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.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Virology - A Study Section (VIRA)
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Stemmy, Erik J
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Loyola University Chicago
Schools of Medicine
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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
Báez-Santos, Yahira M; Barraza, Scott J; Wilson, Michael W et al. (2014) X-ray structural and biological evaluation of a series of potent and highly selective inhibitors of human coronavirus papain-like proteases. J Med Chem 57:2393-412
Mielech, Anna M; Chen, Yafang; Mesecar, Andrew D et al. (2014) Nidovirus papain-like proteases: multifunctional enzymes with protease, deubiquitinating and deISGylating activities. Virus Res 194:184-90
Deng, Xufang; Agnihothram, Sudhakar; Mielech, Anna M et al. (2014) A chimeric virus-mouse model system for evaluating the function and inhibition of papain-like proteases of emerging coronaviruses. J Virol 88:11825-33
Ratia, Kiira; Kilianski, Andrew; Baez-Santos, Yahira M et al. (2014) Structural Basis for the Ubiquitin-Linkage Specificity and deISGylating activity of SARS-CoV papain-like protease. PLoS Pathog 10:e1004113
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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Deng, Xufang; StJohn, Sarah E; Osswald, Heather L et al. (2014) Coronaviruses resistant to a 3C-like protease inhibitor are attenuated for replication and pathogenesis, revealing a low genetic barrier but high fitness cost of resistance. J Virol 88:11886-98
Kilianski, Andy; Baker, Susan C (2014) Cell-based antiviral screening against coronaviruses: developing virus-specific and broad-spectrum inhibitors. Antiviral Res 101:105-12
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

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