Summary Viperin (Virus Inhibitory Protein; Endoplasmic Reticulum-associated, Interferon iNducible) is a radical SAM enzyme found in all 6 kingdoms of life. It constitutes an ancient defense mechanism against viruses and is one of very few radical SAM enzymes conserved in higher animals, including humans. Viperin has been shown to restrict the infectivity of a number of important human viruses including influenza A, HIV, cytomegalovirus and hepatitis C. Viperin has two facets to its antiviral activity. First, it synthesizes the antiviral nucleotide 3’-deoxy- 3’,4’-didehydro-CTP (ddhCTP) by dehydration of CTP. ddhCTP acts as a chain-terminating inhibitor that is mis- incorporated by some, but not all, viral RNA-dependent RNA polymerases to disrupt the replication of RNA virus genomes. Second, in higher animals viperin is also centrally integrated into the broader cellular antiviral response through a wide-ranging network of protein-protein interactions. Through these interactions, viperin down-regulates various cellular pathways important for viral replication, which explains how it is able to restrict replication of DNA viruses and retroviruses such as cytomegalovirus and HIV. In the last grant cycle, we identified two important biochemical pathways that viperin modulates: protein ubiquitination and cholesterol biosynthesis, and further identified specific enzymes within those pathways that interact with viperin. Our goal now is to understand at the molecular level how interactions between viperin and its partner enzymes regulate the activity of both the partner enzyme and viperin. These studies aim to uncover the mechanisms that underpin viperin’s seemingly disparate interactions with different proteins and which contribute to its antiviral properties. Our studies aim to answer the following questions: i) How does viperin activate E3 ubiquitin ligases in the protein ubiquitination system? ii) How does viperin regulate enzymes involved in cholesterol biosynthesis? iii). How do changes to the structure/activity of viperin alter its antiviral activity? To answer these questions, we will reconstitute the complexes of viperin with the various in enzymes in vitro using purified enzymes and undertake detailed enzyme kinetic analyses, combined with biophysical measurements of protein and ligand binding, to study to study the mechanism(s) of activation or inhibition. Concurrently, we will use crystallography and/or cryo-EM, as appropriate, to determine structures for the complexes of viperin with these enzymes. Using what we learn from these studies, we will test our understanding of viperin’s antiviral properties in cell-based models of viral infection.