Impairment of the deISGylation activity of foot-and-mouth disease Lpro causes viral attenuation without affecting interferon expression during viral infection.

Authors: MEDINA, GISSELLE - Kansas State University; Azzinaro, Paul; RAMIREZ-MEDINA, ELIZABETH - University Of Connecticut; Gutkoska, Joseph; FANG, YING - Kansas State University; De Los Santos, Teresa; Diaz San Segundo, Fayna

Submitted to: Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/8/2020
Publication Date: 4/15/2020
Citation: Medina, G., Azzinaro, P.A., Ramirez-Medina, E., Gutkoska, J.R., Fang, De Los Santos, T.B. 2020. Impairment of the deISGylation activity of foot-and-mouth disease Lpro causes viral attenuation without affecting interferon expression during viral infection. Virology. https://doi.org/10.1128/JVI.00341-20.
DOI: https://doi.org/10.1128/JVI.00341-20

Interpretive Summary: Foot and mouth disease virus (FMDV) causes a devastating animal disease that is currently present in many parts of the world including Asia and Africa and pose a risk for disease-free countries such as USA, Australia and European countries. We had previously identified that one of the viral proteins called Lpro is critical for the ability of FMDV to cause disease in animals. In this manuscript we describe a new function for Lpro. Interestingly the impairment of this function did not kill the virus but made it weaker. This discovery could be used toward the development of novel vaccines needed in regions in which the disease is currently present and those free of the disease.

Technical Abstract: Foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) affects several pathways of the host innate immunity. Previous studies have demonstrated that deletion (LLV, leaderless) or point mutations in the SAP conserved domain of Lpro results in increased expression of interferon (IFN) and IFN- induced genes (ISG) including among others, the ubiquitin-like protein modifier ISG15 and the ubiquitin specific peptidase USP18. ISG15 can reversibly attach to a diverse pool of target proteins in a process referred as ISGylation, and similarly to ubiquitination, it has been related to the control of several signaling pathways of innate immunity. It has been demonstrated that FMDV Lpro possesses deUbiquitinase and deISGylase activities. In this study we performed structural analysis and molecular modeling of Lpro coupled to a fragment of the C-terminal region in ISG15, in order to determine specific residues that might be involved in their interaction. We identified a hydrophobic tryptophan residue (Trp/W105) conserved among all FMDV serotypes that may play a role in Lpro-ISG15 molecular interaction. Mutation of this hydrophobic W105 residue to neutral alanine (Ala/A) rendered bacterial expressed Lpro, unable to cleave synthetic pro-ISG15, despite preservation of canonical eIF4G cleavage. Construction of an FMDV infectious clone carrying the LproW105A mutation resulted in a viable virus that grew to similar titers with a plaque phenotype as wild type (WT) FMDV in BHK-21 cells but displayed clear attenuation in IFN competent porcine epithelial cells (SK6). Overexpression of ISG15 and the ISGylation machinery in the same SK6 cells resulted in moderate inhibition of FMDV replication along with a decrease of the overall state of ISGylation upon infection with WT or W105A but not with leaderless virus. Interestingly, the expression of IFN and ISGs mRNA was only reduced upon infection with WT virus. Altogether, our studies indicate that abolishing/reducing the deISGylase 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