The N-terminal Amphipathic α-Helix of Viperin Mediates Localization to the Cytosolic Face of the Endoplasmic Reticulum and Inhibits Protein Secretion

Viperin is an evolutionarily conserved interferon-inducible protein that localizes to the endoplasmic reticulum (ER) and inhibits a number of DNA and RNA viruses. In this study, we report that viperin specifically localizes to the cytoplasmic face of the ER and that an amphipathic α-helix at its N terminus is necessary for the ER localization of viperin and sufficient to promote ER localization of a reporter protein, dsRed. Overexpression of intact viperin but not the amphipathic α-helix fused to dsRed induced crystalloid ER. Consistent with other proteins that induce crystalloid ER, viperin self-associates, and it does so independently of the amphipathic α-helix. Viperin expression also affected the transport of soluble but not membrane-associated proteins. Expression of intact viperin or an N-terminal α-helix-dsRed fusion protein significantly reduced secretion of soluble alkaline phosphatase and reduced its rate of ER-to-Golgi trafficking. Similarly, viperin expression inhibited bulk protein secretion and secretion of endogenous α1-antitrypsin and serum albumin from HepG2 cells. Converting hydrophobic residues in the N-terminal α-helix to acidic residues partially or completely restored normal transport of soluble alkaline phosphatase, suggesting that the extended amphipathic nature of the N-terminal α-helical domain is essential for inhibiting protein secretion. Viperin is an evolutionarily conserved interferon-inducible protein that localizes to the endoplasmic reticulum (ER) and inhibits a number of DNA and RNA viruses. In this study, we report that viperin specifically localizes to the cytoplasmic face of the ER and that an amphipathic α-helix at its N terminus is necessary for the ER localization of viperin and sufficient to promote ER localization of a reporter protein, dsRed. Overexpression of intact viperin but not the amphipathic α-helix fused to dsRed induced crystalloid ER. Consistent with other proteins that induce crystalloid ER, viperin self-associates, and it does so independently of the amphipathic α-helix. Viperin expression also affected the transport of soluble but not membrane-associated proteins. Expression of intact viperin or an N-terminal α-helix-dsRed fusion protein significantly reduced secretion of soluble alkaline phosphatase and reduced its rate of ER-to-Golgi trafficking. Similarly, viperin expression inhibited bulk protein secretion and secretion of endogenous α1-antitrypsin and serum albumin from HepG2 cells. Converting hydrophobic residues in the N-terminal α-helix to acidic residues partially or completely restored normal transport of soluble alkaline phosphatase, suggesting that the extended amphipathic nature of the N-terminal α-helical domain is essential for inhibiting protein secretion. Type I interferons are the first line of defense against viral infections. The significance of the interferon pathway is illustrated by the susceptibility of interferon signaling mutants to infection and by viral mechanisms that counteract this pathway (1Katze M.G. He Y. Gale Jr., M. Nat. Rev. Immunol. 2002; 2: 675-687Crossref PubMed Scopus (898) Google Scholar, 2Haller O. Kochs G. Weber F. Virology. 2006; 344: 119-130Crossref PubMed Scopus (543) Google Scholar). Although many genes are induced upon interferon stimulation, very few of these genes have been functionally characterized. Viperin is highly induced by both Type I and II interferons and has a broad range of antiviral activity, inhibiting DNA viruses, notably human cytomegalovirus (3Chin K.C. Cresswell P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15125-15130Crossref PubMed Scopus (319) Google Scholar); RNA viruses such as influenza, hepatitis C virus (HCV), 2The abbreviations used are: HCV, hepatitis C virus; ER, endoplasmic reticulum; HA, hemagglutinin; SeAP, secreted alkaline phosphatase; WT, wild-type. and alphaviruses (4Wang X. Hinson E.R. Cresswell P. Cell Host Microbe. 2007; 2: 96-105Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar, 5Helbig K.J. Lau D.T. Semendric L. Harley H.A. Beard M.R. Hepatology. 2005; 42: 702-710Crossref PubMed Scopus (201) Google Scholar, 6Zhang Y. Burke C.W. Ryman K.D. Klimstra W.B. J. Virol. 2007; 81: 11246-11255Crossref PubMed Scopus (192) Google Scholar); and retroviruses such as human immunodeficiency virus (7Rivieccio M.A. Suh H.S. Zhao Y. Zhao M.L. Chin K.C. Lee S.C. Brosnan C.F. J. Immunol. 2006; 177: 4735-4741Crossref PubMed Scopus (123) Google Scholar). Upon expression, viperin localizes to the endoplasmic reticulum (ER), where it interacts with farnesyl-diphosphate synthase, an enzyme involved in lipid biosynthesis. This interaction appears to result in the disruption of lipid raft microdomains and prevention of influenza virus from budding from the plasma membrane (4Wang X. Hinson E.R. Cresswell P. Cell Host Microbe. 2007; 2: 96-105Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). Although recent studies have explored the antiviral functions of viperin, the general biochemical properties of this protein remain largely undefined. Viperin is highly conserved across both mammals and lower vertebrates and shares homology with the MoaA family of “radical S-adenosylmethionine” enzymes that bind Fe-S clusters (3Chin K.C. Cresswell P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15125-15130Crossref PubMed Scopus (319) Google Scholar, 8Jiang D. Guo H. Xu C. Chang J. Gu B. Wang L. Block T.M. Guo J.T. J. Virol. 2008; 82: 1665-1678Crossref PubMed Scopus (229) Google Scholar). In addition to a putative Fe-S cluster-binding domain, viperin has a 42-amino acid residue N-terminal amphipathic α-helix, and similar domains in other proteins have been shown to bind membranes and induce membrane curvature (9Antonny B. Curr. Opin. Cell Biol. 2006; 18: 386-394Crossref PubMed Scopus (123) Google Scholar, 10McMahon H.T. Gallop J.L. Nature. 2005; 438: 590-596Crossref PubMed Scopus (1615) Google Scholar). In this study, we examined the role of the viperin N-terminal α-helical domain in both cellular localization and ER membrane morphology and analyzed the biochemical properties of viperin. We discovered that viperin forms dimers and induces a tightly ordered, visually striking array of ER membranes, known as crystalloid ER(11Fukuda M. Yamamoto A. Mikoshiba K. J. Biol. Chem. 2001; 276: 41112-41119Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 12Yamamoto A. Masaki R. Tashiro Y. J. Cell Sci. 1996; 109: 1727-1738Crossref PubMed Google Scholar, 13Snapp E.L. Hegde R.S. Francolini M. Lombardo F. Colombo S. Pedrazzini E. Borgese N. Lippincott-Schwartz J. J. Cell Biol. 2003; 163: 257-269Crossref PubMed Scopus (364) Google Scholar), upon overexpression. In addition, viperin expression impedes the secretion of a variety of soluble proteins. Although the N-terminal amphipathic α-helix is not sufficient to induce crystalloid ER formation, it is both necessary and sufficient to mediate ER localization and to inhibit protein secretion. Cells, Antibodies, and Constructs—HepG2, HeLa, and 293T cells were maintained in Dulbecco's modified Eagle's medium containing 5% bovine calf serum. The following antibodies were purchased commercially: anti-placental alkaline phosphatase (ab11299), anti-α1-antitrypsin (ab7633), anti-human serum albumin (ab18079), and anti-Myc (ab9106) (Abcam) and anti-hemagglutinin (HA) tag (HA.11; Covance). MaP.VIP and rabbit anti-calnexin antibody (4Wang X. Hinson E.R. Cresswell P. Cell Host Microbe. 2007; 2: 96-105Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar) and the mouse anti-tapasin monoclonal antibody PaSta1(14Peaper D.R. Wearsch P.A. Cresswell P. EMBO J. 2005; 24: 3613-3623Crossref PubMed Scopus (145) Google Scholar) were described previously. Goat anti-rabbit and anti-mouse Ig secondary antibodies were purchased commercially from Molecular Probes. All viperin constructs were generated by PCR amplification and then cloned into pcDNA3.1. The dsRed gene cassette was excised from pDsRed-Monomer (Clontech) using restriction enzyme digestion and then cloned into pcDNA3.1 with or without residues 1-42 of viperin. The pRSVPAP construct, which encodes placental alkaline phosphatase, was purchased from American Type Culture Collection. The construct encoding TAP1 (transporter associated with antigen processing subunit 1) fused to Cherry at its C terminus was a kind gift from Dr. David Stepensky (Ben-Gurion University, Beersheba, Israel). Constructs encoding secreted alkaline phosphatase (SeAP) and Sar1 and Arf1 dominant negatives were kind gifts from Dr. Jon Kagan (Harvard University). The expression construct encoding the vesicular stomatitis virus glycoprotein temperature-sensitive ts045 mutant (15Presley J.F. Cole N.B. Schroer T.A. Hirschberg K. Zaal K.J. Lippincott-Schwartz J. Nature. 1997; 389: 81-85Crossref PubMed Scopus (3) Google Scholar) was provided by Dr. Jennifer Lippincott-Schwartz (National Institutes of Health). Transfection—Cells were transiently transfected using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Western Blotting—Cells were harvested, washed once in phosphate-buffered saline, and lysed in 1% Triton X-100 in Tris-buffered saline (0.15 m NaCl and 0.01 m Tris, pH 7.4) containing a protease inhibitor mixture (Roche Applied Science). Whole cell lysates were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes (Millipore), and then probed with the indicated antibodies. Immunofluorescence—HeLa cells or 293T cells transiently expressing wild-type (WT) viperin, viperin lacking the N-terminal amphipathic α-helix (viperin-(Δ1-42)), or viperin bearing mutations in the α-helix were plated onto glass coverslips. Forty-eight hours post-transfection, the cells were fixed in 4% formaldehyde, washed, permeabilized with 0.05% saponin or 0.1% Triton X-100, and stained with the indicated antibodies. For selective plasma membrane permeabilization, cells were treated with 22 μg/ml streptolysin O (Aalto) in the presence of 0.3 mm dithiothreitol for 15 min on ice, washed in intracellular transport buffer (50 mm HEPES, 78 mm KCl, 4 mm MgCl2, 8.37 mm CaCl2, and 10 mm EGTA), and incubated for 5 min at 37 °C. After permeabilization, cells were fixed in 4% formaldehyde and then stained with the indicated antibodies in intracellular transport buffer. Electron Microscopy—293T cells transiently expressing the vector control, WT viperin, or viperin-(1-42)-dsRed were examined by electron microscopy as described previously (4Wang X. Hinson E.R. Cresswell P. Cell Host Microbe. 2007; 2: 96-105Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). For immunoelectron microscopy, cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) in 0.25 m HEPES, pH 7.4, for 1 h at room temperature and then in 8% paraformaldehyde in 0.25 m HEPES, pH 7.4, overnight at 4 °C. Samples were prepared for immunochemistry as described previously (16Folsch H. Pypaert M. Maday S. Pelletier L. Mellman I. J. Cell Biol. 2003; 163: 351-362Crossref PubMed Scopus (172) Google Scholar) and stained with MaP.VIP and 10 nm of protein A-gold (Cell Microscopy Center, Utrecht University, The Netherlands). Sections were examined with a Tecnai 12 Biotwin electron microscope, and images were captured using a charge-coupled device camera (Morada, Olympus). SeAP Assay—293T cells were cotransfected with SeAP and the indicated viperin constructs. Twenty-four hours post-transfection, cell supernatants were harvested and analyzed for SeAP secretion by an alkaline phosphatase assay using the Phospha-Light system (Applied Biosystems) and a luminometer. Radiolabeling and Pulse-Chase Analysis—Transiently transfected 293T cells or HepG2 cells were harvested, washed once in phosphate-buffered saline, and starved for 1 h in Dulbecco's modified Eagle's medium without methionine or cysteine (Sigma) containing 3% dialyzed fetal bovine serum. Cells were labeled for the indicated times with [35S]methionine (PerkinElmer Life Sciences) and then chased with Dulbecco's modified Eagle's medium containing 10% bovine calf serum and excess methionine and cysteine. Cells and supernatants were harvested at the indicated times and stored at -20 °C until detergent lysis. Immunoprecipitation—Cells were lysed in 1% Triton X-100 in Tris-buffered saline containing protease inhibitors for 30 min on ice. For radiolabeled cells, extracts were precleared with protein G-Sepharose (GE Healthcare) and normal rabbit serum and then immunoprecipitated with the indicated antibodies and protein G-Sepharose. Immunoprecipitates were washed, eluted in reducing sample buffer, run on SDS-polyacrylamide gels, dried, and exposed to PhosphorImager screens for quantification with ImageQuant software. For viperin co-immunoprecipitations, cell extracts were precleared with protein G-Sepharose, immunoprecipitated with anti-HA or control antibody, and then blotted with anti-Myc antibody. The N-terminal Amphipathic α-Helix Is Necessary and Sufficient to Localize Viperin to the Cytoplasmic Face of the ER—Although previous studies have shown that viperin localizes to the ER, the precise topology and the ER localization signal of viperin were not identified. To determine whether viperin localizes to the cytosolic or lumenal face of the ER, we used streptolysin O to selectively permeabilize the plasma membrane, followed by staining with the anti-viperin monoclonal antibody MaP.VIP. An anti-tapasin antibody that recognizes a lumenal ER epitope and an anti-calnexin antibody that recognizes a cytosolic epitope were used as controls. Under these conditions, viperin and calnexin staining was readily observed, whereas the ER lumenal epitope of tapasin was detected only after saponin permeabilization This that viperin was to the cytosolic face of the ER. To the domain we on the N-terminal α-helix it has a hydrophobic face that is of that not only bind to the ER but also induce membrane to determine whether the α-helix is necessary for ER we used to cells expressing WT viperin or a mutant lacking this domain WT viperin with ER human TAP1 fused to the protein tag Cherry and but the α-helix viperin to the the N-terminal α-helix of viperin to dsRed was also sufficient to this reporter protein from the and to the ER Viperin Overexpression the ER localization of viperin, we used immunoelectron microscopy to 293T cells expressing viperin. We that these cells highly similar to previously described crystalloid ER, by the of ER membranes into a A. Masaki R. Tashiro Y. J. Cell Sci. 1996; 109: 1727-1738Crossref PubMed Google Scholar, 13Snapp E.L. Hegde R.S. Francolini M. Lombardo F. Colombo S. Pedrazzini E. Borgese N. Lippincott-Schwartz J. J. Cell Biol. 2003; 163: 257-269Crossref PubMed Scopus (364) Google Scholar, L. J.L. Full Text PDF PubMed Scopus Google Scholar). the amphipathic α-helix-dsRed fusion protein not induce these that the α-helical domain is not sufficient to induce crystalloid ER. microscopy that viperin was highly in these in addition to normal ER of cells by using Triton X-100 saponin for permeabilization that expression of WT viperin but not or the α-helix-dsRed fusion protein induced ER by the presence of calnexin Viperin of the N-terminal Amphipathic that induce crystalloid ER are E.L. Hegde R.S. Francolini M. Lombardo F. Colombo S. Pedrazzini E. Borgese N. Lippincott-Schwartz J. J. Cell Biol. 2003; 163: 257-269Crossref PubMed Scopus (364) Google Scholar), and we to determine whether viperin is of and viperin expression we these proteins in 293T cells and studies on detergent viperin with viperin but not with a control protein, protein To determine whether this the N-terminal amphipathic α-helix, we similar with and The viperin mutants were also of that viperin or independently of the amphipathic α-helical domain The N-terminal Amphipathic α-Helix Is Necessary and Sufficient to determine whether the affected ER we examined protein in cells. Viperin expression in HepG2 cells a in protein as by the of proteins secreted into the to the of proteins in cell Although expression of the protein control on protein secretion to the vector control, viperin expression reduced protein secretion from HepG2 cells to with in cells expressing Sar1 and Arf1 and previously shown to protein secretion R. J. Cell Sci. PubMed Scopus Google Scholar, C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). and from cell supernatants and we specifically examined the secretion of endogenous albumin α1-antitrypsin and not by HepG2 cells and that secretion of of these soluble proteins was significantly reduced in cells. To determine whether the in protein secretion was a of crystalloid ER or a associated with the N-terminal amphipathic α-helix, we cotransfected constructs into 293T cells with the secreted of placental alkaline phosphatase lacking the membrane (SeAP) J. J. R. R. PubMed Scopus Google Scholar). SeAP secretion was and intracellular transport was using and The N-terminal amphipathic α-helix was to SeAP secretion to with WT viperin, whereas secretion in cells expressing was with that in cells expressing the vector control or the control protein of SeAP that that the amphipathic α-helical domain was both necessary and sufficient to inhibit the ER-to-Golgi of SeAP, by the rate of of the N-terminal amphipathic α-helix fused to dsRed also inhibited SeAP secretion and significantly the of with the vector control or dsRed C and Although viperin expression affected the secretion of soluble was on the intracellular of proteins. a temperature-sensitive of vesicular stomatitis virus glycoprotein we that the rate at which vesicular stomatitis virus glycoprotein at the cell after to the temperature was in and control cells Similarly, the rate at which placental alkaline phosphatase the protein that is the domain to the soluble SeAP reporter J. J. R. R. PubMed Scopus Google Scholar), at the cell was in cells transport was significantly by expression of the of viperin expression not the ER-to-Golgi of vesicular stomatitis virus glycoprotein not or placental alkaline phosphatase, as by the of in whereas of Sar1 and Arf1 reduced the transport rate in the Amphipathic α-Helix of the N-terminal domain of viperin into a the amphipathic of with hydrophobic on an extended face of the α-helix To determine whether this face of the α-helix is involved in inhibiting protein hydrophobic residues were to or acid in and residues were to in Although hydrophobic residues to or residues to not the of viperin to inhibit protein secretion not hydrophobic residues to acid partially or completely restored protein secretion Although of the acid affected the ER localization of viperin hydrophobic residues to acid ER In addition, in the α-helix ER in the of viperin to the Viperin has a broad range of antiviral and is highly conserved in suggesting that it is functionally is the for other antiviral the precise of of viperin largely To the only an of viperin expression on lipid raft microdomains and a role for the conserved putative in infection (4Wang X. Hinson E.R. Cresswell P. Cell Host Microbe. 2007; 2: 96-105Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar, 8Jiang D. Guo H. Xu C. Chang J. Gu B. Wang L. Block T.M. Guo J.T. J. Virol. 2008; 82: 1665-1678Crossref PubMed Scopus (229) Google Scholar). In this study, we and examined the properties of the domains of viperin. We that a in the domain is for protein and that the N-terminal amphipathic α-helical domain is for ER localization and with the secretion of soluble proteins. Overexpression of viperin induced in ER morphology that are of crystalloid ER. ER is induced by a number of membrane-associated proteins M. Yamamoto A. Mikoshiba K. J. Biol. Chem. 2001; 276: 41112-41119Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 13Snapp E.L. Hegde R.S. Francolini M. Lombardo F. Colombo S. Pedrazzini E. Borgese N. Lippincott-Schwartz J. J. Cell Biol. 2003; 163: 257-269Crossref PubMed Scopus (364) Google Scholar). for crystalloid ER that ER is the cytoplasmic domains of proteins dimers and membranes to a of M. Yamamoto A. Mikoshiba K. J. Biol. Chem. 2001; 276: 41112-41119Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 12Yamamoto A. Masaki R. Tashiro Y. J. Cell Sci. 1996; 109: 1727-1738Crossref PubMed Google Scholar, 13Snapp E.L. Hegde R.S. Francolini M. Lombardo F. Colombo S. Pedrazzini E. Borgese N. Lippincott-Schwartz J. J. Cell Biol. 2003; 163: 257-269Crossref PubMed Scopus (364) Google Scholar). Consistent with this we that viperin to to The interaction in viperin mutants lacking the amphipathic α-helix, that it is of the ER localization not with viperin lacking the N-terminal α-helical domain that the of is in membrane the to by to the of the We not that viperin expression and crystalloid ER induced the protein on of processing not M. H. F. D. Nature. 2002; PubMed Scopus Google Scholar). Although crystalloid ER has on ER that it is not for the in protein secretion that we upon viperin We also to on or localization of a number of ER and not a fusion protein containing the amphipathic α-helix N-terminal to dsRed not induce crystalloid ER but inhibited protein secretion to a with that with WT viperin. We that the N-terminal amphipathic α-helix induces membrane curvature that is upon viperin its C terminus to crystalloid ER have shown that amphipathic membrane curvature (9Antonny B. Curr. Opin. Cell Biol. 2006; 18: 386-394Crossref PubMed Scopus (123) Google Scholar, 10McMahon H.T. Gallop J.L. Nature. 2005; 438: 590-596Crossref PubMed Scopus (1615) Google Scholar, J. Nat. Rev. Cell Biol. 2006; PubMed Scopus Google Scholar). amphipathic with hydrophobic notably to the ER and induce membrane curvature (9Antonny B. Curr. Opin. Cell Biol. 2006; 18: 386-394Crossref PubMed Scopus (123) Google Scholar, L. S. E. M. R. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). hydrophobic residues on the Sar1 were to the mutant Sar1 proteins induced membrane and affected the of generated in in with Sar1 L. S. E. M. R. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). of the amphipathic α-helix of viperin that were to an on secretion in that the hydrophobic residues to to acid residues This the amphipathic α-helix of viperin, which of with residues in the of has a extended hydrophobic with this is the that to or at hydrophobic residues to to acid to induce of viperin from the ER ER upon viperin expression is with an in membrane curvature of the ER, which by the amphipathic α-helix and by viperin is to that the in membrane curvature is also for the in protein secretion that is upon expression of viperin or the amphipathic α-helix to the protein dsRed. Although not the of viperin with protein II and an of membrane curvature by the α-helix the of the that a in the of induced by a in curvature the of soluble to a which the of viperin expression on the transport of soluble but not membrane-associated proteins. to WT viperin were and we were to membrane curvature of in the the amphipathic of viperin and Sar1 a similar of ER localization and similar on membrane for a in soluble protein secretion but not membrane-associated proteins For viperin with bearing soluble or of this is whether the on the ER to its antiviral Viperin inhibit the of soluble and cellular proteins necessary for viral and viruses also membranes from the ER for viral and the J. 2005; PubMed Scopus Google Scholar, S. J. Nat. Rev. 2008; PubMed Scopus Google Scholar). of the antiviral functions of viperin to or the of these viral or previous report that viperin inhibits K.J. Lau D.T. Semendric L. Harley H.A. Beard M.R. Hepatology. 2005; 42: 702-710Crossref PubMed Scopus (201) Google Scholar), which is known to a from ER studies viperin specifically of the and Although viperin shares homology with the MoaA family of enzymes and a conserved to we have been to that viperin of and of soluble viperin in cells of or other not a previous that these to the of viperin D. Guo H. Xu C. Chang J. Gu B. Wang L. Block T.M. Guo J.T. J. Virol. 2008; 82: 1665-1678Crossref PubMed Scopus (229) Google Scholar). is that viperin Fe-S clusters or transiently or that expression were to to viperin. these for To these studies such viperin mutants for antiviral against other viruses. We David Stepensky and Jon Kagan for the Dr. Pypaert for with immunoelectron microscopy, Dr. University, and Dr. of for and Dr. Wang for and for with

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