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Principal Investigator: ANNE GOLDFELD
Organization: BOSTON CHILDREN'S HOSPITAL
Fiscal Year: 2020
Award: $270,500
Funding agency: National Institute of Allergy and Infectious Diseases
Ebola virus (EBOV) and Marburg virus (MARV) are highly lethal hemorrhagic fever viruses of
the Filoviridae family of viruses. Currently, there are no effective oral or easily scalable therapies for
either virus. Both EBOV and MARV manifest vigorous anti-host evasion strategies focused on blocking
the host antiviral interferon response. Both viruses interfere with host cell sensing of infection via their
structural protein VP35, which inhibits viral dsRNA sensing and triggering of the host antiviral response.
Therapeutic strategies that enhance host pathways that are inhibited by these viruses could therefore
be of significant value. We have discovered that the well-tolerated FDA-approved oral anti-parasitic
drug nitazoxanide (NTZ) inhibits infectious EBOV replication in human A549 cells in vitro. Furthermore,
we find that NTZ enhances the RIG-I-like receptor (RLR) proteins retinoic-acid-inducible protein I (RIG-
I) and Melanoma Differentiation-Associated protein 5 (MDA5), their common adaptor protein
mitochondrial antiviral signaling protein (MAVS), the IFN-inducible double-stranded (ds) RNA sensor
protein kinase R (PKR), and interferon signaling pathways. In addition, using deadCas9/CRISPR
editing, we ablated RIG-I and PKR in A549 cells, and found that EBOV growth was significantly
increased and in parallel NTZ's inhibitory effects were significantly attenuated. These results thus
indicate that RIG-I and PKR are both required and non-redundant host restriction factors for EBOV.
Moreover, PKR and RIG-I are both targeted by and necessary for NTZ's action indicating that NTZ
counteracts the EBOV VP35 protein's ability to block RIG-I and PKR sensing of EBOV infection. In Aim
1 of this proposal, we will test the hypothesis that NTZ also inhibits MARV in vitro, which we strongly
expect given the importance of MARV's VP-35 protein in its pathogenesis. In Aim 1, we will also test
our hypothesis that NTZ inhibits both EBOV and MARV in vivo in the guinea pig model. In Aim 2, we
will test the impact of MDA5 or MAVS inhibition, in addition to studying RIG-I, PKR or GADD34
depletion on MARV replication. We will compare these results to experiments evaluating EBOV
replication in monocytic THP-1 cells. These experiments will evaluate the role of these discrete
sensors in NTZ's inhibitory effects upon EBOV and MARV and it will test the hypothesis that NTZ works
through PKR and RIG-I in inhibition of MARV as it does in EBOV infection. Building on these studies,
using an unbiased RNA-seq approach, we will determine transcriptomic signatures of cellular
perturbations induced by NTZ in the setting of EBOV and MARV infection allowing us to define host
immune regulatory circuits involved in cytoplasmic sensing of EBOV and MARV and the impact of NTZ.
We anticipate that these studies will provide scientific underpinning for repurposing NTZ as a therapy
for EBOV and MARV and uncover key innate immune molecules that control their pathogenesis.
Terms: <2-(Acetolyloxy)-N-(5-nitro-2-thiazolyl)benzamide><A549><ATP-protein phosphotransferase><ATRA><Adaptor Protein><Adaptor Protein Gene><Adaptor Signaling Protein><Adaptor Signaling Protein Gene><Africa><Animal Model><Animal Models and Related Studies><Animals><Anti-Viral Response><Antiparasitic Agents><Antiparasitic Drugs><Antiparasitics><Antiviral Agents><Antiviral Drugs><Antiviral Response><Antivirals><Attenuated><Bleeding><Blood monocyte><CRISPR><CRISPR/Cas system><Cavia><Cell Body><Cells><Clustered Regularly Interspaced Short Palindromic Repeats><Data><Development><Disease Outbreaks><Double-Stranded RNA><Drugs><EBOV><Ebola virus><Ebola virus VP35 protein><FDA approved><Family><Filoviridae><Filoviridae Infections><Filovirus><Foundations><Frankfurt-Marburg Syndrome Virus><Future><Gene Action Regulation><Gene Expression Regulation><Gene Regulation><Gene Regulation Process><Gene Transcription><Generalized Growth><Genes><Genetic Transcription><Goals><Growth><Guinea Pigs><Guinea Pigs Mammals><Hemorrhage><Host Factor><Host Factor Protein><Human><IFN><IFN-regulatory factor 3><IRF-3 protein><IRF3><IRF3 gene><Immune><Immune Evasion><Immune response><Immunes><Immunochemical Immunologic><Immunologic><Immunological><Immunological response><Immunologically><Immunologics><In Vitro><Infection><Institutes><Integration Host Factors><Interferon Regulatory Factor 3><Interferons><Kinase Family Gene><Knock-out><Knockout><Laboratories><Malignant Melanoma><Marburg><Marburg virus><Marrow monocyte><Medication><Messenger RNA><Mitochondria><Mitochondrial Proteins><Modeling><Modern Man><Non-Polyadenylated RNA><Oral><Outbreaks><Parasiticides><Pathogenesis><Pathway interactions><Pharmaceutic Preparations><Pharmaceutical Preparations><Phosphatases><Phosphohydrolases><Phosphomonoesterases><Phosphoric Monoester Hydrolases><Preventative vaccine><Preventive vaccine><Prophylactic vaccine><Protein Inhibition><Protein Kinase><Proteins><RNA><RNA Expression><RNA Gene Products><RNA Seq><RNA Viruses><RNA amplification><RNA sequencing><RNAseq><Receptor Protein><Retinoic Acid><Ribonucleic Acid><Role><Signal Pathway><Signaling Factor Proto-Oncogene><Signaling Pathway Gene><Signaling Protein><Structural Protein><Testing><Therapeutic><Tissue Growth><Trans Vitamin A Acid><Transcription><Tretinoin><Tretinoinum><VSV><Vesicular Stomatitis Virus><Vesicular stomatitis Indiana virus><Viral><Viral Diseases><Viral Pathogenesis><Virus><Virus Diseases><Virus Replication><Vitamin A Acid><Work><adapter protein><all-trans-Retinoic Acid><all-trans-Vitamin A acid><anti-viral agents><anti-viral drugs><anti-virals><blood loss><developmental><drug/agent><dsRNA><ebolavirus><experiment><experimental research><experimental study><filovirus infections><glycogen synthase a kinase><hemorrhagic fever virus><host response><hydroxyalkyl protein kinase><immunoresponse><in vivo><insight><mRNA><melanoma><mitochondrial><model of animal><model organism><monocyte><new drug target><new druggable target><new pharmacotherapy target><new therapeutic target><new therapy target><nitazoxanide><novel drug target><novel druggable target><novel pharmacotherapy target><novel therapeutic target><novel therapy target><ontogeny><pathway><phosphorylase b kinase kinase><prevent><preventing><protein kinase R><receptor><response><sensor><social role><therapeutic target><trans-Retinoic Acid><transcriptome sequencing><transcriptomics><viral infection><viral multiplication><viral replication><virus infection><virus multiplication><virus pathogenesis><virus-induced disease>