Foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) affects several pathways of the host innate immune response. Previous studies in bovine cells have demonstrated that deletion (LLV, leaderless) or point mutations in Lpro results in increased expression of interferon (IFN) and IFN-stimulated genes (ISG) 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 bacterial-expressed Lpro unable to cleave ... More
Foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) affects several pathways of the host innate immune response. Previous studies in bovine cells have demonstrated that deletion (LLV, leaderless) or point mutations in Lpro results in increased expression of interferon (IFN) and IFN-stimulated genes (ISG) 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 bacterial-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 WT infected cells. In contrast, reduced deISGylation was observed upon infection with W105A and leaderless virus. Reduction in the levels of deubiquitination were also observed in cells infected with 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 expression of IFN and ISGs mRNA. Furthermore, our studies highlight the potential of ISG15 for development as a novel biotherapeutic against FMD.IMPORTANCE In this study, we have identified an aromatic hydrophobic residue in FMDV 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 when compared to WT. Impaired deISGylase activity in Lpro correlated with viral attenuation in vitro and in vivo and did not affect Lpro ability to block expression of type I IFN and other IFN-stimulated genes. Moreover, overexpression of ISG15 resulted in reduction of FMDV viral titers. Thus, our study highlights the potential of Lpro mutants with modified deISGylase activity for the development of live attenuated vaccine candidates and novel biotherapeutics against FMD.
