PROJECT SUMMARY Human Kaposi’s Sarcoma-associated Herpesvirus (KSHV) is the etiological agent of multiple cancers. However, the molecular details concerning the tumorigenesis of KSHV are not well understood. Our recent studies indicate that herpesviruses employ protein deamidation to evade innate immune response. To probe the role of protein deamidation in fundamental biological processes, we performed a focused screen targeting cellular glutamine amidotransferases (GAT), a potential protein deamidases family. We identified one glutamine amidotransferase as a negative regulator of NF-κB activation. In our preliminary studies, we found that the GAT possessed intrinsic protein deamidating ability to deamidate NF-κB transcription factor to dampen its ability to transactivate NF-κB genes. Remarkably, deamidation promoted aerobic glycolysis to promote cell proliferation and tumorigenesis. Furthermore, KSHV hijacks the cellular mechanism to induce NF-κB deamidation and promote cell proliferation, thereby inducing tumor formation. Our findings support the overarching hypothesis a nucleotide biosynthetic enzyme deamidates a NF-κB subunit to reprogram metabolism, thus promoting cell proliferation and tumorigenesis, and that KSHV hijacks this mechanism to achieve persistent infection. To test this central hypothesis, I propose three aims in this project: 1) Elucidate the GAT-mediated deamidation of a NF-κB subunit and downregulation of NF-κB activation; 2) Delineate the metabolic reprogramming by GAT-mediated deamidation; and 3) Characterize a viral mechanism that hijacks the cellular deamidation to promote proliferation and tumor formation during KSHV latent infection. In the K99 phase, I will achieve the following three sub aims: 1) characterize the molecular detail of the NF-κB deamidation (Aim 1A); 2) examine the role of deamidation in innate immune defense (Aim 1B); and 3) dissect the mechanism of deamidation-mediated metabolic reprogramming (Aim 2A). In the R00 phase, I will complete Aim 2 by defining the role of NF-κB deamidation in proliferation and tumorigenesis of diverse cancer cell lines. Furthermore, I will investigate the mechanism by which KSHV promotes NF-κB deamidation and define its role in metabolism and tumorigenesis during KSHV infection. In summary, the K99/R00 project will characterize novel functions of a cellular metabolic enzyme (in nucleotide synthesis) and a key transcription factor (a NF-κB subunit), and elucidate a new mechanism governing metabolic reprogramming to drive cell proliferation, thereby offering fresh insight into the metabolic regulation during KSHV infection. It will establish the foundation for my long-term career goals to study protein deamidations in cellular metabolism, KSHV-associated tumorigenesis and translational applications to antiviral/antitumor therapies.