E. Neil G. Marsh

Many antiviral and anticancer drugs are based on the structure of pyrimidine nucleosides. Once phosphorylated, they function by interfering with either DNA or RNA replication or transcription. The antiviral enzyme, viperin, catalyzes the dehydration of CTP to produce 3′-deoxy-3′,4′-didehydroCTP (ddhCTP), which functions as a natural antiviral nucleotide. Various drugs mimic the structure of CTP, and furthermore, viperin is upregulated in some cancers and autoimmune diseases. Therefore, we examined whether such drugs may be substrates for viperin. A panel of eight commercially available nucleosides, including several clinically used drugs, were enzymatically converted to their triphosphate forms and investigated as substrates for viperin. Five of the nucleotides were not substrates, indicating that viperin has generally high specificity for CTP. Two nucleotides, 3′-deoxy-3′-fluoroCTP and cytarabine triphosphate elicited reductive cleavage of SAM and were converted to their dehydrated products by viperin. The last compound, gemcitabine triphosphate, elicited reductive cleavage of SAM, but no dehydrated product was observed, implying that it activates SAM cleavage without undergoing hydrogen atom abstraction. The implications of these results are discussed.

Prenylated-FMN (prFMN) is the cofactor used by the UbiD-like family of decarboxylases that catalyzes the decarboxylation of various aromatic and unsaturated carboxylic acids. prFMN is synthesized from reduced FMN and dimethylallyl phosphate (DMAP) by a specialized prenyl transferase, UbiX. UbiX catalyzes the sequential formation of two bonds, the first between N5 of the flavin and C1 of DMAP, and the second between C6 of the flavin and C3 of DMAP. We have examined the reaction of UbiX with both FMN and riboflavin. Although UbiX converts FMN to prFMN, we show that significant amounts of the N5-dimethylallyl-FMN intermediate are released from the enzyme during catalysis. With riboflavin as the substrate, UbiX catalyzes only a partial reaction, resulting in only N5-dimethylallyl-riboflavin being formed. Purification of the N5-dimethylallyl-FMN adduct allowed its structure to be verified by ¹H NMR spectroscopy and its reactivity to be investigated. Surprisingly, whereas reduced prFMN oxidizes in seconds to form the stable prFMN semiquinone radical when exposed to air, N5-dimethylallyl-FMN oxidizes much more slowly over several hours; in this case, oxidation is accompanied by spontaneous hydrolysis to regenerate FMN. These studies highlight the important contribution that cyclization of the prenyl-derived ring of prFMN makes to the cofactor's biological activity.

H/D exchange between substrate and solvent D₂O provides a sensitive test for substrate activation in prenylated-flavin dependent enzymes. We show that a remarkably diverse range of molecules can be activated by one enzyme, ferulic acid decarboxylase.

Viperin is an antiviral protein that exhibits a remarkably broad spectrum of antiviral activity. Viperin-like proteins are found all kingdoms of life, suggesting it is an ancient component of the innate immune system. However, viruses have developed strategies to counteract viperin’s effects. Here, we describe a feedback loop between viperin and viral hemorrhagic septicemia virus (VHSV), a common fish pathogen. We show that Lateolabrax japonicus viperin ( Lj viperin) is induced by both IFN-independent and IFN-dependent pathways, with the C-terminal domain of Lj viperin being important for its anti-VHSV activity. Lj viperin exerts an antiviral effect by binding both the nucleoprotein (N) and phosphoprotein (P) of VHSV and induces their degradation through the autophagy pathway, which is an evolutionarily conserved antiviral mechanism. However, counteracting viperin’s activity, N protein targets and degrades transcription factors that up-regulate Lj viperin expression, interferon regulatory factor (IRF) 1 and IRF9, through ubiquitin-proteasome pathway. Together, our results reveal a previously unknown feedback loop between viperin and virus, providing potential therapeutic targets for VHSV prevention. Importance: Viral hemorrhagic septicaemia (VHS) is a contagious disease caused by the viral hemorrhagic septicaemia virus (VHSV), which poses a threat to over 80 species of marine and freshwater fish. Currently, there are no effective treatments available for this disease. Understanding the mechanisms of VHSV-host interaction is crucial for preventing viral infections. Here, we found that, as an ancient antiviral protein, viperin degrades the N and P proteins of VHSV through the autophagy pathway. Additionally, the N protein also impacts the biological functions of IRF1 and IRF9 through the ubiquitin-proteasome pathway, leading to the suppression of viperin expression. Therefore, the N protein may serve as a potential virulence factor for the development of VHSV vaccines and screening of antiviral drugs. Our research will serve as a valuable reference for the development of strategies to prevent VHSV infections.

Ferulic acid decarboxylase (FDC) catalyzes the reversible carboxylation of various substituted phenylacrylic acids to produce the correspondingly substituted styrenes and CO₂. FDC is a member of the UbiD family of enzymes that use prenylated-FMN (prFMN) to catalyze decarboxylation reactions on aromatic rings and C-C double bonds. Although a growing number of prFMN-dependent enzymes have been identified, the mechanism of the reaction remains poorly understood. Here, we present a detailed pre-steady-state kinetic analysis of the FDC-catalyzed reaction of prFMN with both styrene and phenylacrylic acid. Based on the pattern of reactivity observed, we propose a "two-stroke" kinetic model in which negative cooperativity between the two subunits of the FDC homodimer plays an important and previously unrecognized role in catalysis. In this model, catalysis is initiated at the high-affinity active site, which reacts with phenylacrylate to yield, after decarboxylation, the covalently bound styrene-prFMN cycloadduct. In the second stage of the catalytic cycle, binding of the second substrate molecule to the low-affinity active site drives a conformational switch that interconverts the high-affinity and low-affinity active sites. This switching of affinity couples the energetically unfavorable cycloelimination of styrene from the first site with the energetically favorable cycloaddition and decarboxylation of phenylacrylate at the second site. We note as a caveat that, at this point, the complexity of the FDC kinetics leaves open other mechanistic interpretations and that further experiments will be needed to more firmly establish or refute our proposal.

Viperin is a radical S-adenosylmethionine enzyme that catalyzes the formation of the antiviral ribonucleotide, 3’-deoxy-3’,4’-didehydroCTP. The enzyme is conserved across all kingdoms of life, and in higher animals viperin is localized to the ER-membrane and lipid droplets through an N-terminal extension that forms an amphipathic helix. Evidence suggests that the N-terminal extension plays an important role in viperin’s interactions with other membrane proteins. These interactions serve to modulate the activity of various other enzymes that are important for viral replication and constitute another facet of viperin’s antiviral properties, distinct from its catalytic activity. However, the full-length form of the enzyme, which has proved refractory to expression in E. coli, has not been previously purified. Here we report the purification of the full-length form of viperin from HEK293T cells transfected with viperin. The purification method utilizes nanodiscs to maintain the protein in its membrane-bound state. Unexpectedly, the enzyme exhibits significantly lower catalytic activity once purified, suggesting that interactions with other ER-membrane components may be important to maintain viperin’s activity.