The long term goal of our research is to determine the role of viral protease and interferon antagonism activity in coronavirus replication and pathogenesis. Coronaviruses are positive strand RNA viruses which cause a range of illness, from relatively mild respiratory disease (croup and common cold like symptoms due to HCoV- NL63, 229e or OC43) to severe acute respiratory syndrome (SARS) outbreaks in humans. To date, there are no effective vaccines or antiviral drugs to limit the pathogenesis of any coronavirus infection. Clearly, we need to identify and understand the viral and host factors that contribute to efficient viral replication and pathogenesis to aid in the development of new therapeutics. Previously, we identified the papain-like protease (PLpro) of SARS-CoV as a critical determinant of viral replication and pathogenesis. During coronavirus replication, the input genomic RNA is translated to produce a replicase polyprotein which must be processed by viral papain-like and 3C-like proteases to generate the replication complex. We showed that PLpro processes the replicase polyprotein at three sites using a consensus recognition site of LXGG, similar to the consensus sequence recognized by de-ubiquitinating enzymes (DUBs). We solved the X-ray crystal structure of PLpro and demonstrated that it is indeed a DUB. However, the role of its viral DUB activity in pathogenesis remains to be determined. In addition, we found that PLpro exhibits interferon antagonism activity and that catalytic activity may not be required for full interferon antagonism. We hypothesize that distinct residues within the papain-like protease domain of coronaviruses are critical for mediating substrate specificity and interferon antagonism and that identification of these sites will provide novel targets for antiviral intervention. Here, we propose to investigate the biology of coronavirus papain-like proteases by comparing and contrasting the activity of papain-like proteases of SARS-CoV, HCoV-NL63, and murine coronavirus A59.
Our specific aims are to: 1) Identify sites within the papain-like protease domains that are critical for polyprotein processing, deubiquitinating and deISGylating activity;2) Determine residues within coronavirus papain-like proteases that are important for interferon antagonism and identify cellular proteins that interact with PLPs to block innate immunity;3) Determine the domains, amino acids, and binding energies involved in protein-protein interactions between papain-like proteases and their binding partners;and 4) Determine if mutation of the PLpro or PLP2 domain alters evasion of ubiquitin- or ISG15-dependent innate immune responses. The results from this research will allow us to identify critical sites that mediate specificity of papain-like proteases and provide new insight into viral mechanisms for pathogenesis and evasion of the innate immune response.

Public Health Relevance

The goal of our project is to determine how specific parts of a virus contribute to causing severe acute respiratory syndrome (SARS). We propose to use biochemical and molecular methods to study the SARS coronavirus protease and identify potential Achilles'heels of this protein. The results from our research will allow us to identify sites in the viral protease that may be targeted for antiviral drug development or modified to improve vaccine development.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI085089-04
Application #
8485522
Study Section
Virology - A Study Section (VIRA)
Program Officer
Stemmy, Erik J
Project Start
2010-07-01
Project End
2015-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
4
Fiscal Year
2013
Total Cost
$712,986
Indirect Cost
$115,245
Name
Loyola University Chicago
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
791277940
City
Maywood
State
IL
Country
United States
Zip Code
60153
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
Tomar, Sakshi; Johnston, Melanie L; St John, Sarah E et al. (2015) Ligand-induced Dimerization of Middle East Respiratory Syndrome (MERS) Coronavirus nsp5 Protease (3CLpro): IMPLICATIONS FOR nsp5 REGULATION AND THE DEVELOPMENT OF ANTIVIRALS. J Biol Chem 290:19403-22
Chen, Yafang; Savinov, Sergey N; Mielech, Anna M et al. (2015) X-ray Structural and Functional Studies of the Three Tandemly Linked Domains of Non-structural Protein 3 (nsp3) from Murine Hepatitis Virus Reveal Conserved Functions. J Biol Chem 290:25293-306
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
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; 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
Mielech, Anna M; Kilianski, Andy; Baez-Santos, Yahira M et al. (2014) MERS-CoV papain-like protease has deISGylating and deubiquitinating activities. Virology 450-451:64-70
Agnihothram, Sudhakar; Yount Jr, Boyd L; Donaldson, Eric F et al. (2014) A mouse model for Betacoronavirus subgroup 2c using a bat coronavirus strain HKU5 variant. MBio 5:e00047-14
Báez-Santos, Yahira M; Mielech, Anna M; Deng, Xufang et al. (2014) Catalytic function and substrate specificity of the papain-like protease domain of nsp3 from the Middle East respiratory syndrome coronavirus. J Virol 88:12511-27

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