Joshua C. Munger, Ph.D. - US grants

DESCRIPTION (provided by applicant): It has been known for decades that viruses induce dramatic changes to host-cell metabolism and that these changes are important for viral replication. The mechanisms involved, however, have largely remained obscure. A major challenge in dissecting the mechanisms of viral metabolic manipulation has been the technical difficulty associated with measuring diverse metabolic activities in live cells. We have developed a liquid chromatography-tandem mass spectrometry-based (LC-MS/MS) methodology to measure global metabolic activities in live cells. We propose that elucidation of these viral mechanisms will prove fertile ground for the development of novel anti-viral therapeutics. Our results indicate that three metabolic regulatory activities are required for high-titer HCMV replication;AMP-activated kinase (AMPK) and phosphofructokinase-1 (PFK1), both of which regulate glycolytic flux, and acetyl-CoA carboxylase (ACC1), which regulates fatty acid biosynthesis. In uninfected cells, activated AMPK directly inhibits ACC1 activity and thereby, fatty acid biosynthesis. We find that HCMV blocks this regulatory control, maintaining increased ACC1 activity despite activated AMPK. This results in a dramatic activation of both glycolysis and fatty acid biosynthesis. To explore these mechanisms, we will: (I) Elucidate the mechanisms of HCMV-induced AMPK activation and its role in HCMV replication;(II) Elucidate the mechanisms of HCMV- induced ACC1 activation;and (III) Elucidate the mechanisms of HCMV-induced PFK1 activation and how they contribute to viral replication. Through elucidating the viral mechanisms leading to the activation of these metabolic activities we will identify novel anti-viral targets to combat HCMV-associated disease and further explore viral manipulation of these fundamental host-cell pathways. PUBLIC HEALTH RELEVANCE: Human Cytomegalovirus (HCMV) is a widespread opportunistic pathogen that can cause severe disease in various immunosuppressed populations including the elderly, cancer patients receiving immunosuppressive chemotherapy, transplant recipients, and AIDS patients. HCMV is also the leading cause of congenital viral infection, occurring in 1-2% of all live births, which can result in multiple organ system abnormalities with central nervous system damage occurring in the majority of symptomatic newborns. Long term use of current anti-HCMV therapeutics in patients leads to toxic side effects and has resulted in the emergence of drug-resistant viral strains, highlighting the need for additional anti-HCMV therapeutics. Our proposed research aims to elucidate the mechanisms HCMV utilizes to drive the biosynthesis of virion components. Elucidating these mechanisms will present targets to therapeutically block viral replication and attenuate HCMV-associated disease.

Human Cytomegalovirus (HCMV) is a major cause of congenital birth defects and causes severe disease in a wide variety of immunosuppressed patient populations, including hematological cancer patients and transplant recipients. We have found that HCMV institutes a pro-viral metabolic program that drives numerous cellular metabolic activities to support the production of viral progeny. Key aspects of this metabolic reprogramming include targeting the AMP-activated kinase (AMPK) and the calmodulin dependent-kinase kinase (CamKK), both of which we find are critical for successful HCMV infection. Further, our data indicate that the HCMV UL26 protein is an important viral metabolic determinant that activates fatty acid biosynthesis, a metabolic pathway essential for infection. Many questions remain about how these factors contribute to metabolic reprogramming and successful infection. To address these questions, we will pursue the following aims: 1) Elucidate how AMPK contributes to HCMV-mediated metabolic reprogramming; 2) Determine how calmodulin-dependent kinase kinase signaling contributes to HCMV infection; and, 3) Elucidate how the HCMV UL26 protein contributes to viral metabolic reprogramming. We expect the outcome of our research to be the identification of specific mechanisms through which HCMV manipulates metabolic regulation to support infection. The proposed work will broaden our understanding of an important host pathogen interaction, and given that these processes are essential for productive infection, the proposed experiments will highlight novel targets for therapeutic intervention.

Human Cytomegalovirus (HCMV) is a major cause of congenital birth defects and causes severe disease in a wide variety of immunosuppressed patient populations, including hematological cancer patients and transplant recipients. We have found that HCMV institutes a pro-viral metabolic program that drives numerous cellular metabolic activities to support the production of viral progeny. More recently, we find that the HCMV UL38 protein is necessary and sufficient to drive many aspects of HCMV-induced metabolic remodeling, and we hypothesize that UL38 supports infection through its inhibition of the TSC2 tumor suppressor protein to induce metabolic modulation. We will test this hypothesis in Aim 1 by elucidating how UL38-TSC2-mediated metabolic remodeling contributes to HCMV infection. In addition, we find that HCMV-induces the expression of neuronal enolase 2 (ENO2), which we find is important for robust HCMV infection. We hypothesize that ENO2 induction is critical for HCMV-mediated metabolic modulation, which we will test in Aim 2. Lastly, we find that both HCMV infection and UL38 expression sensitizes cells to metabolic perturbations, revealing vulnerabilities that could potentially be targeted therapeutically. We hypothesize that HCMV infection and UL38 expression induces a metabolically rigid state that sensitizes cells to metabolic challenges, a hypothesis we will test in Aim 3. The proposed work will broaden our understanding of an important host pathogen interaction, and given that these processes are essential for productive infection, the proposed experiments will highlight novel targets for therapeutic intervention.