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Principal Investigator: Susan C. Baker
Organization: LOYOLA UNIVERSITY CHICAGO
Fiscal Year: 2019
Award: $705,182
Funding agency: National Institute of Allergy and Infectious Diseases
DESCRIPTION (provided by applicant): The goal of our research is to determine how viral proteases function in the replication and pathogenesis of coronaviruses (CoVs). CoVs are a family of positive strand RNA viruses and include Severe Acute Respiratory Syndrome (SARS) CoV and Middle East Respiratory Syndrome (MERS) CoV which are significant human pathogens with pandemic potential. Previously, we dissected the multifunctional nature of CoV papain-like proteases (PLPs) and found that CoV PLPs cleave the viral replicase polyprotein, act as deubiquitinases (DUBs) and deISGylases (deISGs) by removing ubiquitin (Ub) or ISG15 conjugated to lysine residues on proteins, and that PLPs can antagonize the innate immune response, likely by deubiquitylating signaling molecules. Using detailed biochemical and PLP-Ub co-crystal structural analysis, we identified residues within CoV PLPs that differentially affec enzymatic activity in vitro and in cell-based assays. Our initial studies were performed using SARS-CoV PLpro. Here we provide preliminary in vitro and structural data demonstrating that these results can be extended to the BSL-2 model coronavirus mouse hepatitis virus (MHV-A59) papain-like protease. We hypothesize that multifunctional PLP/DUB activity contributes to viral pathogenesis and that selectively disrupting DUB activity will allow activation of innate immunity and reduced viral pathogenesis. To test this hypothesis, we will determine if a modified PLP/DUB enzymatic activity alters viral replication, innate immune response or pathogenesis. We will use reverse genetics to generate murine CoVs encoding PLPs with distinct enzymatic profiles such as DUB deficient, deISGylation deficient, or hyperactive protease. These novel viruses will be evaluated in cell culture and in mice for kinetics of viral RNA synthesis, production of infectious virus, and kinetics of activation of innate immune responses. To extend these studies to other CoVs, we will determine the enzymatic profile (EP) and enzymatic fingerprint (EF) of alpha- and beta-CoV papain-like proteases including bat CoV PLPs. We will express the PLP domain from 10 different CoV species and determine the peptide cleavage activity, deubiquitinating activity, deISGylating activity, and lysine-linkage preferences for each enzyme. With this profile in hand, we will use existing and new X-ray structures combined to guide mutagenesis experiments to differentially disrupt DUB activity and identify the fingerprint associated with reduced DUB activity. We will also determine the role of differential activity in regulating the innate immune response in bat cells. Also, we identified an
interaction of the CoV ADP-ribose-1"-phosphatase (ADRP) domain with PLP and we will determine the effect of modifying this interaction on enzymatic activity, viral replication and pathogenesis. These studies will reveal new information on viral protease/DUB activity that will be useful for designing antiviral therapies and vaccines for coronaviruses and other protease/DUB-encoding viruses.
Terms: <ADP Ribose><ADPribose><APF-1><ATP-Dependent Proteolysis Factor 1><Adenosine 5'-(trihydrogen diphosphate), P'-5-ester with D-ribose><Adenosine 5'-Diphosphoribose><Adenosine Diphosphate Ribose><Adenosine Diphosphoribose><Affect><Amino Acid Sequence><Animals><Antiviral Agents><Antiviral Drugs><Antiviral Therapy><Antivirals><Assay><Attenuated><Bats><Bioassay><Biochemical><Biologic Assays><Biological Assay><Body Weight decreased><C57BL/6 Mouse><Cell Body><Cell Culture Techniques><Cells><Chiroptera><Cleaved cell><Coronaviridae><Coronavirus><Crystallization><Data><Disease><Disease Outbreaks><Disorder><Enzyme Gene><Enzymes><Esteroproteases><FDA approved><Family><Fingerprint><Funding><General Viruses><Genetic Alteration><Genetic Change><Genetic defect><Genetics-Mutagenesis><Goals><HMG-20><Hand><Health><High Mobility Protein 20><Human><Hyperactive behavior><Hyperactivity><Hyperkinesia><Hyperkinesis><Hyperkinetic Movements><ISG15><ISG15 gene><Immune><Immune response><Immune system><Immunes><Immunological response><In Vitro><Innate Immune Response><Innate Immunity><Interferon-Induced Protein IFI-15K><Intervention><Intervention Strategies><Kinetics><L-Lysine><Liver><Lysine><MERS-CoV><Mice><Mice Mammals><Middle East Respiratory Syndrome><Middle East Respiratory Syndrome Coronavirus><Modeling><Modern Man><Molecular Dynamics Simulation><Monitor><Motor Hyperactivity><Mouse Hepatitis Virus><Murine><Murine Gastroenteritis Virus><Murine hepatitis virus><Mus><Mutagenesis><Mutagenesis Molecular Biology><Mutate><Mutation><Native Immunity><Natural Immunity><Nature><Non-Specific Immunity><Nonspecific Immunity><Outbreaks><Papain><Pathogenesis><Pathogenicity><Pathology><Peptidases><Peptide Hydrolases><Peptides><Phosphatases><Phosphohydrolases><Phosphomonoesterases><Phosphoric Monoester Hydrolases><Pneumonia><Polyproteins><Primary Protein Structure><Production><Protease Domain><Protease Gene><Proteases><Proteinases><Proteins><Proteolytic Domain><Proteolytic Enzymes><RNA Viruses><RNA chemical synthesis><RNA synthesis><Research><Roentgen Rays><Role><SARS><Severe Acute Respiratory Syndrome><Signaling Molecule><Site-Directed Mutagenesis><Site-Specific Mutagenesis><Structure><System><Targeted DNA Modification><Targeted Modification><Testing><Ubiquitin><Vaccines><Viral><Viral Burden><Viral Load><Viral Load result><Viral Pathogenesis><Virus><Virus Replication><Weight Loss><Weight Reduction><Work><X-Radiation><X-Rays><X-Rays Radiation><Xrays><allergic/immunologic body system><allergic/immunologic organ system><anti-viral agents><anti-viral drugs><anti-viral therapy><anti-virals><base><body weight loss><cell culture><cleaved><cytokine><design><designing><experiment><experimental research><experimental study><genome mutation><hepatic body system><hepatic organ system><host response><human pathogen><immunoresponse><in vitro activity><interventional strategy><molecular dynamics><novel virus><pandemic><pandemic disease><preference><protein sequence><public health relevance><replicase><response><reverse genetics><social role><temperature sensitive mutant><viral RNA><viral infectious disease treatment><viral multiplication><viral replication><virus RNA><virus multiplication><virus pathogenesis><wt-loss>