Probing the Mechanism of Viral Inhibition by the Radical S-adenosyl-L-methionine (SAM) Dependent Enzyme- Viperin

Abstract

Viperin (Virus Inhibitory Protein; Endoplasmic Reticulum associated, INterferon inducible) is an endoplasmic reticulum (ER)-associated antiviral responsive protein that is highly up-regulated in eukaryotic cells upon viral infection. Viperin is a radical S-adenosyl-L-methionine (SAM) enzyme, that catalyses the synthesis of antiviral nucleotide 3’-deoxy-3’, 4’-didehydro-CTP (ddhCTP) exploiting radical SAM chemistry. However, the modulation of its catalytic activity by other intracellular proteins is not well understood and needs further investigation. In this dissertation, I use enzymology-based approaches to investigate how viperin’s enzymatic activity is regulated through its interaction with various cellular and viral proteins that are involved in cellular metabolic and signalling pathways and viral replication. I showed that viperin can reduce the intracellular expression level of the cholesterol biosynthetic enzyme, farnesyl pyrophosphate synthase (FPPS). This, in turn perturbing the intracellular cholesterol synthesis, thereby retarding budding of enveloped viruses from cholesterol-rich lipid rafts of host cell membranes. I also undertook a proteomics study that revealed that viperin interacts with several other endogenous cholesterol biosynthetic enzymes. I also demonstrated that viperin promotes the degradation of viral non-structural protein A (NS5A) from hepatitis C virus through proteasome-mediated degradation in the presence of sterol-regulatory protein VAP-33. In turn, co-expression of viperin with VAP-33 and NS5A reduced the specific activity of viperin by ~ 3-fold. Lastly, this study showed that viperin is activated by innate immune signalling proteins kinase IRAK1 and ubiquitin ligase TRAF6, as it facilitates the ubiquitination of IRAK1 by TRAF6. The results provide valuable insights into the mechanism of action of viperin in regulating these target proteins and its significance as a SAM-dependent enzyme.