HCV core protein uses multiple mechanisms to induce oxidative stress in human hepatoma huh7 cells

Viruses

Volumn 7, Issue 6, 2015, Pages 2745-2770

  Ivanov, Alexander V ^a   Smirnova, Olga A ^a   Petrushanko, Irina Y ^a   Ivanova, Olga N ^a   Karpenko, Inna L ^a   Alekseeva, Ekaterina ^b   Sominskaya, Irina ^b   Makarov, Alexander A ^a   Bartosch, Birke ^c,d   Kochetkov, Sergey N ^a   Isaguliants, Maria G ^e,f,g  

^a ENGELHARDT INSTITUTE OF MOLECULAR BIOLOGY   (Russian Federation)

^b LATVIAN BIOMEDICAL RESEARCH AND STUDY CENTRE   (Latvia)

^c CENTRE DE RECHERCHE EN CANCÉROLOGIE DE LYON   (France)

^d DevWeCan Laboratories of Excellence Network (Labex)   (France)

^e D I IVANOVSKY INSTITUTE OF VIROLOGY   (Russian Federation)

^f RIGA STRADINS UNIVERSITY   (Latvia)

^g KAROLINSKA INSTITUTE   (Sweden)

Author keywords

Cytochrome P450; ER oxidoreductin; Hepatitis C virus; NADPH oxidase; Oxidative stress; Reactive oxygen species; Transforming growth factor

Indexed keywords

CORE PROTEIN; CYCLOOXYGENASE 2; CYTOCHROME P450 2E1; DICHLORODIHYDROFLUORESCEIN DIACETATE; FLUORESCEIN ISOTHIOCYANATE; HELIX LOOP HELIX PROTEIN; HYDROGEN PEROXIDE; MESSENGER RNA; OXIDOREDUCTASE; OXIDOREDUCTIN 1; REACTIVE OXYGEN METABOLITE; REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE OXIDASE 1; REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE OXIDASE 4; SUPEROXIDE; TRANSFORMING GROWTH FACTOR BETA1; UNCLASSIFIED DRUG; CALCIUM; CYBB PROTEIN, HUMAN; ERO1L PROTEIN, HUMAN; MEMBRANE PROTEIN; NOX1 PROTEIN, HUMAN; NUCLEOCAPSID PROTEIN, HEPATITIS C VIRUS; PTGS2 PROTEIN, HUMAN; REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE OXIDASE;

ARTICLE; CALCIUM TRANSPORT; CONFOCAL MICROSCOPY; CONTROLLED STUDY; ENDOPLASMIC RETICULUM; FLOW CYTOMETRY; FLUORESCENCE ANALYSIS; GENE EXPRESSION; GENETIC TRANSFECTION; HEPATITIS C VIRUS; HUMAN; HUMAN CELL; LIVER CELL CARCINOMA; NUCLEAR EXPORT SIGNAL; NUCLEAR LOCALIZATION SIGNAL; OXIDATIVE STRESS; PROTEIN EXPRESSION; REAL TIME POLYMERASE CHAIN REACTION; RNA INTERFERENCE; TRANSCRIPTION REGULATION; WESTERN BLOTTING; DNA MUTATIONAL ANALYSIS; HEPACIVIRUS; HOST PATHOGEN INTERACTION; METABOLISM; MITOCHONDRION; PHYSIOLOGY; TUMOR CELL LINE;

HEPATITIS C VIRUS;

CALCIUM; CELL LINE, TUMOR; CYCLOOXYGENASE 2; CYTOCHROME P-450 CYP2E1; DNA MUTATIONAL ANALYSIS; HEPACIVIRUS; HOST-PATHOGEN INTERACTIONS; HUMANS; HYDROGEN PEROXIDE; MEMBRANE GLYCOPROTEINS; MITOCHONDRIA; NADPH OXIDASE; OXIDATIVE STRESS; OXIDOREDUCTASES; REACTIVE OXYGEN SPECIES; SUPEROXIDES; TRANSFORMING GROWTH FACTOR BETA1; VIRAL CORE PROTEINS;

EID: 84930937748     PISSN: None     EISSN: 19994915     Source Type: Journal    
DOI: 10.3390/v7062745     Document Type: Article

References (70)

1. Levrero, M. Viral hepatitis and liver cancer: The case of hepatitis C. Oncogene 2006, 25, 3834–3847.
2. Jin, D.Y. Molecular pathogenesis of hepatitis C virus-associated hepatocellular carcinoma. Front. Biosci. 2007, 12, 222–233.
3. Clement, S.; Pascarella, S.; Negro, F. Hepatitis C virus infection: Molecular pathways to steatosis, insulin resistance and oxidative stress. Viruses 2009, 1, 126–143.
4. Ivanov, A.V.; Bartosch, B.; Smirnova, O.A.; Isaguliants, M.G.; Kochetkov, S.N. HCV and oxidative stress in the liver. Viruses 2013, 5, 439–469.
5. Smirnova, I.S.; Aksenov, N.D.; Kashuba, E.V.; Payakurel, P.; Grabovetsky, V.V.; Zaberezhny, A.D.; Vonsky, M.S.; Buchinska, L.; Biberfeld, P. Hinkula, J.; et al. ; Hepatitis C virus core protein transforms murine fibroblasts by promoting genomic instability. Cell. Oncol. 2006, 28, 177–190.
6. Moriya, K.; Nakagawa, K.; Santa, T.; Shintani, Y.; Fujie, H.; Miyoshi, H.; Tsutsumi, T.; Miyazawa, T.; Ishibashi, K. Horie, T.; et al. ; Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated hepatocarcinogenesis. Cancer Res. 2001, 61, 4365–4370.
7. Benzoubir, N.; Lejamtel, C.; Battaglia, S.; Testoni, B.; Benassi, B.; Gondeau, C.; Perrin-Cocon, L.; Desterke, C.; Thiers, V. Samuel, D.; et al. ; HCV core-mediated activation of latent tgf-beta via thrombospondin drives the crosstalk between hepatocytes and stromal environment. J. Hepatol. 2013, 59, 1160–1168.
8. Waris, G.; Felmlee, D.J.; Negro, F. Siddiqui, A. ; Hepatitis C virus induces proteolytic cleavage of sterol regulatory element binding proteins and stimulates their phosphorylation via oxidative stress. J. Virol. 2007, 81, 8122–8130.
9. Miura, K.; Taura, K.; Kodama, Y.; Schnabl, B.; Brenner, D.A. Hepatitis c virus-induced oxidative stress suppresses hepcidin expression through increased histone deacetylase activity. Hepatology 2008, 48, 1420–1429.
10. Choi, J. Oxidative stress, endogenous antioxidants, alcohol, and hepatitis C: Pathogenic interactions and therapeutic considerations. Free Radic. Biol. Med. 2012, 52, 1135–1150.
11. Yamaguchi, A.; Tazuma, S.; Nishioka, T.; Ohishi, W.; Hyogo, H.; Nomura, S.; Chayama, K. Hepatitis C virus core protein modulates fatty acid metabolism and thereby causes lipid accumulation in the liver. Dig. Dis. Sci. 2005, 50, 1361–1371.
12. Sharma, P.; Jha, A.B.; Dubey, R.S.; Pessarakli, M. Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. J. Bot. 2012, 2012, e217037. [CrossRef]
13. Brault, C.; Levy, P.L.; Bartosch, B. Hepatitis C virus-induced mitochondrial dysfunctions. Viruses 2013, 5, 954–980.
14. Gong, G.; Waris, G.; Tanveer, R.; Siddiqui, A. Human hepatitis C virus ns5a protein alters intracellular calcium levels, induces oxidative stress, and activates stat-3 and nf-kappa b.. Proc. Natl. Acad. Sci USA 2001, 98, 9599–9604.
15. Ivanov, A.V.; Smirnova, O.A.; Ivanova, O.N.; Masalova, O.V.; Kochetkov, S.N.; Isaguliants, M.G. Hepatitis C virus proteins activate nrf2/are pathway by distinct ros-dependent and independent mechanisms in huh7 cells. PLoS ONE 2011, 6, e24957.
16. Korenaga, M.; Wang, T.; Li, Y.; Showalter, L.A.; Chan, T.; Sun, J.; Weinman, S.A. Hepatitis C virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species(ros) production. J. Biol. Chem. 2005, 280, 37481–37488.
17. Seronello, S.; Montanez, J.; Presleigh, K.; Barlow, M.; Park, S.B.; Choi, J. Ethanol and reactive species increase basal sequence heterogeneity of hepatitis C virus and produce variants with reduced susceptibility to antivirals. PLoS ONE 2011, 6, e27436.
18. Casero, R.A.; Pegg, A.E. Polyamine catabolism and disease. Biochem. J. 2009, 421, 323–338
19. Laurindo, F.R.; Pescatore, L.A.; Fernandes Dde, C. Protein disulfide isomerase in redox cell signaling and homeostasis. Free Radic. Biol. Med. 2012, 52, 1954–1969.
20. Wang, T.; Campbell, R.V.; Yi, M.K.; Lemon, S.M.; Weinman, S.A. Role of hepatitis C virus core protein in viral-induced mitochondrial dysfunction. J. Viral Hepat. 2010, 17, 784–793.
21. De Mochel, N.S.; Seronello, S.; Wang, S.H.; Ito, C.; Zheng, J.X.; Liang, T.J.; Lambeth, J.D.; Choi, J. Hepatocyte nad(p)h oxidases as an endogenous source of reactive oxygen species during hepatitis C virus infection. Hepatology 2010, 52, 47–59.
22. Boudreau, H.E.; Emerson, S.U.; Korzeniowska, A.; Jendrysik, M.A.; Leto, T.L. Hepatitis C virus (HCV) proteins induce nadph oxidase 4 expression in a transforming growth factor beta-dependent manner: A new contributor to HCV-induced oxidative stress. J. Virol. 2009, 83, 12934–12946.
23. Benali-Furet, N.L.; Chami, M.; Houel, L.; de Giorgi, F.; Vernejoul, F.; Lagorce, D.; Buscail, L.; Bartenschlager, R.; Ichas, F.; Rizzuto, R.; et al. Hepatitis c virus core triggers apoptosis in liver cells by inducing er stress and er calcium depletion. Oncogene 2005, 24, 4921–4933.
24. McLauchlan, J. Properties of the hepatitis C virus core protein: A structural protein that modulates cellular processes. J. Viral Hepat. 2000, 7, 2–14.
25. Boulant, S.; Montserret, R.; Hope, R.G.; Ratinier, M.; Targett-Adams, P.; Lavergne, J.P.; Penin, F.; McLauchlan, J. Structural determinants that target the hepatitis C virus core protein to lipid droplets. J. Biol. Chem. 2006, 281, 22236–22247.
26. Suzuki, R.; Sakamoto, S.; Tsutsumi, T.; Rikimaru, A.; Tanaka, K.; Shimoike, T.; Moriishi, K.; Iwasaki, T.; Mizumoto, K. Matsuura, Y.; et al. ; Molecular determinants for subcellular localization of hepatitis C virus core protein. J. Virol. 2005, 79, 1271–1281.
27. 2+ uniporter activity. FASEB J. 2007, 21, 2474–2485.
28. Isaguliants, M.G.; Iakimtchouk, K.; Petrakova, N.V.; Yermalovich, M.A.; Zuber, A.K.; Kashuba, V.I.; Belikov, S.V.; Andersson, S.; Kochetkov, S.N. Klinman, D.M.; et al. ; Gene immunization may induce secondary antibodies reacting with DNA. Vaccine 2004, 22, 1576–1585.
29. Alekseeva, E.; Sominskaya, I.; Skrastina, D.; Egorova, I.; Starodubova, E.; Kushners, E.; Mihailova, M.; Petrakova, N.; Bruvere, R. Kozlovskaya, T.; et al. ; Enhancement of the expression of HCV core gene does not enhance core-specific immune response in DNA immunization: Advantages of the heterologous DNA prime, protein boost immunization regimen. Genet. Vaccines Ther. 2009, 7, e7.
30. Zielonka, J.; Vasquez-Vivar, J.; Kalyanaraman, B. Detection of 2-hydroxyethidium in cellular systems: A unique marker product of superoxide and hydroethidine. Nat. Protoc. 2008, 3, 8–21.
31. Mitkevich, V.A.; Petrushanko, I.Y.; Kretova, O.V.; Zelenikhin, P.V.; Prassolov, V.S.; Tchurikov, N.A.; Ilinskaya, O.N.; Makarov, A.A. Oncogenic c-kit transcript is a target for binase. Cell Cycle 2010, 9, 2674–2678.
32. Anelli, T.; Bergamelli, L.; Margittai, E.; Rimessi, A.; Fagioli, C.; Malgaroli, A.; Pinton, P.; Ripamonti, M.; Rizzuto, R.; Sitia, R. Ero1alpha regulates Ca(2+) fluxes at the endoplasmic reticulum-mitochondria interface (mam). Antioxid. Redox Signal. 2012, 16, 1077–1087.
33. Sancho, P.; Martin-Sanz, P.; Fabregat, I. Reciprocal regulation of nadph oxidases and the cyclooxygenase-2 pathway. Free Radic. Biol. Med. 2011, 51, 1789–1798.
34. Cerutti, A.; Maillard, P.; Minisini, R.; Vidalain, P.O.; Roohvand, F.; Pecheur, E.I.; Pirisi, M.; Budkowska, A. Identification of a functional, crm-1-dependent nuclear export signal in hepatitis C virus core protein. PLoS ONE 2011, 6, e25854.
35. Chen, C.M.; You, L.R.; Hwang, L.H.; Lee, Y.H. Direct interaction of hepatitis C virus core protein with the cellular lymphotoxin-beta receptor modulates the signal pathway of the lymphotoxin-beta receptor. J. Virol. 1997, 71, 9417–9426.
36. Lin, W.; Kim, S.S.; Yeung, E.; Kamegaya, Y.; Blackard, J.T.; Kim, K.A.; Holtzman, M.J.; Chung, R.T. Hepatitis C virus core protein blocks interferon signaling by interaction with the stat1 sh2 domain. J. Virol. 2006, 80, 9226–9235.
37. Sun, C.; Pager, C.T.; Luo, G.; Sarnow, P.; Cate, J.H. Hepatitis c virus core-derived peptides inhibit genotype 1b viral genome replication via interaction with ddx3x. PLoS ONE 2010, 5, e12826.
38. Kalyanaraman, B.; Darley-Usmar, V.; Davies, K.J.; Dennery, P.A.; Forman, H.J.; Grisham, M.B.; Mann, G.E.; Moore, K.; Roberts, L.J., 2nd; Ischiropoulos, H. Measuring reactive oxygen and nitrogen species with fluorescent probes: Challenges and limitations. Free Radic. Biol. Med. 2012, 52, 1–6.
39. Markvicheva, K.N.; Bogdanova, E.A.; Staroverov, D.B.; Lukyanov, S.; Belousov, V.V. Imaging of intracellular hydrogen peroxide production with hyper upon stimulation of hela cells with epidermal growth factor. Methods Mol. Biol. 2009, 476, 76–83.
40. Knockaert, L.; Fromenty, B.; Robin, M.A. Mechanisms of mitochondrial targeting of cytochrome p450 2e1: Physiopathological role in liver injury and obesity. FEBS J. 2011, 278, 4252–4260.
41. Lu, Y.; Cederbaum, A.I. Cyp2e1 and oxidative liver injury by alcohol.. Free Radic. Biol Med. 2008, 44, 723–738.
42. Wu, D.; Cederbaum, A.I. Ethanol cytotoxicity to a transfected hepg2 cell line expressing human cytochrome p4502e1. J. Biol. Chem. 1996, 271, 23914–23919.
43. Li, G.; Mongillo, M.; Chin, K.T.; Harding, H.; Ron, D.; Marks, A.R.; Tabas, I. Role of ero1-alpha-mediated stimulation of inositol 1,4,5-triphosphate receptor activity in endoplasmic reticulum stress-induced apoptosis. J. Cell Biol. 2009, 186, 783–792.
44. Takac, I.; Schroder, K.; Zhang, L.; Lardy, B.; Anilkumar, N.; Lambeth, J.D.; Shah, A.M.; Morel, F.; Brandes, R.P. The e-loop is involved in hydrogen peroxide formation by the nadph oxidase nox4. J. Biol. Chem. 2011, 286, 13304–13313.
45. Spencer, N.Y.; Yan, Z.; Boudreau, R.L.; Zhang, Y.; Luo, M.; Li, Q.; Tian, X.; Shah, A.M.; Davisson, R.L.; Davidson, B.; et al. Control of hepatic nuclear superoxide production by glucose 6-phosphate dehydrogenase and nadph oxidase-4. J. Biol. Chem. 2011, 286, 8977–8987.
46. Nisimoto, Y.; Diebold, B.A.; Constentino-Gomes, D.; Lambeth, J.D. Nox4: A hydrogen peroxide-generating oxygen sensor. Biochemistry 2014, 53, 5111–5120.
47. Helmcke, I.; Heumuller, S.; Tikkanen, R.; Schroder, K.; Brandes, R.P. Identification of structural elements in nox1 and nox4 controlling localization and activity. Antioxid. Redox Signal. 2009, 11, 1279–1287.
48. Anilkumar, N.; San Jose, G.; Sawyer, I.; Santos, C.X.; Sand, C.; Brewer, A.C.; Warren, D.; Shah, A.M. A 28-kda splice variant of nadph oxidase-4 is nuclear-localized and involved in redox signaling in vascular cells. Arterioscler. Thromb. Vasc. Biol. 2013, 33, e104–e112.
49. Nakai, K.; Tanaka, H.; Hanada, K.; Ogata, H.; Suzuki, F.; Kumada, H.; Miyajima, A.; Ishida, S.; Sunouchi, M. Habano, W.; et al. ; Decreased expression of cytochromes P450 1A2, 2E1, and 3A4 and drug transporters Na+-taurocholate-cotransporting polypeptide, organic cation transporter 1, and organic anion-transporting peptide-c correlates with the progression of liver fibrosis in chronic hepatitis C patients.. Drug Metab. Dispos. 2008, 36, 1786–1793.
50. Burdette, D.; Olivarez, M.; Waris, G. Activation of transcription factor Nrf2 by hepatitis C virus induces the cell-survival pathway. J. Gen. Virol. 2010, 91, 681–690.
51. Hayes, J.D.; Dinkova-Kostova, A.T. The Nrf2 regulatory network provides an interface between redox and intermediary metabolism. Trends Biochem. Sci. 2014, 39, 199–218.
52. Miyamoto, N.; Izumi, H.; Miyamoto, R.; Kondo, H.; Tawara, A.; Sasaguri, Y. Kohno, K. ; Quercetin induces the expression of peroxiredoxins 3 and 5 via the Nrf2/NRF1 transcription pathway. Investig. Ophthalmol. Vis. Sci. 2011, 52, 1055–1063.
53. Diamond, D.L.; Syder, A.J.; Jacobs, J.M.; Sorensen, C.M.; Walters, K.A.; Proll, S.C.; McDermott, J.E.; Gritsenko, M.A.; Zhang, Q. Zhao, R.; et al. ; Temporal proteome and lipidome profiles reveal hepatitis C virus-associated reprogramming of hepatocellular metabolism and bioenergetics. PLoS Pathog. 2010, 6, e1000719.
54. Brault, C.; Levy, P.; Duponchel, S.; Michelet, M.; Salle, A.; Pecheur, E.I.; Plissonnier, M.L.; Parent, R.; Vericel, E.; Ivanov, A.V. et al. Glutathione peroxidase 4 is reversibly induced by HCV to control lipid peroxidation and to increase virion infectivity. Gut 2014.
55. Choi, J.; Corder, N.L.; Koduru, B.; Wang, Y. Oxidative stress and hepatic nox proteins in chronic hepatitis C and hepatocellular carcinoma. Free Radic. Biol. Med. 2014, 72, 267–284.
56. Sancho, P.; Mainez, J.; Crosas-Molist, E.; Roncero, C.; Fernandez-Rodriguez, C.M.; Pinedo, F.; Huber, H.; Eferl, R.; Mikulits, W.; Fabregat, I. Nadph oxidase nox4 mediates stellate cell activation and hepatocyte cell death during liver fibrosis development. PLoS ONE 2012, 7, e45285.
57. Jiang, J.X.; Chen, X.; Serizawa, N.; Szyndralewiez, C.; Page, P.; Schroder, K.; Brandes, R.P.; Devaraj, S.; Torok, N.J. Liver fibrosis and hepatocyte apoptosis are attenuated by GKT137831, a novel NOX4/NOX1 inhibitor in vivo. Free Radic. Biol. Med. 2012, 53, 289–296.
58. Otani, K.; Korenaga, M.; Beard, M.R.; Li, K.; Qian, T.; Showalter, L.A.; Singh, A.K.; Wang, T.; Weinman, S.A. Hepatitis c virus core protein, cytochrome P450 2e1, and alcohol produce combined mitochondrial injury and cytotoxicity in hepatoma cells. Gastroenterology 2005, 128, 96–107.
59. Nieto, N.; Friedman, S.L.; Cederbaum, A.I. Stimulation and proliferation of primary rat hepatic stellate cells by cytochrome P450 2e1-derived reactive oxygen species. Hepatology 2002, 35, 62–73.
60. Nieto, N.; Cederbaum, A.I. Increased Sp1-dependent transactivation of the lamgamma 1 promoter in hepatic stellate cells co-cultured with hepg2 cells overexpressing cytochrome p450 2e1. J. Biol. Chem. 2003, 278, 15360–15372.
61. Haufroid, V.; Ligocka, D.; Buysschaert, M.; Horsmans, Y.; Lison, D. Cytochrome p4502e1 (cyp2e1) expression in peripheral blood lymphocytes: Evaluation in hepatitis C and diabetes. Eur. J. Clin. Pharmacol. 2003, 59, 29–33.
62. Haufroid, V.; Ligocka, D.; Wallemacq, P.; Lison, D.; Horsmans, Y. Comparison of cytochrome p4502e1 (cyp2e1) activity and hepatic and lymphocyte mrna expression in patients with chronic hepatitis C. Toxicol. Lett. 2005, 155, 171–177.
63. Gochee, P.A.; Jonsson, J.R.; Clouston, A.D.; Pandeya, N.; Purdie, D.M.; Powell, E.E. Steatosis in chronic hepatitis C: Association with increased messenger rna expression of collagen I, tumor necrosis factor-alpha and cytochrome p450 2e1. J. Gastroenterol. Hepatol. 2003, 18, 386–392.
64. Liu, T.; Wang, P.; Cong, M.; Xu, Y.; Jia, J.; You, H. The CYP2E1 inhibitor DDC up-regulates MMP-1 expression in hepatic stellate cells via an ERK1/2- and Akt-dependent mechanism. Biosci. Rep. 2013, 33, e00041.
65. Garofalo, R.P.; Kolli, D.; Casola, A. Respiratory syncytial virus infection: Mechanisms of redox control and novel therapeutic opportunities. Antioxid. Redox Signal. 2013, 18, 186–217.
66. Grandvaux, N.; Soucy-Faulkner, A.; Fink, K. Innate host defense: Nox and Duox on phox’s tail. Biochimie 2007, 89, 1113–1122.
67. Kutomi, G.; Tamura, Y.; Tanaka, T.; Kajiwara, T.; Kukita, K.; Ohmura, T.; Shima, H.; Takamaru, T.; Satomi, F. Suzuki, Y.; et al. ; Human endoplasmic reticulum oxidoreductin 1-alpha is a novel predictor for poor prognosis of breast cancer. Cancer Sci. 2013, 104, 1091–1096.
68. Reshi, M.L.; Su, Y.C.; Hong, J.R. RNA viruses: Ros-mediated cell death. Int. J. Cell Biol. 2014, 2014, e467452.
69. Olinski, R.; Styczynski, J.; Olinska, E.; Gackowski, D. Viral infection-oxidative stress/DNA damage-aberrant DNA methylation: Separate or interrelated events responsible for genetic instability and childhood all development? Biochim. Biophys. Acta 2014, 1846, 226–231.
70. Tubbs, E.; Theurey, P.; Vial, G.; Bendridi, N.; Bravard, A.; Chauvin, M.A.; Ji-Cao, J.; Zoulim, F.; Bartosch, B. Ovize, M.; et al.; Mitochondria-associated endoplasmic reticulum membrane (mam) integrity is required for insulin signaling and is implicated in hepatic insulin resistance. Diabetes 2014, 63, 3279–3294.