DEFINING THE FUNCTIONS OF HUMAN SIRTUIN 2 IN CELLULAR HOUSEKEEPING AND DURING VIRAL INFECTION

Abstract

The cellular proteome is regulated at multiple levels, including transcription, translation, post-translational modifications, and degradation. This regulatory complexity allows for finely-tuned maintenance of intracellular homeostasis. NAD+- dependent deacetylases sirtuins (SIRTs) are key regulators of such processes. They are positioned to receive information about intracellular energy states and to relay this information to downstream pathways by modulating their substrates. Sirtuins have been linked to various human diseases, including cancer, neurodegeneration, and viral infection. More recently, we found that sirtuins act in antiviral response against several DNA and RNA viruses. However, how sirtuins exert these functions remains largely unknown. In my thesis work, I integrated virology, proteomics, bioinformatics, and biochemistry to investigate one of the least characterized sirtuins, the cytoplasmic SIRT2, and its cooperative functions with nuclear SIRT1. First, I describe how proteomics approaches using label-free and metabolic-labeling mass spectrometry can be applied to studying protein functions through investigation of protein-protein interactions. Next, I used these methods to investigate SIRT2, its dynamic protein interactions, and the impact of SIRT2 on the proteome of human fibroblasts. From these studies, I discovered that SIRT2 is positioned to function in intracellular trafficking through its interactions and localization at the intersection of the ER-Golgi trafficking pathways, as well as through regulation of proteins in membrane trafficking routes. Next, to assess its roles during infection, we tested the impact of sirtuin activation or inhibition on viral protein levels at different stages of infection with human cytomegalovirus (HCMV). A reduction in early and late HCMV protein levels was observed upon treatment with small molecules that activate sirtuins. This sirtuin antiviral function is evolutionarily conserved, as we show that the sirtuin homologue in E.Coli, CobB, acts against bacteriophage infections. Finally, we provide mechanistic insights into the individual and cooperative functions of SIRT1 and SIRT2 during HCMV infection. We show that simultaneous inhibition of SIRT1 and SIRT2 decreases HCMV replication in part through a p53-dependent mechanism. I also characterize SIRT2 interactions and localization during HCMV infection time course, which point to a possible contribution to regulation of viral gene expression and virion assembly.