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.

Public Health Relevance

Coronaviruses can emerge from animal reservoirs and infect humans resulting in outbreaks of pneumonia, such as Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS). We want to identify how coronaviruses evade the early warning system of the immune response. These studies will teach us how coronaviruses, and potentially other viruses, slip under the radar of our immune defenses and cause disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI085089-07
Application #
9096719
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Stemmy, Erik J
Project Start
2010-07-01
Project End
2020-06-30
Budget Start
2016-07-01
Budget End
2017-06-30
Support Year
7
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Loyola University Chicago
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
791277940
City
Maywood
State
IL
Country
United States
Zip Code
60153
O'Brien, Amornrat; Mettelman, Robert C; Volk, Aaron et al. (2018) Characterizing replication kinetics and plaque production of type I feline infectious peritonitis virus in three feline cell lines. Virology 525:1-9
Deng, Xufang; Baker, Susan C (2018) An ""Old"" protein with a new story: Coronavirus endoribonuclease is important for evading host antiviral defenses. Virology 517:157-163
Clasman, Jozlyn R; Báez-Santos, Yahira M; Mettelman, Robert C et al. (2017) X-ray Structure and Enzymatic Activity Profile of a Core Papain-like Protease of MERS Coronavirus with utility for structure-based drug design. Sci Rep 7:40292
Daczkowski, Courtney M; Dzimianski, John V; Clasman, Jozlyn R et al. (2017) Structural Insights into the Interaction of Coronavirus Papain-Like Proteases and Interferon-Stimulated Gene Product 15 from Different Species. J Mol Biol 429:1661-1683
Deng, Xufang; Hackbart, Matthew; Mettelman, Robert C et al. (2017) Coronavirus nonstructural protein 15 mediates evasion of dsRNA sensors and limits apoptosis in macrophages. Proc Natl Acad Sci U S A 114:E4251-E4260
St John, Sarah E; Anson, Brandon J; Mesecar, Andrew D (2016) X-Ray Structure and Inhibition of 3C-like Protease from Porcine Epidemic Diarrhea Virus. Sci Rep 6:25961
St John, Sarah E; Tomar, Sakshi; Stauffer, Shaun R et al. (2015) Targeting zoonotic viruses: Structure-based inhibition of the 3C-like protease from bat coronavirus HKU4--The likely reservoir host to the human coronavirus that causes Middle East Respiratory Syndrome (MERS). Bioorg Med Chem 23:6036-48
Báez-Santos, Yahira M; St John, Sarah E; Mesecar, Andrew D (2015) The SARS-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds. Antiviral Res 115:21-38
Mielech, Anna M; Deng, Xufang; Chen, Yafang et al. (2015) Murine coronavirus ubiquitin-like domain is important for papain-like protease stability and viral pathogenesis. J Virol 89:4907-17
St John, Sarah E; Therkelsen, Matthew D; Nyalapatla, Prasanth R et al. (2015) X-ray structure and inhibition of the feline infectious peritonitis virus 3C-like protease: Structural implications for drug design. Bioorg Med Chem Lett 25:5072-7

Showing the most recent 10 out of 28 publications