Deciphering the role of ddhCTP in viperin-related functions

Viperin (virus-inhibitory protein, endoplasmic reticulum-associated, interferon (IFN) inducible) is an interferon inducible protein that inhibits and/or is involved in the restriction of a remarkable range of both RNA and DNA viruses1-3. Viperin is a member of the radical S-adenosylmethionine (RS) superfamily of enzymes, all of which possess an oxygen-sensitive iron-sulfur cluster that is indispensable for catalytic activity. The mechanisms by which this 42 kDa enzyme elicits broad- spectrum antiviral activity remained a mystery until we recently discovered that viperin catalyzes the conversion of CTP to 3ʹ-deoxy-3′,4ʹ-didehydro-CTP (ddhCTP) through a radical-based mechanism. ddhCTP is a previously undescribed ribonucleotide, which acts as a chain terminator in vitro against several flavivirus RNA-dependent RNA polymerases (RDRP), including Dengue (DNV), West Nile (WNV), and Zika virus. Furthermore, we demonstrated that ddhCTP inhibited Zika virus replication in vivo. The discovery of ddhCTP provided, for the first time, a potential mechanism for some of viperin’s antiviral activities. However, one important caveat is that picornavirus RDRPs are not sensitive to ddhCTP, despite viperin serving as a restriction factor for these microbes. Viperin has also been implicated in several additional cellular processes important for viral replication and innate immunity, including cholesterol metabolism, fatty acid b- oxidation, protein turnover and ribosomal protein translation. These observations underly our major driving hypothesis that ddhCTP possesses additional critical roles in cellular metabolism that impact viral replication as well as other physiological processes. The objectives of this proposal are to provide biochemical, structural, and mechanistic insights into the roles that ddhCTP plays in the broader context of viperin-related functions.