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Human Subtilase SKI-1/S1P Is a Master Regulator of the HCV Lifecycle and a Potential Host Cell Target for Developing Indirect-Acting Antiviral Agents

DOI: 10.1371/journal.ppat.1002468

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HCV infection is a major risk factor for liver cancer and liver transplantation worldwide. Overstimulation of host lipid metabolism in the liver by HCV-encoded proteins during viral infection creates a favorable environment for virus propagation and pathogenesis. In this study, we hypothesize that targeting cellular enzymes acting as master regulators of lipid homeostasis could represent a powerful approach to developing a novel class of broad-spectrum antivirals against infection associated with human Flaviviridae viruses such as hepatitis C virus (HCV), whose assembly and pathogenesis depend on interaction with lipid droplets (LDs). One such master regulator of cholesterol metabolic pathways is the host subtilisin/kexin-isozyme-1 (SKI-1) – or site-1 protease (S1P). SKI-1/S1P plays a critical role in the proteolytic activation of sterol regulatory element binding proteins (SREBPs), which control expression of the key enzymes of cholesterol and fatty-acid biosynthesis. Here we report the development of a SKI-1/S1P-specific protein-based inhibitor and its application to blocking the SREBP signaling cascade. We demonstrate that SKI-1/S1P inhibition effectively blocks HCV from establishing infection in hepatoma cells. The inhibitory mechanism is associated with a dramatic reduction in the abundance of neutral lipids, LDs, and the LD marker: adipose differentiation-related protein (ADRP)/perilipin 2. Reduction of LD formation inhibits virus assembly from infected cells. Importantly, we confirm that SKI-1/S1P is a key host factor for HCV infection by using a specific active, site-directed, small-molecule inhibitor of SKI-1/S1P: PF-429242. Our studies identify SKI-1/S1P as both a novel regulator of the HCV lifecycle and as a potential host-directed therapeutic target against HCV infection and liver steatosis. With identification of an increasing number of human viruses that use host LDs for infection, our results suggest that SKI-1/S1P inhibitors may allow development of novel broad-spectrum biopharmaceuticals that could lead to novel indirect-acting antiviral options with the current standard of care.


[1]  Manes S, del Real G, Lacalle RA, Lucas P, Gomez-Mouton C, et al. (2000) Membrane raft microdomains mediate lateral assemblies required for HIV-1 infection. EMBO Rep 1: 190–196.
[2]  Sakamoto H, Okamoto K, Aoki M, Kato H, Katsume A, et al. (2005) Host sphingolipid biosynthesis as a target for hepatitis C virus therapy. Nat Chem Biol 1: 333–337.
[3]  Nguyen DH, Hildreth JE (2000) Evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts. J Virol 74: 3264–3272.
[4]  Shi ST, Lee KJ, Aizaki H, Hwang SB, Lai MM (2003) Hepatitis C virus RNA replication occurs on a detergent-resistant membrane that cofractionates with caveolin-2. J Virol 77: 4160–4168.
[5]  Olofsson SO, Bostrom P, Andersson L, Rutberg M, Perman J, et al. (2009) Lipid droplets as dynamic organelles connecting storage and efflux of lipids. Biochim Biophys Acta 1791: 448–458.
[6]  Cheung W, Gill M, Esposito A, Kaminski CF, Courousse N, et al. (2010) Rotaviruses associate with cellular lipid droplet components to replicate in viroplasms, and compounds disrupting or blocking lipid droplets inhibit viroplasm formation and viral replication. J Virol 84: 6782–6798.
[7]  Samsa MM, Mondotte JA, Iglesias NG, Assuncao-Miranda I, Barbosa-Lima G, et al. (2009) Dengue virus capsid protein usurps lipid droplets for viral particle formation. PLoS Pathog 5: e1000632.
[8]  Miyanari Y, Atsuzawa K, Usuda N, Watashi K, Hishiki T, et al. (2007) The lipid droplet is an important organelle for hepatitis C virus production. Nat Cell Biol 9: 1089–1097.
[9]  Boulant S, Targett-Adams P, McLauchlan J (2007) Disrupting the association of hepatitis C virus core protein with lipid droplets correlates with a loss in production of infectious virus. J Gen Virol 88: 2204–2213.
[10]  Shavinskaya A, Boulant S, Penin F, McLauchlan J, Bartenschlager R (2007) The lipid droplet binding domain of hepatitis C virus core protein is a major determinant for efficient virus assembly. J Biol Chem 282: 37158–37169.
[11]  Herker E, Harris C, Hernandez C, Carpentier A, Kaehlcke K, et al. (2010) Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1. Nat Med 16: 1295–1298.
[12]  Shepard CW, Finelli L, Alter MJ (2005) Global epidemiology of hepatitis C virus infection. Lancet Infect Dis 5: 558–567.
[13]  Grebely J, Matthews GV, Dore GJ (2011) Treatment of acute HCV infection. Nat Rev Gastroenterol Hepatol 8: 265–274.
[14]  Robertson B, Myers G, Howard C, Brettin T, Bukh J, et al. (1998) Classification, nomenclature, and database development for hepatitis C virus (HCV) and related viruses: proposals for standardization. International Committee on Virus Taxonomy. Arch Virol 143: 2493–2503.
[15]  Moradpour D, Penin F, Rice CM (2007) Replication of hepatitis C virus. Nat Rev Microbiol 5: 453–463.
[16]  Boulant S, Douglas MW, Moody L, Budkowska A, Targett-Adams P, et al. (2008) Hepatitis C virus core protein induces lipid droplet redistribution in a microtubule- and dynein-dependent manner. Traffic 9: 1268–1282.
[17]  Moradpour D, Englert C, Wakita T, Wands JR (1996) Characterization of cell lines allowing tightly regulated expression of hepatitis C virus core protein. Virology 222: 51–63.
[18]  Bartenschlager R, Penin F, Lohmann V, Andre P (2011) Assembly of infectious hepatitis C virus particles. Trends Microbiol 19: 95–103.
[19]  Waris G, Felmlee DJ, Negro F, Siddiqui A (2007) Hepatitis C virus induces proteolytic cleavage of sterol regulatory element binding proteins and stimulates their phosphorylation via oxidative stress. J Virol 81: 8122–8130.
[20]  Kim K, Kim KH, Ha E, Park JY, Sakamoto N, et al. (2009) Hepatitis C virus NS5A protein increases hepatic lipid accumulation via induction of activation and expression of PPARgamma. FEBS Lett 583: 2720–2726.
[21]  Perlemuter G, Sabile A, Letteron P, Vona G, Topilco A, et al. (2002) Hepatitis C virus core protein inhibits microsomal triglyceride transfer protein activity and very low density lipoprotein secretion: a model of viral-related steatosis. FASEB J 16: 185–194.
[22]  Moriya K, Yotsuyanagi H, Shintani Y, Fujie H, Ishibashi K, et al. (1997) Hepatitis C virus core protein induces hepatic steatosis in transgenic mice. J Gen Virol 78(Pt 7): 1527–1531.
[23]  Bach N, Thung SN, Schaffner F (1992) The histological features of chronic hepatitis C and autoimmune chronic hepatitis: a comparative analysis. Hepatology 15: 572–577.
[24]  Adinolfi LE, Gambardella M, Andreana A, Tripodi MF, Utili R, et al. (2001) Steatosis accelerates the progression of liver damage of chronic hepatitis C patients and correlates with specific HCV genotype and visceral obesity. Hepatology 33: 1358–1364.
[25]  Antunez I, Aponte N, Fernandez-Carbia A, Rodriguez-Perez F, Toro DH (2004) Steatosis as a predictive factor for treatment response in patients with chronic hepatitis C. P R Health Sci J 23: 57–60.
[26]  Soresi M, Tripi S, Franco V, Giannitrapani L, Alessandri A, et al. (2006) Impact of liver steatosis on the antiviral response in the hepatitis C virus-associated chronic hepatitis. Liver Int 26: 1119–1125.
[27]  Negro F, Sanyal AJ (2009) Hepatitis C virus, steatosis and lipid abnormalities: clinical and pathogenic data. Liver Int 29: Suppl 226–37.
[28]  Alvisi G, Madan V, Bartenschlager R (2011) Hepatitis C virus and host cell lipids: an intimate connection. RNA Biol 8: 258–269.
[29]  Syed GH, Amako Y, Siddiqui A (2010) Hepatitis C virus hijacks host lipid metabolism. Trends Endocrinol Metab 21: 33–40.
[30]  Park CY, Jun HJ, Wakita T, Cheong JH, Hwang SB (2009) Hepatitis C virus nonstructural 4B protein modulates sterol regulatory element-binding protein signaling via the AKT pathway. J Biol Chem 284: 9237–9246.
[31]  Oem JK, Jackel-Cram C, Li YP, Zhou Y, Zhong J, et al. (2008) Activation of sterol regulatory element-binding protein 1c and fatty acid synthase transcription by hepatitis C virus non-structural protein 2. J Gen Virol 89: 1225–1230.
[32]  Kim KH, Hong SP, Kim K, Park MJ, Kim KJ, et al. (2007) HCV core protein induces hepatic lipid accumulation by activating SREBP1 and PPARgamma. Biochem Biophys Res Commun 355: 883–888.
[33]  Brown MS, Goldstein JL (2009) Cholesterol feedback: from Schoenheimer's bottle to Scap's MELADL. J Lipid Res 50 Suppl. pp. S15–27.
[34]  Yang T, Espenshade PJ, Wright ME, Yabe D, Gong Y, et al. (2002) Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER. Cell 110: 489–500.
[35]  Nohturfft A, DeBose-Boyd RA, Scheek S, Goldstein JL, Brown MS (1999) Sterols regulate cycling of SREBP cleavage-activating protein (SCAP) between endoplasmic reticulum and Golgi. Proc Natl Acad Sci U S A 96: 11235–11240.
[36]  Brown MS, Goldstein JL (1999) A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood. Proc Natl Acad Sci U S A 96: 11041–11048.
[37]  Sakai J, Rawson RB, Espenshade PJ, Cheng D, Seegmiller AC, et al. (1998) Molecular identification of the sterol-regulated luminal protease that cleaves SREBPs and controls lipid composition of animal cells. Mol Cell 2: 505–514.
[38]  Rawson RB, Zelenski NG, Nijhawan D, Ye J, Sakai J, et al. (1997) Complementation cloning of S2P, a gene encoding a putative metalloprotease required for intramembrane cleavage of SREBPs. Mol Cell 1: 47–57.
[39]  Hua X, Yokoyama C, Wu J, Briggs MR, Brown MS, et al. (1993) SREBP-2, a second basic-helix-loop-helix-leucine zipper protein that stimulates transcription by binding to a sterol regulatory element. Proc Natl Acad Sci U S A 90: 11603–11607.
[40]  Briggs MR, Yokoyama C, Wang X, Brown MS, Goldstein JL (1993) Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter. I. Identification of the protein and delineation of its target nucleotide sequence. J Biol Chem 268: 14490–14496.
[41]  Sato R (2010) Sterol metabolism and SREBP activation. Arch Biochem Biophys 501: 177–181.
[42]  Nakamuta M, Yada R, Fujino T, Yada M, Higuchi N, et al. (2009) Changes in the expression of cholesterol metabolism-associated genes in HCV-infected liver: a novel target for therapy? Int J Mol Med 24: 825–828.
[43]  Chang ML, Yeh CT, Chen JC, Huang CC, Lin SM, et al. (2008) Altered expression patterns of lipid metabolism genes in an animal model of HCV core-related, nonobese, modest hepatic steatosis. BMC Genomics 9: 109.
[44]  Molloy SS, Anderson ED, Jean F, Thomas G (1999) Bi-cycling the furin pathway: from TGN localization to pathogen activation and embryogenesis. Trends Cell Biol 9: 28–35.
[45]  Thomas G (2002) Furin at the cutting edge: from protein traffic to embryogenesis and disease. Nat Rev Mol Cell Biol 3: 753–766.
[46]  Seidah NG (2011) The proprotein convertases, 20 years later. Methods Mol Biol 768: 23–57.
[47]  Bodvard K, Mohlin J, Knecht W (2007) Recombinant expression, purification, and kinetic and inhibitor characterisation of human site-1-protease. Protein Expr Purif 51: 308–319.
[48]  Pasquato A, Burri DJ, Traba EG, Hanna-El-Daher L, Seidah NG, et al. (2011) Arenavirus envelope glycoproteins mimic autoprocessing sites of the cellular proprotein convertase subtilisin kexin isozyme-1/site-1 protease. Virology 417: 18–26.
[49]  Schechter I, Berger A (1967) On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun 27: 157–162.
[50]  Lenz O, ter Meulen J, Klenk HD, Seidah NG, Garten W (2001) The Lassa virus glycoprotein precursor GP-C is proteolytically processed by subtilase SKI-1/S1P. Proc Natl Acad Sci U S A 98: 12701–12705.
[51]  Beyer WR, Popplau D, Garten W, von Laer D, Lenz O (2003) Endoproteolytic processing of the lymphocytic choriomeningitis virus glycoprotein by the subtilase SKI-1/S1P. J Virol 77: 2866–2872.
[52]  Bergeron E, Vincent MJ, Nichol ST (2007) Crimean-Congo hemorrhagic fever virus glycoprotein processing by the endoprotease SKI-1/S1P is critical for virus infectivity. J Virol 81: 13271–13276.
[53]  Eberle D, Hegarty B, Bossard P, Ferre P, Foufelle F (2004) SREBP transcription factors: master regulators of lipid homeostasis. Biochimie 86: 839–848.
[54]  Jean F, Stella K, Thomas L, Liu G, Xiang Y, et al. (1998) alpha1-Antitrypsin Portland, a bioengineered serpin highly selective for furin: application as an antipathogenic agent. Proc Natl Acad Sci U S A 95: 7293–7298.
[55]  Jean F, Thomas L, Molloy SS, Liu G, Jarvis MA, et al. (2000) A protein-based therapeutic for human cytomegalovirus infection. Proc Natl Acad Sci U S A 97: 2864–2869.
[56]  Whisstock JC, Silverman GA, Bird PI, Bottomley SP, Kaiserman D, et al. (2010) Serpins flex their muscle: II. Structural insights into target peptidase recognition, polymerization, and transport functions. J Biol Chem 285: 24307–24312.
[57]  Huntington JA, Read RJ, Carrell RW (2000) Structure of a serpin-protease complex shows inhibition by deformation. Nature 407: 923–926.
[58]  Richer MJ, Keays CA, Waterhouse J, Minhas J, Hashimoto C, et al. (2004) The Spn4 gene of Drosophila encodes a potent furin-directed secretory pathway serpin. Proc Natl Acad Sci U S A 101: 10560–10565.
[59]  Pelham HR (1990) The retention signal for soluble proteins of the endoplasmic reticulum. Trends Biochem Sci 15: 483–486.
[60]  Arnberg N (2009) Adenovirus receptors: implications for tropism, treatment and targeting. Rev Med Virol 19: 165–178.
[61]  Zhong J, Gastaminza P, Cheng G, Kapadia S, Kato T, et al. (2005) Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci U S A 102: 9294–9299.
[62]  Richer MJ, Juliano L, Hashimoto C, Jean F (2004) Serpin mechanism of hepatitis C virus nonstructural 3 (NS3) protease inhibition: induced fit as a mechanism for narrow specificity. J Biol Chem 279: 10222–10227.
[63]  Silverman GA, Whisstock JC, Bottomley SP, Huntington JA, Kaiserman D, et al. (2010) Serpins flex their muscle: I. Putting the clamps on proteolysis in diverse biological systems. J Biol Chem 285: 24299–24305.
[64]  Hawkins JL, Robbins MD, Warren LC, Xia D, Petras SF, et al. (2008) Pharmacologic inhibition of site 1 protease activity inhibits sterol regulatory element-binding protein processing and reduces lipogenic enzyme gene expression and lipid synthesis in cultured cells and experimental animals. J Pharmacol Exp Ther 326: 801–808.
[65]  Hay BA, Abrams B, Zumbrunn AY, Valentine JJ, Warren LC, et al. (2007) Aminopyrrolidineamide inhibitors of site-1 protease. Bioorg Med Chem Lett 17: 4411–4414.
[66]  DeBose-Boyd RA, Brown MS, Li WP, Nohturfft A, Goldstein JL, et al. (1999) Transport-dependent proteolysis of SREBP: relocation of site-1 protease from Golgi to ER obviates the need for SREBP transport to Golgi. Cell 99: 703–712.
[67]  Wu WW, Pante N (2009) The directionality of the nuclear transport of the influenza A genome is driven by selective exposure of nuclear localization sequences on nucleoprotein. Virol J 6: 68.
[68]  Labonte P, Begley S, Guevin C, Asselin MC, Nassoury N, et al. (2009) PCSK9 impedes hepatitis C virus infection in vitro and modulates liver CD81 expression. Hepatology 50: 17–24.
[69]  Owen DM, Huang H, Ye J, Gale M Jr (2009) Apolipoprotein E on hepatitis C virion facilitates infection through interaction with low-density lipoprotein receptor. Virology. 394: 99–108.
[70]  Goldstein JL, Brown MS (2009) The LDL receptor. Arterioscler Thromb Vasc Biol 29: 431–438.
[71]  Jeong HJ, Lee HS, Kim KS, Kim YK, Yoon D, et al. (2008) Sterol-dependent regulation of proprotein convertase subtilisin/kexin type 9 expression by sterol-regulatory element binding protein-2. J Lipid Res 49: 399–409.
[72]  Sato R, Inoue J, Kawabe Y, Kodama T, Takano T, et al. (1996) Sterol-dependent transcriptional regulation of sterol regulatory element-binding protein-2. J Biol Chem 271: 26461–26464.
[73]  Sakai J, Duncan EA, Rawson RB, Hua X, Brown MS, et al. (1996) Sterol-regulated release of SREBP-2 from cell membranes requires two sequential cleavages, one within a transmembrane segment. Cell 85: 1037–1046.
[74]  Brasaemle DL, Barber T, Wolins NE, Serrero G, Blanchette-Mackie EJ, et al. (1997) Adipose differentiation-related protein is an ubiquitously expressed lipid storage droplet-associated protein. J Lipid Res 38: 2249–2263.
[75]  Blight KJ, McKeating JA, Rice CM (2002) Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J Virol 76: 13001–13014.
[76]  Prussia A, Thepchatri P, Snyder JP, Plemper RK (2011) Systematic Approaches towards the Development of Host-Directed Antiviral Therapeutics. Int J Mol Sci 12: 4027–4052.
[77]  Jones DM, McLauchlan J (2010) Hepatitis C virus: assembly and release of virus particles. J Biol Chem 285: 22733–22739.
[78]  Brown MS, Goldstein JL (1997) The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89: 331–340.
[79]  Agnello V, Abel G, Elfahal M, Knight GB, Zhang QX (1999) Hepatitis C virus and other flaviviridae viruses enter cells via low density lipoprotein receptor. Proc Natl Acad Sci U S A 96: 12766–12771.
[80]  Mazumdar B, Banerjee A, Meyer K, Ray R (2011) Hepatitis C virus E1 envelope glycoprotein interacts with apolipoproteins in facilitating entry into hepatocytes. Hepatology 54: 1149–1156.
[81]  Xu G, Sztalryd C, Lu X, Tansey JT, Gan J, et al. (2005) Post-translational regulation of adipose differentiation-related protein by the ubiquitin/proteasome pathway. J Biol Chem 280: 42841–42847.
[82]  Straub BK, Stoeffel P, Heid H, Zimbelmann R, Schirmacher P (2008) Differential pattern of lipid droplet-associated proteins and de novo perilipin expression in hepatocyte steatogenesis. Hepatology 47: 1936–1946.
[83]  Motomura W, Inoue M, Ohtake T, Takahashi N, Nagamine M, et al. (2006) Up-regulation of ADRP in fatty liver in human and liver steatosis in mice fed with high fat diet. Biochem Biophys Res Commun 340: 1111–1118.
[84]  Urata S, Yun N, Pasquato A, Paessler S, Kunz S, et al. (2011) Antiviral activity of a small-molecule inhibitor of arenavirus glycoprotein processing by the cellular site 1 protease. J Virol 85: 795–803.
[85]  Georgel P, Schuster C, Zeisel MB, Stoll-Keller F, Berg T, et al. (2010) Virus-host interactions in hepatitis C virus infection: implications for molecular pathogenesis and antiviral strategies. Trends Mol Med 16: 277–286.
[86]  Gelman MA, Glenn JS (2010) Mixing the right hepatitis C inhibitor cocktail. Trends Mol Med. E-pub ahead of print.
[87]  Whisstock JC, Bottomley SP (2006) Molecular gymnastics: serpin structure, folding and misfolding. Curr Opin Struct Biol 16: 761–768.
[88]  Tan SL, Pause A, Shi Y, Sonenberg N (2002) Hepatitis C therapeutics: current status and emerging strategies. Nat Rev Drug Discov 1: 867–881.
[89]  Ye J (2011) Cell biology. Protease sets site-1 on lysosomes. Science 333: 50–51.
[90]  Hinson ER, Cresswell P (2009) The antiviral protein, viperin, localizes to lipid droplets via its N-terminal amphipathic alpha-helix. Proc Natl Acad Sci U S A 106: 20452–20457.
[91]  Kato T, Furusaka A, Miyamoto M, Date T, Yasui K, et al. (2001) Sequence analysis of hepatitis C virus isolated from a fulminant hepatitis patient. J Med Virol 64: 334–339.
[92]  Han J, Zhang H, Min G, Kemler D, Hashimoto C (2000) A novel Drosophila serpin that inhibits serine proteases. FEBS Lett 468: 194–198.
[93]  Maxwell KN, Fisher EA, Breslow JL (2005) Overexpression of PCSK9 accelerates the degradation of the LDLR in a post-endoplasmic reticulum compartment. Proc Natl Acad Sci U S A 102: 2069–2074.
[94]  Takehara S, Onda M, Zhang J, Nishiyama M, Yang X, et al. (2009) The 2.1-A crystal structure of native neuroserpin reveals unique structural elements that contribute to conformational instability. J Mol Biol 388: 11–20.
[95]  Martin MM, Condotta SA, Fenn J, Olmstead AD, Jean F (2011) In-cell selectivity profiling of membrane-anchored and replicase-associated hepatitis C virus NS3-4A protease reveals a common, stringent substrate recognition profile. Biol Chem 392: 927–935.
[96]  Condotta SA, Martin MM, Boutin M, Jean F (2010) Detection and in-cell selectivity profiling of the full-length West Nile virus NS2B/NS3 serine protease using membrane-anchored fluorescent substrates. Biol Chem 391: 549–559.
[97]  Martin MM, Jean F (2006) Single-cell resolution imaging of membrane-anchored hepatitis C virus NS3/4A protease activity. Biol Chem 387: 1075–1080.
[98]  Hamill P, Hudson D, Kao RY, Chow P, Raj M, et al. (2006) Development of a red-shifted fluorescence-based assay for SARS-coronavirus 3CL protease: identification of a novel class of anti-SARS agents from the tropical marine sponge Axinella corrugata. Biol Chem 387: 1063–1074.
[99]  Elagoz A, Benjannet S, Mammarbassi A, Wickham L, Seidah NG (2002) Biosynthesis and cellular trafficking of the convertase SKI-1/S1P: ectodomain shedding requires SKI-1 activity. J Biol Chem 277: 11265–11275.
[100]  Seidah NG, Mowla SJ, Hamelin J, Mamarbachi AM, Benjannet S, et al. (1999) Mammalian subtilisin/kexin isozyme SKI-1: A widely expressed proprotein convertase with a unique cleavage specificity and cellular localization. Proc Natl Acad Sci U S A 96: 1321–1326.
[101]  Wang X, Briggs MR, Hua X, Yokoyama C, Goldstein JL, et al. (1993) Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter. II. Purification and characterization. J Biol Chem 268: 14497–14504.


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