Investigating Human Mitochondrial Sirtuin 3 and its Anti-Viral Functions

Abstract

Human sirtuins are NAD + -dependent deacetylases that have been predominantly associated with aging and human diseases, including cancer and diabetes. More recently, research has shown that sirtuins play critical roles in host cell defense against infection with DNA and RNA viruses such as human cytomegalovirus and influenza A, respectively. The mechanisms through which they accomplish this function remain to be elucidated. My thesis research examined the mitochondrial sirtuin 3 (SIRT3). Although SIRT3 has not been previously investigated during viral infection, this enzyme is thought to regulate the acetylation levels of numerous mitochondrial proteins with critical roles in regulating cellular metabolism. Therefore, I hypothesized that SIRT3 acts in host defenseby interacting with host factors in metabolic pathways that are necessary for viral replication. To test this hypothesis, I investigated SIRT3 functions during infection with HCMV, a beta-herpes virus that is an important wide-spread human pathogen. I first characterized a SIRT3 construct tagged with green fluorescence protein (GFP) and confirmed that it is functional and well-suited for SIRT3 protein interaction and functional assays. Based on our preliminary proteomics studies of SIRT3 interactions, I next focused on several associations that were significantly reduced upon infection when compared to uninfected cells. Two of these interactions were with proteins that are key enzymes in fatty acid oxidation and the Urea cycle, ACAD10 and CPS1, respectively. Knockdown studies demonstrated that these proteins indeed play important roles during HCMV infection, their knockdowns resulting in significant decreases in virus titers. Other SIRT3 interactions were upregulated during infection, including those with ATP5O and GSTK1 which are proteins associated with regulation of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS). This led me to ask whether MMPand ROS are dynamically regulated during infection. Given the contribution of mitochondrial pH to both MMP and non-enzymatic acetylation events, I optimized protocols for testing changes in mitochondrial pH during infection. I next investigated ROS levels and MMP during infection, finding that increased SIRT3 levels correlate withincreased MMP in both uninfected and infected cells. Lastly, to place these findings in the broader context of changes occurring during HCMV infection, I investigated the mitochondrial proteome at different time points during HCMV infection (24, 48, and 72 hours post infection). Overall, my work expands the current knowledge of mitochondrial regulation and SIRT3 functions during HCMV infection. Further investigation of the antiviral roles of SIRT3 may help in the design of future antiviral therapies.