Research Project: Elucidating the Roles of CMPK2 in Mitochondrial Homeostasis and Antiviral Immunity
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PROJECT SUMMARY Cytidine/uridine monophosphate kinase 2 (CMPK2) is an interferon-regulated enzyme that was originally reported to catalyze the ATP-dependent phosphorylation of dCMP and dUMP to diphosphate forms in vitro. Due to a putative mitochondrial targeting sequence, CMPK2 was postulated to function in the mitochondrial deoxyribonucleotide salvage pathway necessary for the synthesis and maintenance of mitochondrial DNA (mtDNA). However, more recent data have revealed that CMPK2 prefers ribonucleotide diphosphate substrates in vitro and functions to restrict HIV and other RNA viruses in cell based assays. Beyond these seemingly contrasting findings, no other studies have addressed the cellular localization and tissue expression patterns of CMPK2 or utilized genetic knockouts to determine true biological activity. Therefore, the overall objective of this proposal is to close these knowledge gaps and mechanistically advance understanding of CMPK2 in mitochondrial function, tissue homeostasis, and antiviral innate immunity using a diverse toolkit of cell and animal models. The central hypothesis is that by maintaining mitochondrial homeostasis, CMPK2 boosts cell-intrinsic innate immunity and limits runaway inflammation triggered by mitochondrial stressors and viral infection. In support of this hypothesis, ongoing studies have revealed that CMPK2 localizes strongly to mitochondria and that ectopic overexpression of CMPK2 is sufficient to protect cells from RNA virus infection. Moreover, after systemic challenge with innate immune agonists, CMPK2 is markedly upregulated in the lungs and liver, and CMPK2 knockout mice exhibit elevated expression of proinflammatory cytokines and type I interferon (IFN-I) responses after Toll-like receptor (TLR) stimulation. To gain additional insight into how CMPK2 functions in mitochondria and antiviral immunity, two related, but independent aims are proposed. Aim 1 will elucidate the molecular mechanisms by which CMPK2 maintains mitochondrial homeostasis at rest and during stress. Here, CMPK2 knockout cells, novel lines reconstituted with mutant CMPK2 vectors lacking nucleotide kinase activity or mitochondrial targeting, and whole body CMPK2 knockout mice will be utilized. Aim 2 will determine that the mitochondrial activity of CMPK2 restricts coronavirus replication and maintains mitochondrial function during infection. Here, an intranasal mouse hepatitis virus challenge protocol that models acute respiratory distress syndrome and closely mirrors coronavirus pneumonia in humans will be employed. This research will fundamentally advance our understanding of how CMPK2 functions in mitochondrial homeostasis and antiviral innate immunity at both the cellular and organismal levels. Moreover, it may have a positive impact on public health by revealing novel CMPK2-centered strategies to maintain mitochondrial homeostasis, boost antiviral immunity, and limit damaging inflammation during coronavirus infection.
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