Uncovering and harnessing connected metabolic pathways essential to virus infection. - Project Summary / Abstract Polyamines are small, abundant molecules found in all eukaryotic cells that function in a variety of cellular processes, including transcription and translation. These molecules are critical for cells, but RNA viruses also require these molecules for their replication. We previously showed that RNA viruses of diverse families, including enteroviruses, alphaviruses, and bunyaviruses, rely on polyamines to facilitate virus genome packaging, enzyme activity, and attachment to susceptible cells. Thus, viruses usurp the cellular polyamine resource for their own replication. However, the cell responds to infection by depleting polyamines, by acetylating them and causing their degradation in peroxisomes. We also find that polyamines coordinate cellular metabolism on a global levels, mediating levels of cellular nucleotides, lipids, and cholesterol, all important resources for RNA viruses. Given the importance of polyamines for viruses, cells, and metabolism, we hypothesize that viruses subvert cellular metabolism through polyamines to support virus replication. To test this hypothesis, we will (1) determine how viruses use polyamines, specifically for binding to and entering susceptible cells, (2) measure the impact of polyamines in cell stress responses – including virus infection – and their contribution to cell survival, and (3) identify ways that polyamines interconnect with other metabolites in the cellular metabolic network to mediate cellular homeostasis and virus infection. We use several model systems to study polyamines and metabolites in virus infection. We use Coxsackievirus B3 (CVB3), Rift Valley fever virus (RVFV), and chikungunya virus (CHIKV) as our primary model systems because (1) they have robust, well-described in vitro systems, (2) my laboratory has significant experience with these models, and (3) they represent diverse viruses such that we can compare and contrast between similar and dissimilar phenotypes. In addition, we have model systems for poxviruses, coronaviruses, arenaviruses, and flaviviruses that we regularly use. We showed that each of these viruses relies on polyamines, but each virus appears to use polyamines differently, from packaging to entry to enzyme activity. Here, we will expand our investigation, building on our significant work during the previous funding period, to determine how polyamines function specifically during viral entry. We will also determine how cells use polyamines in the response to stress, especially translational stress, in order to survive the stress. Finally, we will determine how polyamines connect to other metabolites within the cell and how all of these metabolites are used to facilitate RNA virus infection, with a particular interest in lipids. This work will illuminate the connectedness of polyamine biosynthesis to other metabolic pathways and how viruses rely on these interconnected pathways for successful replication. This work will highlight fundamental roles for polyamines in virus replication and in cellular metabolism.
