INVESTIGATING THE FUNCTION OF HUMAN SIRTUIN 3 IN REGULATING MITOCHONDRIAL MORPHOLOGY AND FUNCTION DURING VIRAL INFECTION

Abstract

The regulation of mitochondrial structure and function in mammalian cells is central to many distinct viral infections as means to modulate cellular metabolism and immune responses. Indeed, alterations in mitochondria morphology and functions can be observed across evolutionarily distinct viral infections. Among these viruses is the human cytomegalovirus (HCMV), a ubiquitous β-herpesvirus that has an ancient history of co-evolution with its host. Current perspectives of HCMV host-pathogen interactions point towards a role for protein acetylation within the mitochondria as a toggle between pro- and anti-virus host responses, with the mitochondrial deacetylase sirtuin 3 (SIRT3) being found to antagonize HCMV.In my thesis work, I investigated the antiviral functions of SIRT3 and, more broadly, the temporal regulation and functions of protein acetylation events during HCMV infection. By integrating proteomics, virology, bioinformatics, and microscopy, I defined the temporal landscape of protein acetylation during the course of HCMV infection in human fibroblasts. This study revealed acetylation occurring on both viral and host proteins that highlighted pro- and anti-viral functions of this modification. Although alterations in protein acetylation were found in many subcellular organelles, the most striking change that I uncovered was a global upregulation of hundreds of acetylation sites on mitochondrial proteins. Moreover, numerous known or predicted SIRT3 substrates displayed dynamic acetylations during infection. Indeed, I demonstrate that the deacetylase activity of SIRT3 is necessary for its ability to suppress virus production. To understand the mechanisms underlying SIRT3 antiviral function, I defined the temporality of SIRT3-substrate interactions during infection. Concomitant with changes in acetylation levels, SIRT3 dynamically associated with the mitochondrial fusion factor Optic Atrophy (OPA1). By employing microscopy and virology assays, I demonstrate that the acetylation state of OPA1 modulates mitochondrial morphology of infected cells and inhibits HCMV production. In addition to the known K834 site on OPA1, I discover another acetylation site, K931, is targeted by the SIRT3 deacetylase. Indeed, I show that OPA1 K931 acetylation restricts HCMV infection. Together with the finding that SIRT3 is critical for regulating mitochondrial pH, membrane potential, and structure along with a comprehensive annotation of SIRT3-substrate interactions during infection these data indicate a role for SIRT3 in the regulation of cellular metabolism. For example, I demonstrate that SIRT3 loses its association with the acetyl-CoA acyltransferase 2 (ACAA2), a protein functioning in fatty acid beta-oxidation, concomitant with increased ACAA2 acetylation that also impacts HCMV infection. Finally, to gain a broader view of the impact of HCMV infection of the regulation of proteins and their interactions, we employed thermal proximity coaggregation (TPCA) mass spectrometry. This approach allowed us to monitor at a system level the maintenance, dissociation and association of functional protein complexes during the HCMV replication cycle. These dynamic interactions reflected organelle remodeling events, as well as functional movements of proteins. For example, by examining our interaction dataset and performing functional follow-up assays, we discovered that the HCMV co-receptor integrin beta 1 (ITGB1) dissociates from extracellular matrix proteins, becoming internalized with the tetraspanin CD63, which is necessary for virus production. In conclusion, my thesis study has characterized the temporal and spatial regulation of protein acetylation on both host and viral proteins. By integrating multiple datasets and functional assays, I uncovered two of the SIRT3 substrates that mediate the antiviral function of SIRT3. This work provides insight into the regulation of protein acetylation and protein-protein interactions during viral infection, and may facilitate the development of antiviral treatment.