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J. Virol. doi:10.1128/JVI.02747-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Proteolytic Processing and Deubiquitinating Activity of Papain-Like Proteases of Human Coronavirus NL63

Department of Microbiology and Immunology, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois; Department of Biochemistry and Molecular Biology, Beijing Institute of Radiation Medicine, Beijing, China; Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois; and Department of Biochemistry, Emory University School of Medicine, Atlanta, GA

^* To whom correspondence should be addressed. Email: sbaker1{at}lumc.edu.

	Abstract

Human coronavirus NL63 (HCoV-NL63), a common human respiratory pathogen, is associated with both upper and lower respiratory tract disease in children and adults. Currently, no antiviral drugs are available to treat coronavirus infections, thus potential drug targets need to be identified and characterized. Here, we identify HCoV-NL63 replicase gene products and characterize two viral papain-like proteases, PLP1 and PLP2, that process the viral replicase polyprotein. We generated polyclonal antisera directed against two of the predicted replicase nonstructural proteins (nsp3 and nsp4) and detected replicase proteins from HCoV-NL63-infected LLC-MK2 cells by immunofluorescence, immunoprecipitation, and western blot assays. We found that HCoV-NL63 replicase products can be detected at 24 hours post-infection and that these proteins accumulate in perinuclear sites, consistent with membrane-associated replication complexes. To determine which viral proteases are responsible for processing these products, we generated constructs representing the amino-terminal end of the HCoV-NL63 replicase gene, and established protease cis-cleavage assays. We found that PLP1 processes cleavage site 1 to release nsp1, whereas PLP2 is responsible for processing both cleavage sites 2 and 3 to release nsp2 and nsp3. We expressed and purified PLP2 and used a peptide-based assay to identify the cleavage sites recognized by this enzyme. Furthermore, by using K48-linked hexa-ubiquitin substrate and ubiquitin-vinylsulfone inhibitor specific for deubiquitinating enzymes (DUBs), we confirmed that, like severe acute respiratory syndrome (SARS) coronavirus PLpro, HCoV-NL63 PLP2 has DUB activity. The identification of the replicase products and characterization of HCoV-NL63 papain-like protease-deubiquitinating activity will facilitate comparative studies of coronavirus proteases and aid in the development of novel antiviral reagents directed against human pathogens such as HCoV-NL63 and SARS-CoV.

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