Replication of positive-strand RNA viruses requires processing of virus-encoded polyproteins by viral proteases. Proteases mediate temporal and hierarchical processing of constantly changing polyprotein precursors, which in turn determine the successful viral replication and pathogenesis. Proteases of RNA viruses therefore represent important targets for development of broadly applicable inhibitors of virus replication. Rational design of protease inhibitors has targeted conserved structure/function elements such as active-site cavities and substrate-binding domains. However, RNA viruses have shown the capacity to select for resistance to these types of inhibitors. Thus, it is important to identify determinants of protease activity that are independent from catalytic or substrate sites, and which can be targeted by inhibitors that can prevent emergence of resistance. The goals of this proposal are to use the coronavirus murine hepatitis nsp5 protease as a model to: 1) test the role of nsp5 precursors, and non-catalytic, non-substrate binding structure-function determinants, in protease activity and specificity;and 2) identify and test the role of novel intramolecular residue networks in nsp5 polyprotein processing in vitro and during virus replication in culture. Coronaviruses are positive-strand RNA viruses, important pathogens of humans, and express a polyprotein composed of sixteen nonstructural replicase protein domains (nsp1-16), of which nsp4-16 are processed by the nsp5 protease (3CLpro, Mpro) at eleven cleavage sites. Nsp5 possesses two chymotrypsin-like domains, whose interface comprises the active-site cavity and substrate-binding regions, and a unique domain 3 of unknown function. In vitro biochemical and structural studies have concluded that functional nsp5 dimeric, and have identified structure/function determinants of dimerization and catalysis, but none of these have been tested in a replicating virus.
Specific Aims 1 and 2 will test predicted nsp5 structure/function determinants in the context of intermediate precursors and membrane-associated complexes. Experiments in Specific Aim 3 will perform bioinformatic analysis of extensive sequence and structure datasets of nsp5 and related proteases, in order to predict intramolecular residue networks based on: a) iterative mutation and reversion of conditional temperature sensitive mutant viruses;b) co-evolution and structural proximity;and c) protein flexibility and movement. The role of predicted network residues will be tested in reverse genetic replication studies of mutant viruses, and by in vitro biochemical assays of purified nsp5. The proposed studies will answer fundamental questions in coronavirus polyprotein processing and will define critical new intramolecular networks, communication pathways, and determinants of the evolution and function of nidovirus nsp5 and orthologs. The outcome of the proposed experiments will be of high impact and significance by establishing universally applicable systems for identification and testing of novel non-catalytic RNA virus protease targets for inhibition or attenuation of virus replication.

Public Health Relevance

RNA viruses such as coronaviruses are important pathogens of humans, and RNA virus proteases are important targets for development of inhibitors. Coronavirus nsp5 protease is essential for virus replication and mediates processing of the replicase polyprotein.
The Aims of this proposal will define novel conserved structure and sequence determinants of coronavirus nsp5 during replication, and identify and test the role of intramolecular networks of residues as regulators of RNA virus protease activity and specificity during replication.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Virology - B Study Section (VIRB)
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Park, Eun-Chung
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Vanderbilt University Medical Center
Schools of Medicine
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Athmer, Jeremiah; Fehr, Anthony R; Grunewald, Matthew et al. (2017) In Situ Tagged nsp15 Reveals Interactions with Coronavirus Replication/Transcription Complex-Associated Proteins. MBio 8:
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
Mounce, Bryan C; Cesaro, Teresa; Moratorio, Gonzalo et al. (2016) Inhibition of Polyamine Biosynthesis Is a Broad-Spectrum Strategy against RNA Viruses. J Virol 90:9683-9692
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
Smith, Everett Clinton; Case, James Brett; Blanc, Hervé et al. (2015) Mutations in coronavirus nonstructural protein 10 decrease virus replication fidelity. J Virol 89:6418-26
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
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
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
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
Jacobs, Jon; Grum-Tokars, Valerie; Zhou, Ya et al. (2013) Discovery, synthesis, and structure-based optimization of a series of N-(tert-butyl)-2-(N-arylamido)-2-(pyridin-3-yl) acetamides (ML188) as potent noncovalent small molecule inhibitors of the severe acute respiratory syndrome coronavirus (SARS-CoV) 3CL pr J Med Chem 56:534-46

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