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Viral mechanisms of metabolic network manipulation
Viruses are obligate parasites that depend on the metabolic resources provided by the host to supply the energy and biochemical building blocks necessary for virion production. Many anti-viral compounds, such as nucleotide analogs, target viral utilization of macro-molecular precursors and have proven to be clinically successful. Despite these successes, very little is known about the mechanisms governing viral manipulation of the small molecule metabolic network. Our laboratory’s goal is to elucidate these mechanisms and thereby identify potential anti-viral therapeutic targets.
Currently our work focuses on human cytomegalovirus (HCMV), a herpes virus, which is the leading cause of congenital viral infection, resulting in central nervous system damage in the majority of symptomatic newborns. HCMV infection also poses a serious health risk to immunosuppressed individuals, such as the elderly, and patients receiving immunosuppressive chemotherapy, including cancer patients, transplant recipients, and AIDS patients.
In order to elucidate virally-induced changes to the metabolic network, we utilize liquid chromatography/ tandem mass spectrometry (LC-MS/MS) to measure the concentrations of specific metabolite pools. Utilizing this technology, we can measure the concentrations of a wide variety of metabolites in tissue culture, covering a large proportion of metabolite chemical diversity.
In addition to measuring metabolite concentrations, we also measure the rate of molecular conversion from one metabolite to another, i.e. metabolic flux. The speed of metabolic flux is crucial to understanding metabolic network behavior and how it is affected by a cellular perturbation, e.g. viral infection. We measure metabolic flux by feeding cells with stable-isotope-labeled nutrient, e.g. 13C glucose, which because of its heavier composition (as compared to 12C) produces a unique mass signature. As 13C incorporates into the metabolic network it can be distinguished from 12C metabolites by mass spectrometry. By measuring the speed of 13C incorporation into various metabolite pools we can estimate metabolic flux values and how they are affected by various perturbations such as viral infection.
We have found that HCMV infection activates several metabolic pathways including various aspects of central carbon metabolism and nucleotide biosynthesis. Inhibition of certain virally-up-regulated pathways has been found to block normal viral replication. In addition to screening for metabolic pathways important for viral infection, we utilize genetic techniques such as bacterial artificial chromosome based mutagenesis and retroviral transduction to identify the viral and host cell factors responsible for virally-induced metabolic changes. Increased understanding of the mechanisms of viral metabolic manipulation and the role they play during viral infection will continue to illuminate potential therapeutic avenues.
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