Mechanisms of Enzyme Regulation by Viperin in the Cellular Antiviral Response

Project Summary Eukaryotic cells have developed sophisticated defenses aimed at limiting viral replication and thereby preventing infection from escalating to other cells. This R35 MIRA application will support studies to understand how at the biochemical level various cellular metabolic pathways are regulated as part of the innate immune response to viral infection to disrupt virus replication. Such studies are essential for developing more effective anti-viral therapeutics. Our studies will center around a key enzyme in the antiviral response: viperin (Virus Inhibitory Protein; Endoplasmic Reticulum-associated, INterferon-inducible), which is strongly up-regulated in viral infections and has been shown to restrict the infectivity of a number of important human viruses including influenza A, HIV and hepatitis C. Viperin catalyzes the synthesis of the antiviral nucleotide 3’-deoxy-3’,4’-didehydro-CTP (ddhCTP) from CTP by a radical SAM-dependent mechanism. ddhCTP has been shown to have antiviral properties against some RNA viruses, e.g. flaviviruses, where it acts as a chain-terminating inhibitor of the viral RNA polymerase. However, this observation does not explain the much broader antiviral properties of viperin that are associated with its network of protein-protein interactions with viral proteins, cellular proteins important for viral replication, and components of innate immune signaling pathways. Building on the results of our previous NIH-supported research, we will investigate how viperin acts to regulate various cellular metabolic and signaling pathways that are important for establishing the antiviral state. Our strategy is to focus on examples of viperin’s interactions with other enzymes for which there is both a well- validated biological response and a clear biochemical rational underpinning the response. Our experimental approach will combine detailed mechanistic and structural studies on purified enzymes with functional studies of enzymes transfected in mammalian cell lines. As part of these studies, we will investigate how viperin engages with ubiquitin ligases to activate innate immune signaling pathways and proteasomal degradation of enzymes necessary for viral replication. We will investigate how viperin down-regulates fatty acid b-oxidation through its interactions with the mitochondrial trifunctional enzyme (protein) complex. We will also investigate how viperin interacts with enzymes in the cholesterol biosynthetic pathway to decrease cellular cholesterol, which is necessary for enveloped viruses to bud from the cell membrane. The anticipated outcome of this project is a unified understanding of the biochemical mechanisms underpinning viperin’s diverse antiviral functions.