Purification of the full-length, membrane-associated form of the antiviral enzyme viperin utilizing nanodiscs.

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.

The antiviral enzyme viperin inhibits cholesterol biosynthesis.

Many enveloped viruses bud from cholesterol-rich lipid rafts on the cell membrane. Depleting cellular cholesterol impedes this process and results in viral particles with reduced viability. Viperin (Virus Inhibitory Protein, Endoplasmic Reticulum-associated, Interferon iNducible) is an endoplasmic reticulum membrane-associated enzyme that exerts broad-ranging antiviral effects, including inhibiting the budding of some enveloped viruses. However, the relationship between viperin expression and the retarded budding of virus particles from lipid rafts on the cell membrane is unclear. Here, we investigated the effect of viperin expression on cholesterol biosynthesis using transiently expressed genes in the human cell line human embryonic kidney 293T (HEK293T). We found that viperin expression reduces cholesterol levels by 20% to 30% in these cells. Following this observation, a proteomic screen of the viperin interactome identified several cholesterol biosynthetic enzymes among the top hits, including lanosterol synthase (LS) and squalene monooxygenase (SM), which are enzymes that catalyze key steps in establishing the sterol carbon skeleton. Coimmunoprecipitation experiments confirmed that viperin, LS, and SM form a complex at the endoplasmic reticulum membrane. While coexpression of viperin was found to significantly inhibit the specific activity of LS in HEK293T cell lysates, coexpression of viperin had no effect on the specific activity of SM, although did reduce SM protein levels by approximately 30%. Despite these inhibitory effects, the coexpression of neither LS nor SM was able to reverse the viperin-induced depletion of cellular cholesterol levels, possibly because viperin is highly expressed in transfected HEK293T cells. Our results establish a link between viperin expression and downregulation of cholesterol biosynthesis that helps explain viperin's antiviral effects against enveloped viruses.

The Antiviral Enzyme, Viperin, Activates Protein Ubiquitination by the E3 Ubiquitin Ligase, TRAF6.

Viperin is a broadly conserved radical SAM enzyme that synthesizes the antiviral nucleotide ddhCTP. In higher animals, viperin expression also accelerates the degradation of various cellular and viral proteins necessary for viral replication; however, the details of this process remain largely unknown. Here, we show that viperin activates a component of the protein ubiquitination machinery, which plays an important role in both protein degradation and immune signaling pathways. We demonstrate that viperin binds the E3 ubiquitin ligase, TRAF6, which catalyzes K63-linked ubiquitination associated with immune signaling pathways. Viperin activates ubiquitin transfer by TRAF6-2.5-fold and causes a significant increase in polyubiquitinated forms of TRAF6 that are important for mediating signal transduction. Our observations both imply a role for viperin as an agonist of immune signaling and suggest that viperin may activate other K48-linked E3-ligases involved in targeting proteins for proteasomal degradation.

Interactions between Viperin, Vesicle-Associated Membrane Protein A, and Hepatitis C Virus Protein NS5A Modulate Viperin Activity and NS5A Degradation.

The radical SAM enzyme, viperin, exerts a wide range of antiviral effects through both the synthesis of the antiviral nucleotide 3'-deoxy-3',4'-didehydro-CTP (ddhCTP) and through its interactions with various cellular and viral proteins. Here we investigate the interaction of viperin with hepatitis C virus nonstructural protein 5A (NS5A) and the host sterol regulatory protein, vesicle-associated membrane protein A (VAP-33). NS5A and VAP-33 form part of the viral replication complex that is essential for replicating the RNA genome of the hepatitis C virus. Using transfected enzymes in HEK293T cells, we show that viperin binds independently to both NS5A and the C-terminal domain of VAP-33 (VAP-33C) and that this interaction is dependent on the proteins being colocalized to the ER membrane. Coexpression of VAP-33C and NS5A resulted in changes to the catalytic activity of viperin that depended upon viperin being colocalized to the ER membrane. The viperin-NS5A-VAP-33C complex exhibited the lowest specific activity, indicating that NS5A may inhibit viperin's ability to synthesize ddhCTP. Coexpression of viperin with NS5A was also found to significantly reduce cellular NS5A levels, most likely by increasing the rate of proteasomal degradation. An inactive mutant of viperin, unable to bind the iron-sulfur cluster, was similarly effective at reducing cellular NS5A levels.

Per- and polyfluoroalkyl substances and fluorinated alternatives in urine and serum by on-line solid phase extraction-liquid chromatography-tandem mass spectrometry.

Per- and polyfluoroalkyl substances (PFAS), man-made chemicals with variable length carbon chains containing the perfluoroalkyl moiety (CnF2n+1-), are used in many commercial applications. Since 1999-2000, several long-chain PFAS, including perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA), have been detected at trace levels in the blood of most participants of the National Health and Nutrition Examination Survey (NHANES)-representative samples of the U.S. general population-while short-chain PFAS have not. Lower detection frequencies and concentration ranges may reflect lower exposure to short-chain PFAS than to PFOS or PFOA or that, in humans, short-chain PFAS efficiently eliminate in urine. We developed on-line solid phase extraction-HPLC-isotope dilution-MS/MS methods for the quantification in 50 µL of urine or serum of 15 C3-C11 PFAS (C3 only in urine), and three fluorinated alternatives used as PFOA or PFOS replacements: GenX (ammonium salt of 2,3,3,3,-tetrafluoro-2-(1,1,2,2,3,3,3-heptafluoropropoxy)-propanoate, also known as HFPO-DA), ADONA (ammonium salt of 4,8-dioxa-3H-perfluorononanoate), and 9Cl-PF3ONS (9-chlorohexadecafluoro-3-oxanonane-1-sulfonate), main component of F53-B. Limit of detection for all analytes was 0.1 ng/mL. To validate the method, we analyzed 50 commercial urine/serum paired samples collected in 2016 from U.S. volunteers with no known exposure to the chemicals. In serum, detection frequency and concentration patterns agreed well with those from NHANES. By contrast, except for perfluorobutanoate, we did not detect long-chain or short-chain PFAS in urine. Also, we did not detect fluorinated alternatives in either urine or serum. Together, these results suggest limited exposure to both short-chain PFAS and select fluorinated alternatives in this convenience population.