Abstract
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.
Highlights
IMPORTANCE In this study, we identified an aromatic hydrophobic residue in footand-mouth disease virus (FMDV) leader proteinase (Lpro) (W105) that is involved in the interaction with ISG15
By comparing interacting Lpro residues that are conserved across different Foot-and-mouth disease virus (FMDV) serotypes [27] and that bind to ISG15 from multiple known host species, we identified FMDV Lpro residues that might be important for the interaction with ISG15
Other Lpro residues mediating its interaction with ISG15 have been recently identified [24], we propose that LproW105 is important for modulating viral infection kinetics
Summary
IMPORTANCE In this study, we identified an aromatic hydrophobic residue in footand-mouth disease virus (FMDV) leader proteinase (Lpro) (W105) that is involved in the interaction with ISG15. Understanding virus-host interactions should help to identify novel cellular factors and mechanisms that participate in antiviral immunity against FMDV and could provide alternatives for therapeutic discovery. FMDV Lpro is a papain-like protease (PLP) known to block the cellular innate immune response, at both the transcriptional and translational level by utilizing different mechanisms, including (i) shutting down translation of host capped mRNAs through the cleavage of the translation initiation factor eIF4G [5, 6]; (ii) downregulating IFN mRNA expression by causing degradation of NF-B, IRF-3, IRF-7, and LGP2 [7,8,9,10]; (iii) targeting the chromatin remodeling machinery to disrupt the expression of IFN and ISG mRNAs [11]; and (iv) targeting of G3BP1/2 to block stress granule formation [12]. Unlike Ub, ISG15 and the ISGylation machinery are robustly induced by type I IFN [13] and can be upregulated upon viral infection [14]
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