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Impairment of the DeISGylation Activity of Foot-and-Mouth Disease Virus Lpro Causes Attenuation In Vitro and In Vivo
Foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) affects several pathways of the host innate immune response. Previous studies in bovine cells demonstrated that deletions (leaderless [LLV]) or point mutations in Lpro result in increased expression of interferon (IFN) and IFN-stimulated g...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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American Society for Microbiology
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7307143/ https://www.ncbi.nlm.nih.gov/pubmed/32295921 http://dx.doi.org/10.1128/JVI.00341-20 |
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author | Medina, Gisselle N. Azzinaro, Paul Ramirez-Medina, Elizabeth Gutkoska, Joseph Fang, Ying Diaz-San Segundo, Fayna de los Santos, Teresa |
author_facet | Medina, Gisselle N. Azzinaro, Paul Ramirez-Medina, Elizabeth Gutkoska, Joseph Fang, Ying Diaz-San Segundo, Fayna de los Santos, Teresa |
author_sort | Medina, Gisselle N. |
collection | PubMed |
description | Foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) affects several pathways of the host innate immune response. Previous studies in bovine cells demonstrated that deletions (leaderless [LLV]) or point mutations in Lpro result in increased expression of interferon (IFN) and IFN-stimulated genes (ISGs), including, among others, the ubiquitin-like protein modifier ISG15 and the ubiquitin specific peptidase USP18. In addition to its conventional papain-like protease activity, Lpro acts as a deubiquitinase (DUB) and deISGylase. In this study, we identified a conserved residue in Lpro that is involved in its interaction with ISG15. Mutation W105A rendered Escherichia coli-expressed Lpro unable to cleave the synthetic substrate pro-ISG15 while preserving cellular eIF4G cleavage. Interestingly, mutant FMDV W105A was viable. Overexpression of ISG15 and the ISGylation machinery in porcine cells resulted in moderate inhibition of FMDV replication, along with a decrease of the overall state of ISGylation in wild-type (WT)-infected cells. In contrast, reduced deISGylation was observed upon infection with W105A and leaderless virus. Reduction in the levels of deubiquitination was also observed in cells infected with the FMDV LproW105A mutant. Surprisingly, similarly to WT, infection with W105A inhibited IFN/ISG expression despite displaying an attenuated phenotype in vivo in mice. Altogether, our studies indicate that abolishing/reducing the deISGylase/DUB activity of Lpro causes viral attenuation independently of its ability to block the expression of IFN and ISG mRNA. Furthermore, our studies highlight the potential of ISG15 to be developed as a novel biotherapeutic molecule against FMD. IMPORTANCE In this study, we identified an aromatic hydrophobic residue in foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) (W105) that is involved in the interaction with ISG15. Mutation in Lpro W105 (A12-LproW105A) resulted in reduced deISGylation in vitro and in porcine-infected cells. Impaired deISGylase activity correlated with viral attenuation in vitro and in vivo and did not affect the ability of Lpro to block expression of type I interferon (IFN) and other IFN-stimulated genes. Moreover, overexpression of ISG15 resulted in the reduction of FMDV viral titers. Thus, our study highlights the potential use of Lpro mutants with modified deISGylase activity for development of live attenuated vaccine candidates, and ISG15 as a novel biotherapeutic against FMD. |
format | Online Article Text |
id | pubmed-7307143 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | American Society for Microbiology |
record_format | MEDLINE/PubMed |
spelling | pubmed-73071432020-07-10 Impairment of the DeISGylation Activity of Foot-and-Mouth Disease Virus Lpro Causes Attenuation In Vitro and In Vivo Medina, Gisselle N. Azzinaro, Paul Ramirez-Medina, Elizabeth Gutkoska, Joseph Fang, Ying Diaz-San Segundo, Fayna de los Santos, Teresa J Virol Virus-Cell Interactions Foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) affects several pathways of the host innate immune response. Previous studies in bovine cells demonstrated that deletions (leaderless [LLV]) or point mutations in Lpro result in increased expression of interferon (IFN) and IFN-stimulated genes (ISGs), including, among others, the ubiquitin-like protein modifier ISG15 and the ubiquitin specific peptidase USP18. In addition to its conventional papain-like protease activity, Lpro acts as a deubiquitinase (DUB) and deISGylase. In this study, we identified a conserved residue in Lpro that is involved in its interaction with ISG15. Mutation W105A rendered Escherichia coli-expressed Lpro unable to cleave the synthetic substrate pro-ISG15 while preserving cellular eIF4G cleavage. Interestingly, mutant FMDV W105A was viable. Overexpression of ISG15 and the ISGylation machinery in porcine cells resulted in moderate inhibition of FMDV replication, along with a decrease of the overall state of ISGylation in wild-type (WT)-infected cells. In contrast, reduced deISGylation was observed upon infection with W105A and leaderless virus. Reduction in the levels of deubiquitination was also observed in cells infected with the FMDV LproW105A mutant. Surprisingly, similarly to WT, infection with W105A inhibited IFN/ISG expression despite displaying an attenuated phenotype in vivo in mice. Altogether, our studies indicate that abolishing/reducing the deISGylase/DUB activity of Lpro causes viral attenuation independently of its ability to block the expression of IFN and ISG mRNA. Furthermore, our studies highlight the potential of ISG15 to be developed as a novel biotherapeutic molecule against FMD. IMPORTANCE In this study, we identified an aromatic hydrophobic residue in foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) (W105) that is involved in the interaction with ISG15. Mutation in Lpro W105 (A12-LproW105A) resulted in reduced deISGylation in vitro and in porcine-infected cells. Impaired deISGylase activity correlated with viral attenuation in vitro and in vivo and did not affect the ability of Lpro to block expression of type I interferon (IFN) and other IFN-stimulated genes. Moreover, overexpression of ISG15 resulted in the reduction of FMDV viral titers. Thus, our study highlights the potential use of Lpro mutants with modified deISGylase activity for development of live attenuated vaccine candidates, and ISG15 as a novel biotherapeutic against FMD. American Society for Microbiology 2020-06-16 /pmc/articles/PMC7307143/ /pubmed/32295921 http://dx.doi.org/10.1128/JVI.00341-20 Text en Copyright © 2020 Medina et al. https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Virus-Cell Interactions Medina, Gisselle N. Azzinaro, Paul Ramirez-Medina, Elizabeth Gutkoska, Joseph Fang, Ying Diaz-San Segundo, Fayna de los Santos, Teresa Impairment of the DeISGylation Activity of Foot-and-Mouth Disease Virus Lpro Causes Attenuation In Vitro and In Vivo |
title | Impairment of the DeISGylation Activity of Foot-and-Mouth Disease Virus Lpro Causes Attenuation In Vitro and In Vivo |
title_full | Impairment of the DeISGylation Activity of Foot-and-Mouth Disease Virus Lpro Causes Attenuation In Vitro and In Vivo |
title_fullStr | Impairment of the DeISGylation Activity of Foot-and-Mouth Disease Virus Lpro Causes Attenuation In Vitro and In Vivo |
title_full_unstemmed | Impairment of the DeISGylation Activity of Foot-and-Mouth Disease Virus Lpro Causes Attenuation In Vitro and In Vivo |
title_short | Impairment of the DeISGylation Activity of Foot-and-Mouth Disease Virus Lpro Causes Attenuation In Vitro and In Vivo |
title_sort | impairment of the deisgylation activity of foot-and-mouth disease virus lpro causes attenuation in vitro and in vivo |
topic | Virus-Cell Interactions |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7307143/ https://www.ncbi.nlm.nih.gov/pubmed/32295921 http://dx.doi.org/10.1128/JVI.00341-20 |
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