title: A chimeric virus-mouse model system for evaluating the function and inhibition of papain-like proteases of emerging coronaviruses.
abstract: To combat emerging coronaviruses, developing safe and efficient platforms to evaluate viral protease activities and the efficacy of protease inhibitors is a high priority. Here, we exploit a biosafety level 2 (BSL-2) chimeric Sindbis virus system to evaluate protease activities and the efficacy of inhibitors directed against the papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus (SARS-CoV), a biosafety level 3 (BSL-3) pathogen. We engineered Sindbis virus to coexpress PLpro and a substrate, murine interferon-stimulated gene 15 (ISG15), and found that PLpro mediates removal of ISG15 (deISGylation) from cellular proteins. Mutation of the catalytic cysteine residue of PLpro or addition of a PLpro inhibitor blocked deISGylation in virus-infected cells. Thus, deISGylation is a marker of PLpro activity. Infection of alpha/beta interferon receptor knockout (IFNAR(-/-)) mice with these chimeric viruses revealed that PLpro deISGylation activity removed ISG15-mediated protection during viral infection. Importantly, administration of a PLpro inhibitor protected these mice from lethal infection, demonstrating the efficacy of a coronavirus protease inhibitor in a mouse model. However, this PLpro inhibitor was not sufficient to protect the mice from lethal infection with SARS-CoV MA15, suggesting that further optimization of the delivery and stability of PLpro inhibitors is needed. We extended the chimeric-virus platform to evaluate the papain-like protease/deISGylating activity of Middle East respiratory syndrome coronavirus (MERS-CoV) to provide a small-animal model to evaluate PLpro inhibitors of this recently emerged pathogen. This platform has the potential to be universally adaptable to other viral and cellular enzymes that have deISGylating activities. Importance: Evaluating viral protease inhibitors in a small-animal model is a critical step in the path toward antiviral drug development. We modified a biosafety level 2 chimeric virus system to facilitate evaluation of inhibitors directed against highly pathogenic coronaviruses. We used this system to demonstrate the in vivo efficacy of an inhibitor of the papain-like protease of severe acute respiratory syndrome coronavirus. Furthermore, we demonstrate that the chimeric-virus system can be adapted to study the proteases of emerging human pathogens, such as Middle East respiratory syndrome coronavirus. This system provides an important tool to rapidly assess the efficacy of protease inhibitors targeting existing and emerging human pathogens, as well as other enzymes capable of removing ISG15 from cellular proteins.
authors.affiliation: Department of Microbiology and Immunology, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois, USA.
authors.forename: Xufang
authors.initials: X
authors.lastname: Deng
authors.affiliation: Departments of Epidemiology and Microbiology and Immunology, University of North Carolina, Chapel Hill, North Carolina, USA.
authors.forename: Sudhakar
authors.initials: S
authors.lastname: Agnihothram
authors.affiliation: Department of Microbiology and Immunology, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois, USA.
authors.forename: Anna M
authors.initials: AM
authors.lastname: Mielech
authors.affiliation: Department of Microbiology and Immunology, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois, USA.
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Nichols
authors.affiliation: Vahlteich Medicinal Chemistry Core, College of Pharmacy, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Wilson
authors.affiliation: Departments of Biological Science and Chemistry, Purdue University, West Lafayette, Indiana, USA.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: StJohn
authors.affiliation: Vahlteich Medicinal Chemistry Core, College of Pharmacy, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Scott D
authors.initials: SD
authors.lastname: Larsen
authors.affiliation: Departments of Biological Science and Chemistry, Purdue University, West Lafayette, Indiana, USA.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Mesecar
authors.affiliation: Department of Internal Medicine and Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Deborah J
authors.initials: DJ
authors.lastname: Lenschow
authors.affiliation: Departments of Epidemiology and Microbiology and Immunology, University of North Carolina, Chapel Hill, North Carolina, USA.
authors.forename: Ralph S
authors.initials: RS
authors.lastname: Baric
authors.affiliation: Department of Microbiology and Immunology, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois, USA sbaker1@luc.edu.
authors.forename: Susan C
authors.identifier: http://orcid.org/0000-0001-6485-8143
authors.initials: SC
authors.lastname: Baker
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D017934:Coronavirus / Q000201:enzymology / Q000502:physiology*; D006224:Cricetinae; D004195:Disease Models, Animal; D051379:Mice; D010206:Papain / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D014709:Vero Cells
chemical_list: D010447:Peptide Hydrolases; D010206:Papain
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