DEFINING MECHANISMS UNDERLYING HOST-VIRUS INTERPLAY IN METABOLIC AND CELL CYCLE REGULATION DURING HUMAN CYTOMEGALOVIRUS INFECTION

Abstract

As obligate intracellular parasites, viruses rely on mitochondria to produce the biosynthetic precursors and energy necessary for replication. Particularly striking changes in metabolism and mitochondrial structure are observed after infection with the prevalent pathogen human cytomegalovirus (HCMV). However, how HCMV alters mitochondrial structure and function presented a conundrum. Mitochondrial fragmentation is typically associated with decreased bioenergetics, however HCMV induces both mitochondrial fragmentation and respiration. By integrating proteomic datasets with super-resolution confocal microscopy, LC-MS based metabolite profiling and metabolic assays, we discovered a previously uncharacterized viral protein, pUL13, targets the mitochondria to increase respiration during infection. This finding addressed the outstanding question of how HCMV modulates mitochondria to increase bioenergetics and expanded the knowledge of the intricate connection between mitochondrial architecture and electron transport chain function. A consequence of increased metabolic flux driven by virus infection is posttranslational modification (PTM) of cellular proteins with reactive acylations derived from metabolic pathways. This is an emerging and largely unstudied interface of host-virus interaction. Sirtuins (SIRTs) are evolutionary-conserved enzymes that are ubiquitously expressed in mammalian cells and function broadly as deacylases to regulate cellular homeostasis. We previously discovered that SIRTs are broad-spectrum viral restriction factors, protecting host cells against DNA and RNA viruses, including HCMV, demonstrating that regulation of protein acylation is central to viral replication and host defense processes. We recently demonstrated that protein acetylation is dramatically altered during HCMV infection. However, the mechanistic underpinnings of how this PTM is regulated during infection remain unknown. Here, we discovered that inhibition of SIRT2 deacetylase activity hinders an early stage of the HCMV replication cycle, indicating a pro-virus function for SIRT2. To uncover this function, we characterized SIRT2-dependent changes in the temporal acetylome, which we integrated with proteome, interactome, flow cytometry, and molecular virology assays. These analyses demonstrated that SIRT2 functions in a pro-virus capacity by deacetylating key cell cycle proteins, thereby regulating cell cycle progression during HCMV infection. Altogether, this work helps define (1) how HCMV targets mitochondrial structure-function relationships to rewire cellular metabolism during infection, and (2) the function of protein acylation and SIRT proteins at the interface of host-virus interactions.