The antiviral protein, viperin, localizes to lipid droplets via its N-terminal amphipathic alpha-helix

Abstract

Lipid droplets are intracellular lipid-storage organelles that are thought to be derived from the endoplasmic reticulum (ER). Several pathogens, notably hepatitis C virus, use lipid droplets for replication. Numerous questions remain about how lipid droplets are generated and used by viruses. Here we show that the IFN-induced antiviral protein viperin, which localizes to the cytosolic face of the ER and inhibits HCV, localizes to lipid droplets. We show that the N-terminal amphipathic alpha-helix of viperin that is responsible for ER localization is also necessary and sufficient to localize both viperin and the fluorescent protein dsRed to lipid droplets. Point mutations in the alpha-helix that prevent ER association also disrupt lipid droplet association, and sequential deletion mutants indicate that the same number of helical turns are necessary for ER and lipid droplet association. Finally, we show that the N-terminal amphipathic alpha-helix of the hepatitis C viral protein NS5A can localize dsRed and viperin to lipid droplets. These findings indicate that the amphipathic alpha-helices of viperin and NS5A are lipid droplet-targeting domains and suggest that viperin inhibits HCV by localizing to lipid droplets using a domain and mechanism similar to that used by HCV itself.

Fig. 1.

Viperin localizes to lipid droplets. (A and B) Primary mouse bone marrow derived macrophages (BMMΦ) were treated with 200 U/mL IFNα and 400 μM oleic acid overnight to induce viperin expression and lipid droplet formation, respectively. (A) BMMΦ were fixed, permeabilized with saponin, and then examined by immunofluorescence for viperin colocalization with the lipid droplet dye, BODIPY, and an anti-ADRP antibody. (B) BMMΦ from wild-type B6 (WT) or viperin knock-out (KO) mice were subjected to lipid droplet fractionation using a sucrose gradient. The whole cell lysate (WC), membrane (Mem), and lipid droplet (LD) fractions were separated by SDS/PAGE and then analyzed by Western blot analysis for viperin expression using anti-GRp94 and anti-ADRP antibodies as markers for the ER and lipid droplets, respectively. (C) HepG2 cells that had been transiently transfected with viperin were fixed, permeabilized with saponin, and then analyzed by immunofluorescence for localization to lipid droplets using BODIPY and an anti-ADRP antibody as lipid droplet markers.

Fig. 2.

The N-terminal amphipathic α-helix of NS5A is sufficient to localize viperin to lipid droplets. 293T cells were transiently transfected with the vector control, wild-type viperin (WT), NS5A (1–30)Viperin (43–362), or viperin Δ1–42 as diagramed in (A) and then treated with 400 μM oleic acid overnight to induce lipid droplet formation. (B) Transfected and oleic acid–treated 293T cells were examined by immunofluoresence as described in Fig. 1A for localization with the lipid droplet markers BODIPY and ADRP. (C) The 293T cells were subjected to lipid droplet fractionation using a sucrose gradient. The whole cell lysate (WC), membrane (Membrane), and lipid droplet (LD) fractions were separated by SDS/PAGE and then analyzed by Western blot analysis for viperin expression. GRp94, TPP-1, and ADRP served as markers for the ER, lysosomes, and lipid droplets, respectively. Gel Lanes: vector control (1), wild-type viperin (2), NS5A (1–30) Vip (43–362) (3), and viperin Δ1–42 (4).

Fig. 3.

The N-terminal amphipathic α-helices of viperin and NS5A are necessary and sufficient to localize dsRed to lipid droplets. 293T cells were transiently transfected with the noted dsRed constructs and then treated with 400 μM oleic acid overnight to induce lipid droplet formation. (A) 293T cells were examined by immunofluoresence as described in Fig. 1A for localization with the lipid droplet markers BODIPY and ADRP. (B) The cells were subjected to lipid droplet fractionation using a sucrose gradient. The whole cell lysate (WC), membrane (Membrane), and lipid droplet (LD) fractions were separated by SDS/PAGE and then analyzed by Western blot analysis for viperin expression. GRp94, TPP-1, and ADRP served as markers for the ER, lysosomes, and lipid droplets, respectively. Gel Lanes: vector control (1), dsRed (2), and Vip (1–42) dsRed (3).

Fig. 4.

ER association is necessary for viperin to localize to lipid droplets. (A) Helical wheel drawing of the N-terminal amphipathic α-helix of viperin. Amino acid residues 9–42 were diagramed in a helical wheel using a helical wheel projection program (http://rzlab.ucr.edu/scripts/wheel/wheel.cgi?sequence = ABCDEFGHIJLKMNOP&submit =Submit). The circles, diamonds, and pentagons represent hydrophilic, hydrophobic, and potentially positively charged residues, respectively. The hydrophobic and hydrophilic residues are colored in green and red, respectively, with a greater hydrophobicity or hydrophilicity corresponding to a darker, purer color. Neutral residues are represented in yellow and potentially charged residues are in blue. Residues that were mutated to glutamic acid have been outlined in black and the sequences of the amphipathic α-helix for wild-type viperin (WT), W26E/L8E/L1E, and L20E/L27E/L31E are shown below with the mutated residues highlighted in bold. (B) 293T cells were transiently transfected with the noted triple helical point mutants in which the noted hydrophobic residues were mutated to glutamic acid and then examined for ER localization using anti-calnexin as an ER marker. (C and D) 293T cells were transiently transfected with the noted triple helical point mutants and treated with 400 μM oleic acid overnight. The cells were examined for lipid droplet localization using BODIPY and an anti-ADRP antibody as lipid droplet markers as described in Fig. 1A (C) or by Western blot analysis of sucrose gradients as described in Fig. 2C (D). Gel Lanes: vector control (1), wild-type viperin (2), W26E/L8E/L1E (3), and L20E/L27E/L31E (4). (E) 293T cells were transfected with the indicated sequential helical turn deletion mutants in which one (T1), two (T1–2), three (T1–3), etc. helical turns were deleted, and then treated with 400 μM oleic acid overnight. These cells were examined by immunofluorescence as described in Fig. 1A.