The innate immune response involves the induction of hundreds of interferon-stimulated genes (ISGs), many of which play a role in cancer immunosurveillance and antiviral immunity. Therefore, understanding how ISGs function and coordinate with the cellular network is of critical importance. We focus on one such ISG, viperin (virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible). Viperin has been reported to inhibit a broad spectrum of DNA and RNA viruses, including many flaviviruses, human cytomegalovirus (HCMV), Chikungunya virus, influenza A virus, Sindbis virus, vesicular stomatitis virus, tick-borne encephalitis virus, HIV-1 and more. It is highly conserved in evolution, from protozoans to humans. Viperin is a peripheral membrane protein, associating on the cytosolic face of the ER via its N-terminal amphipathic helix. The cytosolic domain contains a radical SAM domain with a [4Fe-4S] cluster coordinated by three cysteine residues in the active site. Although the enzymatic function and substrate of viperin is still under debate, a recent study reveals that viperin catalyzes the conversion of cytidine triphosphate to 3′-deoxy-3′,4′-didehydro-CTP (ddhCTP) via its radical SAM activity. However, a general mechanism for viperin action and its potential roles within the innate immune response remain to be defined. Here, we demonstrate that viperin inhibits global protein translation via its radical SAM-dependent enzymatic activity. Using immortalized mouse macrophages from wild-type and viperin knockout mice we show that, although other ISGs are induced, type I IFN treatment of WT but not viperin KO mouse microphages leads to a global change in polysome profile with an increase in the ribosome fraction and a decrease in actively translating ribosomes. In parallel, the translational regulation function was evaluated in 293T-cells with transient transfection and doxycycline-inducible system. Despite no IFN stimulation and therefore no other ISG expression, viperin effectively represses translation. Furthermore, we find that viperin reduces global protein translation by ~30% in live cells using [35S]-methionine metabolic pulse-labeling or O-propargyl-puromycin (OPP) labelling. The radical SAM enzymatic activity is required for translational inhibition: site-directed mutagenesis of amino residues involved in iron-sulfur cluster and substrate binding abolishes the translational inhibition activity. We also demonstrate that ddhCTP, a recently identified vipern product, inhibits global protein translation in live cells. Viral replication requires the host translation machinery to make viral proteins, and our results suggest that much of its antiviral activity may be attributable to translational regulation. We show that viperin inhibits viral protein synthesis and viral replication of the Kunjin virus, a West Nile Virus variant, in tissue culture cells. Our study suggests a partially unifying mechanism for the broad antiviral function of viperin based on translational regulation.
Citation Format: Chun-Chieh Hsu, Maudry Laurent-Rolle, Peter Cresswell. Regulation of translation by the interferon-induced antiviral protein viperin [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B163.