Citrullination during Human Cytomegalovirus infection: implications for autoimmune diseases

INTRODUCTION: Human Cytomegalovirus (HCMV) is a widespread beta-Herpesvirus carried by 70% up to 90% of the human population. Following primary infection, HCMV establishes a lifelong latency in cells of the myeloid lineage, where reactivation is often driven by inflammation. Autoimmune diseases (AD) are characterized by chronic inflammation due to an abnormal immune response against the body’s own tissue. In genetically predisposed patients, HCMV has been shown to be relevant in the pathogenesis of AD but whether it initiates or supports the development of AD is still not known. Citrullination is a post-translational modification (PTM) catalyzed by peptidyl arginine deiminases (PAD) that convert peptidylarginine into peptidylcitrulline, whose dysregulation has been associated with a spectrum of ADs, cancer, and neurodegenerative disorders. Against this background, the goal of this project is to characterize PTMs such as citrullination during infection with Herpesviruses. MATERIAL AND METHODS: We investigated the PADs expression in Human Foreskin Fibroblasts (HFF) during HCMV infection using both Real Time quantitative PCR and Western Blot analysis. We also used an in vitro antibody-based assay in order to measured the PAD activity. Furthermore we determined the pattern of citrullination during infection using a citrulline-specific rhodamine phenylglyoxal (RhPG)-based probe. RESULTS: We demonstrate that HCMV triggers PAD2 expression in HFF both at mRNA and protein levels. Viral replication rate of the HMCV is strongly impaired in the presence of Cl-amidine, a specific pan-PAD inhibitor, indicating that citrullination is required for HCMV replication. Consistently, the depletion of PAD2 by siRNA technology during infection showed decreased infectious yields compared to the control. We then measured the PAD activity in infected HFFs, a striking increase of PAD2 activity was detected at 48 and 72 hpi that was inhibited by Cl-amidine. Interestingly, using the citrulline-specific probe, we determined the overall pattern of citrullination during infection that changes consistently at different time points during infection. DISCUSSION AND CONCLUSION: These findings may shed light on the role of HCMV in the pathogenesis of ADs and provide possible medical interventions for their treatment.