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Analysis of Coronavirus Temperature-Sensitive Mutants Reveals an Interplay between the Macrodomain and Papain-Like Protease Impacting Replication and Pathogenesis.
J Virol. 2019 06 15; 93(12)JV

Abstract

Analysis of temperature-sensitive (ts) mutant viruses is a classic method allowing researchers to identify genetic loci involved in viral replication and pathogenesis. Here, we report genetic analysis of a ts strain of mouse hepatitis virus (MHV), tsNC11, focusing on the role of mutations in the macrodomain (MAC) and the papain-like protease 2 (PLP2) domain of nonstructural protein 3 (nsp3), a component of the viral replication complex. Using MHV reverse genetics, we generated a series of mutant viruses to define the contributions of macrodomain- and PLP2-specific mutations to the ts phenotype. Viral replication kinetics and efficiency-of-plating analysis performed at permissive and nonpermissive temperatures revealed that changes in the macrodomain alone were both necessary and sufficient for the ts phenotype. Interestingly, mutations in the PLP2 domain were not responsible for the temperature sensitivity but did reduce the frequency of reversion of macrodomain mutants. Coimmunoprecipitation studies are consistent with an interaction between the macrodomain and PLP2. Expression studies of the macrodomain-PLP2 portion of nsp3 indicate that the ts mutations enhance proteasome-mediated degradation of the protein. Furthermore, we found that during virus infection, the replicase proteins containing the MAC and PLP2 mutations were more rapidly degraded at the nonpermissive temperature than were the wild-type proteins. Importantly, we show that the macrodomain and PLP2 mutant viruses trigger production of type I interferon in vitro and are attenuated in mice, further highlighting the importance of the macrodomain-PLP2 interplay in viral pathogenesis.IMPORTANCE Coronaviruses (CoVs) are emerging human and veterinary pathogens with pandemic potential. Despite the established and predicted threat these viruses pose to human health, there are currently no approved countermeasures to control infections with these viruses in humans. Viral macrodomains, enzymes that remove posttranslational ADP-ribosylation of proteins, and viral multifunctional papain-like proteases, enzymes that cleave polyproteins and remove polyubiquitin chains via deubiquitinating activity, are two important virulence factors. Here, we reveal an unanticipated interplay between the macrodomain and the PLP2 domain that is important for replication and antagonizing the host innate immune response. Targeting the interaction of these enzymes may provide new therapeutic opportunities to treat CoV disease.

Authors+Show Affiliations

Department of Microbiology and Immunology, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois, USA.Department of Microbiology and Immunology, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois, USA.Department of Microbiology and Immunology, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois, USA.Department of Microbiology and Immunology, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois, USA.Department of Mathematics and Statistics, Loyola University Chicago, Chicago, Illinois, USA.Department of Microbiology and Immunology, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois, USA sbaker1@luc.edu.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

30918076

Citation

Deng, Xufang, et al. "Analysis of Coronavirus Temperature-Sensitive Mutants Reveals an Interplay Between the Macrodomain and Papain-Like Protease Impacting Replication and Pathogenesis." Journal of Virology, vol. 93, no. 12, 2019.
Deng X, Mettelman RC, O'Brien A, et al. Analysis of Coronavirus Temperature-Sensitive Mutants Reveals an Interplay between the Macrodomain and Papain-Like Protease Impacting Replication and Pathogenesis. J Virol. 2019;93(12).
Deng, X., Mettelman, R. C., O'Brien, A., Thompson, J. A., O'Brien, T. E., & Baker, S. C. (2019). Analysis of Coronavirus Temperature-Sensitive Mutants Reveals an Interplay between the Macrodomain and Papain-Like Protease Impacting Replication and Pathogenesis. Journal of Virology, 93(12). https://doi.org/10.1128/JVI.02140-18
Deng X, et al. Analysis of Coronavirus Temperature-Sensitive Mutants Reveals an Interplay Between the Macrodomain and Papain-Like Protease Impacting Replication and Pathogenesis. J Virol. 2019 06 15;93(12) PubMed PMID: 30918076.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Analysis of Coronavirus Temperature-Sensitive Mutants Reveals an Interplay between the Macrodomain and Papain-Like Protease Impacting Replication and Pathogenesis. AU - Deng,Xufang, AU - Mettelman,Robert C, AU - O'Brien,Amornrat, AU - Thompson,John A, AU - O'Brien,Timothy E, AU - Baker,Susan C, Y1 - 2019/05/29/ PY - 2018/11/30/received PY - 2019/03/18/accepted PY - 2019/3/29/pubmed PY - 2020/6/24/medline PY - 2019/3/29/entrez KW - coronavirus KW - innate immunity KW - interferon KW - macrodomain KW - papain-like protease KW - temperature sensitive KW - viral replication JF - Journal of virology JO - J. Virol. VL - 93 IS - 12 N2 - Analysis of temperature-sensitive (ts) mutant viruses is a classic method allowing researchers to identify genetic loci involved in viral replication and pathogenesis. Here, we report genetic analysis of a ts strain of mouse hepatitis virus (MHV), tsNC11, focusing on the role of mutations in the macrodomain (MAC) and the papain-like protease 2 (PLP2) domain of nonstructural protein 3 (nsp3), a component of the viral replication complex. Using MHV reverse genetics, we generated a series of mutant viruses to define the contributions of macrodomain- and PLP2-specific mutations to the ts phenotype. Viral replication kinetics and efficiency-of-plating analysis performed at permissive and nonpermissive temperatures revealed that changes in the macrodomain alone were both necessary and sufficient for the ts phenotype. Interestingly, mutations in the PLP2 domain were not responsible for the temperature sensitivity but did reduce the frequency of reversion of macrodomain mutants. Coimmunoprecipitation studies are consistent with an interaction between the macrodomain and PLP2. Expression studies of the macrodomain-PLP2 portion of nsp3 indicate that the ts mutations enhance proteasome-mediated degradation of the protein. Furthermore, we found that during virus infection, the replicase proteins containing the MAC and PLP2 mutations were more rapidly degraded at the nonpermissive temperature than were the wild-type proteins. Importantly, we show that the macrodomain and PLP2 mutant viruses trigger production of type I interferon in vitro and are attenuated in mice, further highlighting the importance of the macrodomain-PLP2 interplay in viral pathogenesis.IMPORTANCE Coronaviruses (CoVs) are emerging human and veterinary pathogens with pandemic potential. Despite the established and predicted threat these viruses pose to human health, there are currently no approved countermeasures to control infections with these viruses in humans. Viral macrodomains, enzymes that remove posttranslational ADP-ribosylation of proteins, and viral multifunctional papain-like proteases, enzymes that cleave polyproteins and remove polyubiquitin chains via deubiquitinating activity, are two important virulence factors. Here, we reveal an unanticipated interplay between the macrodomain and the PLP2 domain that is important for replication and antagonizing the host innate immune response. Targeting the interaction of these enzymes may provide new therapeutic opportunities to treat CoV disease. SN - 1098-5514 UR - https://www.unboundmedicine.com/medline/citation/30918076/Analysis_of_Coronavirus_Temperature_Sensitive_Mutants_Reveals_an_Interplay_between_the_Macrodomain_and_Papain_Like_Protease_Impacting_Replication_and_Pathogenesis_ L2 - https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/30918076/ DB - PRIME DP - Unbound Medicine ER -