Does Viperin Function as a Radical S-Adenosyl-l-methionine-dependent Enzyme in Regulating Farnesylpyrophosphate Synthase Expression and Activity?

Caitlyn Makins,Soumi Ghosh,Gabriel D Román-Meléndez,Paige A Malec,Robert T Kennedy,E Neil G Marsh

doi:10.1074/jbc.m116.751040

Abstract

Viperin is an endoplasmic reticulum-associated antiviral responsive protein that is highly up-regulated in eukaryotic cells upon viral infection through both interferon-dependent and independent pathways. Viperin is predicted to be a radical S-adenosyl-l-methionine (SAM) enzyme, but it is unknown whether viperin actually exploits radical SAM chemistry to exert its antiviral activity. We have investigated the interaction of viperin with its most firmly established cellular target, farnesyl pyrophosphate synthase (FPPS). Numerous enveloped viruses utilize cholesterol-rich lipid rafts to bud from the host cell membrane, and it is thought that by inhibiting FPPS activity (and therefore cholesterol synthesis), viperin retards viral budding from infected cells. We demonstrate that, consistent with this hypothesis, overexpression of viperin in human embryonic kidney cells reduces the intracellular rate of accumulation of FPPS but does not inhibit or inactivate FPPS. The endoplasmic reticulum-localizing, N-terminal amphipathic helix of viperin is specifically required for viperin to reduce cellular FPPS levels. However, although viperin reductively cleaves SAM to form 5'-deoxyadenosine in a slow, uncoupled reaction characteristic of radical SAM enzymes, this cleavage reaction is independent of FPPS. Furthermore, mutation of key cysteinyl residues ligating the catalytic [Fe4S4] cluster in the radical SAM domain, surprisingly, does not abolish the inhibitory activity of viperin against FPPS; indeed, some mutations potentiate viperin activity. These observations imply that viperin does not act as a radical SAM enzyme in regulating FPPS.

Highlights

Central to the mechanism of all radical SAM enzymes is the generation of a highly reactive 5Ј-deoxyadenosyl radical (Ado1⁄7) [1, 4, 17]
Viperin appears to be a radical SAM-dependent enzyme [22]. It is one of eight putative radical SAM enzymes identified in humans; four of these appear to be involved in tRNA modifications, two seem to be involved in co-factor biosynthesis, and the remaining two, including viperin, have unknown enzymatic activities [23]
The C87A, C90A, and triple mutant variants proved to be significantly more effective at reducing the intracellular level of farnesyl pyrophosphate synthase (FPPS) (ϳ6-fold) compared with wild-type viperin (ϳ2-fold). These results demonstrate that the iron-sulfur cluster is not required for viperin to reduce FPPS levels and imply that radical SAM chemistry is not involved in the regulation of FPPS by viperin

Summary

Introduction

Central to the mechanism of all radical SAM enzymes is the generation of a highly reactive 5Ј-deoxyadenosyl radical (Ado1⁄7) [1, 4, 17]. Viperin (Virus inhibitory protein, endoplasmic reticulumassociated, interferon inducible) is an interferon-stimulated gene that forms a component of the innate immune response that is activated upon viral infection (19 –21). It is highly conserved in vertebrates and is one of the few interferon-stimulated genes shown to have direct antiviral activity against a number of viruses, including human cytomegalovirus, hepatitis C, influenza A, and HIV. The mechanism by which viperin reduces cellular FPPS activity is still unknown

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