Structure-Guided Mutagenesis Alters Deubiquitinating Activity and Attenuates Pathogenesis of a Murine Coronavirus

Coronaviruses express a multifunctional papain-like protease, termed papain-like protease 2 (PLP2). PLP2 acts as a protease that cleaves the viral replicase polyprotein and as a deubiquitinating (DUB) enzyme which removes ubiquitin (Ub) moieties from ubiquitin-conjugated proteins. Previous studies i...

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Bibliographic Details
Published in:Journal of virology 2020-05, Vol.94 (11)
Main Authors: Deng, Xufang, Chen, Yafang, Mielech, Anna M, Hackbart, Matthew, Kesely, Kristina R, Mettelman, Robert C, O'Brien, Amornrat, Chapman, Mackenzie E, Mesecar, Andrew D, Baker, Susan C
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Coronavirus Infections - virology
Host-Pathogen Interactions
Interferon Type I - metabolism
Macrophages - immunology
Macrophages - metabolism
Macrophages - virology
Mice
Models, Molecular
Murine hepatitis virus - pathogenicity
Murine hepatitis virus - physiology
Mutagenesis
Pathogenesis and Immunity
Protein Conformation
Structure-Activity Relationship
Ubiquitination
Viral Proteins - chemistry
Viral Proteins - genetics
Viral Proteins - metabolism
Virulence
Virus Replication
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Summary:Coronaviruses express a multifunctional papain-like protease, termed papain-like protease 2 (PLP2). PLP2 acts as a protease that cleaves the viral replicase polyprotein and as a deubiquitinating (DUB) enzyme which removes ubiquitin (Ub) moieties from ubiquitin-conjugated proteins. Previous studies implicated PLP2/DUB activity as a negative regulator of the host interferon (IFN) response, but the role of DUB activity during virus infection was unknown. Here, we used X-ray structure-guided mutagenesis and functional studies to identify amino acid substitutions within the ubiquitin-binding surface of PLP2 that reduced DUB activity without affecting polyprotein processing activity. We engineered a DUB mutation (Asp1772 to Ala) into a murine coronavirus and evaluated the replication and pathogenesis of the DUB mutant virus (DUBmut) in cultured macrophages and in mice. We found that the DUBmut virus replicates similarly to the wild-type (WT) virus in cultured cells, but the DUBmut virus activates an IFN response at earlier times compared to the wild-type virus infection in macrophages, consistent with DUB activity negatively regulating the IFN response. We compared the pathogenesis of the DUBmut virus to that of the wild-type virus and found that the DUBmut-infected mice had a statistically significant reduction (  
ISSN:0022-538X
1098-5514
DOI:10.1128/JVI.01734-19