1 and Parkin, appears to be enriched in mitochondrial fractions as compared with uninfected cells, which is indicative of mitochondrial damage. Dynamin related protein 1 (Drp1), a protein that is associated with mitochondrial fission, demonstrated a modest enrichment in mitochondrial fractions of infected cells. Treatment with an inhibitor of mitochondrial fission, Mdivi-1, led to a decrease in caspase cleavage, suggesting that mitochondrial fission was likely to contribute to apoptosis of infected cells. Finally, our data demonstrate that mitophagy ensues in infected cells. In combination, our data suggest that VEEV infection results in significant changes in the mitochondrial landscape that may influence pathological outcomes in the infected cell.
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http://dx.doi.org/10.1080/21505594.2016.1276690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5810500PMC
November 2017

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

Biochim Biophys Acta Gen Subj 2017 Jan 7;1861(1 Pt A):3019-3029. Epub 2016 Sep 7.

School of Systems Biology, and the National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, Virginia, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbagen.2016.09.006DOI Listing
January 2017

The role of signal transducer and activator of transcription 3 in Rift Valley fever virus infection.

Virology 2016 09 16;496:175-185. Epub 2016 Jun 16.

National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, 10650 Pyramid Place, Manassas, VA, United States. Electronic address:

Rift Valley fever (RVF) is a zoonotic disease that can cause severe illness in humans and livestock, triggering spontaneous abortion in almost 100% of pregnant ruminants. In this study, we demonstrate that signal transducer and activator of transcription 3 (STAT3) is phosphorylated on its conserved tyrosine residue (Y705) following RVFV infection. This phosphorylation was dependent on a major virulence factor, the viral nonstructural protein NSs. Loss of STAT3 had little effect on viral replication, but rather resulted in cells being more susceptible to RVFV-induced cell death. Phosphorylated STAT3 translocated to the nucleus, coinciding with inhibition of fos, jun, and nr4a2 gene expression, and the presence of STAT3 and NSs at the nr4a2 promoter. NSs was found predominantly in the cytoplasm of STAT3 null cells, indicating that STAT3 influences NSs nuclear localization. Collectively, these data demonstrate that STAT3 functions in a pro-survival capacity through modulation of NSs localization.
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http://dx.doi.org/10.1016/j.virol.2016.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4969177PMC
September 2016

Venezuelan equine encephalitis virus non-structural protein 3 (nsP3) interacts with RNA helicases DDX1 and DDX3 in infected cells.

Antiviral Res 2016 07 20;131:49-60. Epub 2016 Apr 20.

National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, VA, United States. Electronic address:

The mosquito-borne New World alphavirus, Venezuelan equine encephalitis virus (VEEV) is a Category B select agent with no approved vaccines or therapies to treat infected humans. Therefore it is imperative to identify novel targets that can be targeted for effective therapeutic intervention. We aimed to identify and validate interactions of VEEV nonstructural protein 3 (nsP3) with host proteins and determine the consequences of these interactions to viral multiplication. We used a HA tagged nsP3 infectious clone (rTC-83-nsP3-HA) to identify and validate two RNA helicases: DDX1 and DDX3 that interacted with VEEV-nsP3. In addition, DDX1 and DDX3 knockdown resulted in a decrease in infectious viral titers. Furthermore, we propose a functional model where the nsP3:DDX3 complex interacts with the host translational machinery and is essential in the viral life cycle. This study will lead to future investigations in understanding the importance of VEEV-nsP3 to viral multiplication and apply the information for the discovery of novel host targets as therapeutic options.
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http://dx.doi.org/10.1016/j.antiviral.2016.04.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7113772PMC
July 2016

Lessons from the Ebola Outbreak: Action Items for Emerging Infectious Disease Preparedness and Response.

Ecohealth 2016 Mar 25;13(1):200-12. Epub 2016 Feb 25.

Department of Geography and Geoinformation Science, College of Science, George Mason University, Fairfax, VA, USA.

As the Ebola outbreak in West Africa wanes, it is time for the international scientific community to reflect on how to improve the detection of and coordinated response to future epidemics. Our interdisciplinary team identified key lessons learned from the Ebola outbreak that can be clustered into three areas: environmental conditions related to early warning systems, host characteristics related to public health, and agent issues that can be addressed through the laboratory sciences. In particular, we need to increase zoonotic surveillance activities, implement more effective ecological health interventions, expand prediction modeling, support medical and public health systems in order to improve local and international responses to epidemics, improve risk communication, better understand the role of social media in outbreak awareness and response, produce better diagnostic tools, create better therapeutic medications, and design better vaccines. This list highlights research priorities and policy actions the global community can take now to be better prepared for future emerging infectious disease outbreaks that threaten global public health and security.
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http://dx.doi.org/10.1007/s10393-016-1100-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7087787PMC
March 2016

Phosphorylation of Single Stranded RNA Virus Proteins and Potential for Novel Therapeutic Strategies.

Viruses 2015 Oct 12;7(10):5257-73. Epub 2015 Oct 12.

National Center for Biodefense and Infectious Disease, School of Systems Biology, George Mason University, 10650 Pyramid Place, Manassas, VA 20110, USA.

Post translational modification of proteins is a critical requirement that regulates function. Among the diverse kinds of protein post translational modifications, phosphorylation plays essential roles in protein folding, protein:protein interactions, signal transduction, intracellular localization, transcription regulation, cell cycle progression, survival and apoptosis. Protein phosphorylation is also essential for many intracellular pathogens to establish a productive infection cycle. Preservation of protein phosphorylation moieties in pathogens in a manner that mirrors the host components underscores the co-evolutionary trajectory of pathogens and hosts, and sheds light on how successful pathogens have usurped, either in part or as a whole, the host enzymatic machinery. Phosphorylation of viral proteins for many acute RNA viruses including Flaviviruses and Alphaviruses has been demonstrated to be critical for protein functionality. This review focuses on phosphorylation modifications that have been documented to occur on viral proteins with emphasis on acutely infectious, single stranded RNA viruses. The review additionally explores the possibility of repurposing Food and Drug Administration (FDA) approved inhibitors as antivirals for the treatment of acute RNA viral infections.
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http://dx.doi.org/10.3390/v7102872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4632380PMC
October 2015

Mutation of the BRCA1 SQ-cluster results in aberrant mitosis, reduced homologous recombination, and a compensatory increase in non-homologous end joining.

Oncotarget 2015 Sep;6(29):27674-87

Department of Radiation Oncology, Virginia Commonwealth University, Richmond, VA 23298, USA.

Mutations in the breast cancer susceptibility 1 (BRCA1) gene are catalysts for breast and ovarian cancers. Most mutations are associated with the BRCA1 N- and C-terminal domains linked to DNA double-strand break (DSB) repair. However, little is known about the role of the intervening serine-glutamine (SQ) - cluster in the DNA damage response beyond its importance in regulating cell cycle checkpoints. We show that serine-to-alanine alterations at critical residues within the SQ-cluster known to be phosphorylated by ATM and ATR result in reduced homologous recombination repair (HRR) and aberrant mitosis. While a S1387A BRCA1 mutant - previously shown to abrogate S-phase arrest in response to radiation - resulted in only a modest decrease in HRR, S1387A together with an additional alteration, S1423A (BRCA12P), reduced HRR to vector control levels and similar to a quadruple mutant also including S1457A and S1524A (BRCA14P). These effects appeared to be independent of PALB2. Furthermore, we found that BRCA14P promoted a prolonged and struggling HRR late in the cell cycle and shifted DSB repair from HRR to non-homologous end joining which, in the face of irreparable chromosomal damage, resulted in mitotic catastrophe. Altogether, SQ-cluster phosphorylation is critical for allowing adequate time for completing normal HRR prior to mitosis and preventing cells from entering G1 prematurely resulting in gross chromosomal aberrations.
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http://dx.doi.org/10.18632/oncotarget.4876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4695017PMC
September 2015

Repurposing FDA-approved drugs as therapeutics to treat Rift Valley fever virus infection.

Front Microbiol 2015 8;6:676. Epub 2015 Jul 8.

National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University Manassas, VA, USA.

There are currently no FDA-approved therapeutics available to treat Rift Valley fever virus (RVFV) infection. In an effort to repurpose drugs for RVFV treatment, a library of FDA-approved drugs was screened to determine their ability to inhibit RVFV. Several drugs from varying compound classes, including inhibitors of growth factor receptors, microtubule assembly/disassembly, and DNA synthesis, were found to reduce RVFV replication. The hepatocellular and renal cell carcinoma drug, sorafenib, was the most effective inhibitor, being non-toxic and demonstrating inhibition of RVFV in a cell-type and virus strain independent manner. Mechanism of action studies indicated that sorafenib targets at least two stages in the virus infectious cycle, RNA synthesis and viral egress. Computational modeling studies also support this conclusion. siRNA knockdown of Raf proteins indicated that non-classical targets of sorafenib are likely important for the replication of RVFV.
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http://dx.doi.org/10.3389/fmicb.2015.00676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4495339PMC
July 2015