Japanese Encephalitis Virus Nonstructural Protein NS5 Interacts with Mitochondrial Trifunctional Protein and Impairs Fatty Acid β-Oxidation

PLoS Pathogens

Volumn 11, Issue 3, 2015, Pages 1-26

  Kao, Yu Ting ^a,b   Chang, Bi Lan ^b   Liang, Jian Jong ^b   Tsai, Hang Jen ^a   Lee, Yi Ling ^b   Lin, Ren Jye ^c,d   Lin, Yi Ling ^a,b,e  

^a NATIONAL DEFENSE MEDICAL CENTER   (Taiwan)

^b INSTITUTE OF BIOMEDICAL SCIENCES   (Taiwan)

^c TAIPEI MEDICAL UNIVERSITY   (Taiwan)

^d TAIPEI MEDICAL UNIVERSITY HOSPITAL   (Taiwan)

^e GENOMICS RESEARCH CENTER   (Taiwan)

Author keywords

[No Author keywords available]

Indexed keywords

3 HYDROXYACYL COENZYME A DEHYDROGENASE; INTERLEUKIN 6; LONG CHAIN FATTY ACID; MITOCHONDRIAL TRIFUNCTIONAL PROTEIN; NONSTRUCTURAL PROTEIN 5; PROTEINASE K; REACTIVE OXYGEN METABOLITE; TUMOR NECROSIS FACTOR ALPHA; FATTY ACID; MITOCHONDRIAL TRIFUNCTIONAL PROTEIN ALPHA SUBUNIT; MITOCHONDRIAL TRIFUNCTIONAL PROTEIN BETA SUBUNIT; NS5 PROTEIN, FLAVIVIRUS; VIRAL NONSTRUCTURAL PROTEIN;

ANIMAL EXPERIMENT; ANIMAL MODEL; ARTICLE; CONFOCAL MICROSCOPY; CONTROLLED STUDY; CYTOKINE PRODUCTION; CYTOTOXICITY; ENZYME LINKED IMMUNOSORBENT ASSAY; FATTY ACID OXIDATION; HUMAN; HUMAN CELL; IMMUNOFLUORESCENCE TEST; IMMUNOPRECIPITATION; JAPANESE ENCEPHALITIS VIRUS; MOUSE; MUTAGENESIS; NONHUMAN; OXYGEN CONSUMPTION; PATHOGENESIS; PROTEIN INTERACTION; PROTEIN LOCALIZATION; REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION; VIRUS VIRULENCE; WESTERN BLOTTING; ANIMAL; GENETICS; HEK293S CELL LINE; JAPANESE ENCEPHALITIS; JAPANESE ENCEPHALITIS VIRUS GROUP; METABOLISM; OXIDATION REDUCTION REACTION; POINT MUTATION;

JAPANESE ENCEPHALITIS VIRUS; MUS;

ANIMALS; ENCEPHALITIS VIRUS, JAPANESE; ENCEPHALITIS, JAPANESE; FATTY ACIDS; HEK293 CELLS; HUMANS; MICE; MITOCHONDRIAL TRIFUNCTIONAL PROTEIN, ALPHA SUBUNIT; MITOCHONDRIAL TRIFUNCTIONAL PROTEIN, BETA SUBUNIT; OXIDATION-REDUCTION; POINT MUTATION; VIRAL NONSTRUCTURAL PROTEINS;

EID: 84926505155     PISSN: 15537366     EISSN: 15537374     Source Type: Journal    
DOI: 10.1371/journal.ppat.1004750     Document Type: Article

References (86)

1. Fernandez-Garcia MD, Mazzon M, Jacobs M, Amara A, (2009) Pathogenesis of flavivirus infections: using and abusing the host cell. Cell Host Microbe 5: 318–328. doi: 10.1016/j.chom.2009.04.001 19380111
2. Campbell GL, Hills SL, Fischer M, Jacobson JA, Hoke CH, et al. (2011) Estimated global incidence of Japanese encephalitis: a systematic review. Bull World Health Organ 89: 766–774, 774A–774E. doi: 10.2471/BLT.10.085233 22084515
3. Le Flohic G, Porphyre V, Barbazan P, Gonzalez JP, (2013) Review of climate, landscape, and viral genetics as drivers of the Japanese encephalitis virus ecology. PLoS Negl Trop Dis 7: e2208. doi: 10.1371/journal.pntd.0002208 24069463
4. Mukhopadhyay S, Kuhn RJ, Rossmann MG, (2005) A structural perspective of the flavivirus life cycle. Nat Rev Microbiol 3: 13–22. 15608696
5. Unni SK, Ruzek D, Chhatbar C, Mishra R, Johri MK, et al. (2011) Japanese encephalitis virus: from genome to infectome. Microbes Infect 13: 312–321. doi: 10.1016/j.micinf.2011.01.002 21238600
6. Zhang Y, Wang Z, Chen H, Chen Z, Tian Y, (2014) Antioxidants: potential antiviral agents for Japanese encephalitis virus infection. Int J Infect Dis 24: 30–36. doi: 10.1016/j.ijid.2014.02.011 24780919
7. Lin RJ, Chang BL, Yu HP, Liao CL, Lin YL, (2006) Blocking of interferon-induced Jak-Stat signaling by Japanese encephalitis virus NS5 through a protein tyrosine phosphatase-mediated mechanism. J Virol 80: 5908–5918. 16731929
8. Laurent-Rolle M, Boer EF, Lubick KJ, Wolfinbarger JB, Carmody AB, et al. (2010) The NS5 protein of the virulent West Nile virus NY99 strain is a potent antagonist of type I interferon-mediated JAK-STAT signaling. J Virol 84: 3503–3515. doi: 10.1128/JVI.01161-09 20106931
9. Best SM, Morris KL, Shannon JG, Robertson SJ, Mitzel DN, et al. (2005) Inhibition of interferon-stimulated JAK-STAT signaling by a tick-borne flavivirus and identification of NS5 as an interferon antagonist. J Virol 79: 12828–12839. 16188985
10. Ashour J, Laurent-Rolle M, Shi PY, Garcia-Sastre A, (2009) NS5 of dengue virus mediates STAT2 binding and degradation. J Virol 83: 5408–5418. doi: 10.1128/JVI.02188-08 19279106
11. Miller S, Krijnse-Locker J, (2008) Modification of intracellular membrane structures for virus replication. Nat Rev Microbiol 6: 363–374. doi: 10.1038/nrmicro1890 18414501
12. Belov GA, van Kuppeveld FJ, (2012) (+)RNA viruses rewire cellular pathways to build replication organelles. Curr Opin Virol 2: 740–747. doi: 10.1016/j.coviro.2012.09.006 23036609
13. Lam TK, Schwartz GJ, Rossetti L, (2005) Hypothalamic sensing of fatty acids. Nat Neurosci 8: 579–584. 15856066
14. Kim JJ, Battaile KP, (2002) Burning fat: the structural basis of fatty acid beta-oxidation. Curr Opin Struct Biol 12: 721–728. 12504675
15. Fould B, Garlatti V, Neumann E, Fenel D, Gaboriaud C, et al. (2010) Structural and functional characterization of the recombinant human mitochondrial trifunctional protein. Biochemistry 49: 8608–8617. doi: 10.1021/bi100742w 20825197
16. Angdisen J, Moore VD, Cline JM, Payne RM, Ibdah JA, (2005) Mitochondrial trifunctional protein defects: molecular basis and novel therapeutic approaches. Curr Drug Targets Immune Endocr Metabol Disord 5: 27–40. 15777202
17. Roe CR, Roe DS, Wallace M, Garritson B, (2007) Choice of oils for essential fat supplements can enhance production of abnormal metabolites in fat oxidation disorders. Mol Genet Metab 92: 346–350. 17825594
18. Tonin AM, Grings M, Busanello EN, Moura AP, Ferreira GC, et al. (2010) Long-chain 3-hydroxy fatty acids accumulating in LCHAD and MTP deficiencies induce oxidative stress in rat brain. Neurochem Int 56: 930–936. doi: 10.1016/j.neuint.2010.03.025 20381565
19. Tripathy D, Mohanty P, Dhindsa S, Syed T, Ghanim H, et al. (2003) Elevation of free fatty acids induces inflammation and impairs vascular reactivity in healthy subjects. Diabetes 52: 2882–2887. 14633847
20. Vickers AE, (2009) Characterization of hepatic mitochondrial injury induced by fatty acid oxidation inhibitors. Toxicol Pathol 37: 78–88. doi: 10.1177/0192623308329285 19234235
21. Kapoor RR, James C, Flanagan SE, Ellard S, Eaton S, et al. (2009) 3-Hydroxyacyl-coenzyme A dehydrogenase deficiency and hyperinsulinemic hypoglycemia: characterization of a novel mutation and severe dietary protein sensitivity. J Clin Endocrinol Metab 94: 2221–2225. doi: 10.1210/jc.2009-0423 19417036
22. Sperk A, Mueller M, Spiekerkoetter U, (2010) Outcome in six patients with mitochondrial trifunctional protein disorders identified by newborn screening. Mol Genet Metab 101: 205–207. doi: 10.1016/j.ymgme.2010.07.003 20659813
23. McCrimmon RJ, (2012) Update in the CNS response to hypoglycemia. J Clin Endocrinol Metab 97: 1–8. doi: 10.1210/jc.2011-1927 22223763
24. Dharancy S, Malapel M, Perlemuter G, Roskams T, Cheng Y, et al. (2005) Impaired expression of the peroxisome proliferator-activated receptor alpha during hepatitis C virus infection. Gastroenterology 128: 334–342. 15685545
25. Syed GH, Amako Y, Siddiqui A, (2010) Hepatitis C virus hijacks host lipid metabolism. Trends Endocrinol Metab 21: 33–40. doi: 10.1016/j.tem.2009.07.005 19854061
26. Wu JM, Skill NJ, Maluccio MA, (2010) Evidence of aberrant lipid metabolism in hepatitis C and hepatocellular carcinoma. HPB (Oxford) 12: 625–636. doi: 10.1111/j.1477-2574.2010.00207.x 20961371
27. Roe B, Kensicki E, Mohney R, Hall WW, (2011) Metabolomic profile of hepatitis C virus-infected hepatocytes. PLoS One 6: e23641. doi: 10.1371/journal.pone.0023641 21853158
28. Jonsson JR, Barrie HD, O'Rourke P, Clouston AD, Powell EE, (2008) Obesity and steatosis influence serum and hepatic inflammatory markers in chronic hepatitis C. Hepatology 48: 80–87. doi: 10.1002/hep.22311 18571785
29. Yao D, Kuwajima M, Chen Y, Shiota M, Okumura Y, et al. (2007) Impaired long-chain fatty acid metabolism in mitochondria causes brain vascular invasion by a non-neurotropic epidemic influenza A virus in the newborn/suckling period: implications for influenza-associated encephalopathy. Mol Cell Biochem 299: 85–92. 16896540
30. Erta M, Quintana A, Hidalgo J, (2012) Interleukin-6, a major cytokine in the central nervous system. Int J Biol Sci 8: 1254–1266. doi: 10.7150/ijbs.4679 23136554
31. Terry RL, Getts DR, Deffrasnes C, van Vreden C, Campbell IL, et al. (2012) Inflammatory monocytes and the pathogenesis of viral encephalitis. J Neuroinflammation 9: 270. doi: 10.1186/1742-2094-9-270 23244217
32. Ramesh G, MacLean AG, Philipp MT, (2013) Cytokines and chemokines at the crossroads of neuroinflammation, neurodegeneration, and neuropathic pain. Mediators Inflamm 2013: 480739. doi: 10.1155/2013/480739 23997430
33. Ravi V, Parida S, Desai A, Chandramuki A, Gourie-Devi M, et al. (1997) Correlation of tumor necrosis factor levels in the serum and cerebrospinal fluid with clinical outcome in Japanese encephalitis patients. J Med Virol 51: 132–136. 9021544
34. Winter PM, Dung NM, Loan HT, Kneen R, Wills B, et al. (2004) Proinflammatory cytokines and chemokines in humans with Japanese encephalitis. J Infect Dis 190: 1618–1626. 15478067
35. Zhang J, Nuebel E, Wisidagama DR, Setoguchi K, Hong JS, et al. (2012) Measuring energy metabolism in cultured cells, including human pluripotent stem cells and differentiated cells. Nat Protoc 7: 1068–1085. doi: 10.1038/nprot.2012.048 22576106
36. Randle PJ, Garland PB, Hales CN, Newsholme EA, (1963) The glucose fatty-acid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet 1: 785–789. 13990765
37. Eaton S, Bartlett K, Pourfarzam M, (1996) Mammalian mitochondrial beta-oxidation. Biochem J 320 (Pt 2): 345–357. 8973539
38. Takegami T, Hotta S, (1989) In vitro synthesis of Japanese encephalitis virus (JEV) RNA: membrane and nuclear fractions of JEV-infected cells possess high levels of virus-specific RNA polymerase activity. Virus Res 13: 337–350. 2816040
39. Wang JJ, Liao CL, Yang CI, Lin YL, Chiou CT, et al. (1998) Localizations of NS3 and E proteins in mouse brain infected with mutant strain of Japanese encephalitis virus. Arch Virol 143: 2353–2369. 9930192
40. Egloff MP, Benarroch D, Selisko B, Romette JL, Canard B, (2002) An RNA cap (nucleoside-2'-O-)-methyltransferase in the flavivirus RNA polymerase NS5: crystal structure and functional characterization. EMBO J 21: 2757–2768. 12032088
41. Liang JJ, Liao CL, Liao JT, Lee YL, Lin YL, (2009) A Japanese encephalitis virus vaccine candidate strain is attenuated by decreasing its interferon antagonistic ability. Vaccine 27: 2746–2754. doi: 10.1016/j.vaccine.2009.03.007 19366580
42. Kimura T, Katoh H, Kayama H, Saiga H, Okuyama M, et al. (2013) Ifit1 inhibits Japanese encephalitis virus replication through binding to 5' capped 2'-O unmethylated RNA. J Virol 87: 9997–10003. doi: 10.1128/JVI.00883-13 23824812
43. Zhou Y, Ray D, Zhao Y, Dong H, Ren S, et al. (2007) Structure and function of flavivirus NS5 methyltransferase. J Virol 81: 3891–3903. 17267492
44. Daffis S, Szretter KJ, Schriewer J, Li J, Youn S, et al. (2010) 2'-O methylation of the viral mRNA cap evades host restriction by IFIT family members. Nature 468: 452–456. doi: 10.1038/nature09489 21085181
45. Li SH, Dong H, Li XF, Xie X, Zhao H, et al. (2013) Rational design of a flavivirus vaccine by abolishing viral RNA 2'-O methylation. J Virol 87: 5812–5819. doi: 10.1128/JVI.02806-12 23487465
46. Mackenzie JM, Kenney MT, Westaway EG, (2007) West Nile virus strain Kunjin NS5 polymerase is a phosphoprotein localized at the cytoplasmic site of viral RNA synthesis. J Gen Virol 88: 1163–1168. 17374759
47. Hannemann H, Sung PY, Chiu HC, Yousuf A, Bird J, et al. (2013) Serotype-specific differences in dengue virus non-structural protein 5 nuclear localization. J Biol Chem 288: 22621–22635. doi: 10.1074/jbc.M113.481382 23770669
48. Yang SH, Liu ML, Tien CF, Chou SJ, Chang RY, (2009) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interaction with 3' ends of Japanese encephalitis virus RNA and colocalization with the viral NS5 protein. J Biomed Sci 16: 40. doi: 10.1186/1423-0127-16-40 19368702
49. Ye J, Chen Z, Zhang B, Miao H, Zohaib A, et al. (2013) Heat shock protein 70 is associated with replicase complex of Japanese encephalitis virus and positively regulates viral genome replication. PLoS One 8: e75188. doi: 10.1371/journal.pone.0075188 24086464
50. Hall RA, Tan SE, Selisko B, Slade R, Hobson-Peters J, et al. (2009) Monoclonal antibodies to the West Nile virus NS5 protein map to linear and conformational epitopes in the methyltransferase and polymerase domains. J Gen Virol 90: 2912–2922. doi: 10.1099/vir.0.013805-0 19710254
51. Claros MG, (1995) MitoProt, a Macintosh application for studying mitochondrial proteins. Comput Appl Biosci 11: 441–447. 8521054
52. Claros MG, Vincens P, (1996) Computational method to predict mitochondrially imported proteins and their targeting sequences. Eur J Biochem 241: 779–786. 8944766
53. Hu J, Chu Z, Han J, Zhang Q, Zhang D, et al. (2014) Phosphorylation-dependent mitochondrial translocation of MAP4 is an early step in hypoxia-induced apoptosis in cardiomyocytes. Cell Death Dis 5: e1424. doi: 10.1038/cddis.2014.369 25232678
54. Li M, Zhong Z, Zhu J, Xiang D, Dai N, et al. (2010) Identification and characterization of mitochondrial targeting sequence of human apurinic/apyrimidinic endonuclease 1. J Biol Chem 285: 14871–14881. doi: 10.1074/jbc.M109.069591 20231292
55. Terada K, Kanazawa M, Bukau B, Mori M, (1997) The human DnaJ homologue dj2 facilitates mitochondrial protein import and luciferase refolding. J Cell Biol 139: 1089–1095. 9382858
56. Wang RY, Huang YR, Chong KM, Hung CY, Ke ZL, et al. (2011) DnaJ homolog Hdj2 facilitates Japanese encephalitis virus replication. Virol J 8: 471. doi: 10.1186/1743-422X-8-471 21999493
57. Gerbeth C, Mikropoulou D, Meisinger C, (2013) From inventory to functional mechanisms: regulation of the mitochondrial protein import machinery by phosphorylation. FEBS J 280: 4933–4942. doi: 10.1111/febs.12445 23895388
58. Shiba-Fukushima K, Imai Y, Yoshida S, Ishihama Y, Kanao T, et al. (2012) PINK1-mediated phosphorylation of the Parkin ubiquitin-like domain primes mitochondrial translocation of Parkin and regulates mitophagy. Sci Rep 2: 1002. doi: 10.1038/srep01002 23256036
59. Lu G, Gong P, (2013) Crystal Structure of the full-length Japanese encephalitis virus NS5 reveals a conserved methyltransferase-polymerase interface. PLoS Pathog 9: e1003549. doi: 10.1371/journal.ppat.1003549 23950717
60. Li ZN, Lee BJ, Langley WA, Bradley KC, Russell RJ, et al. (2008) Length requirements for membrane fusion of influenza virus hemagglutinin peptide linkers to transmembrane or fusion peptide domains. J Virol 82: 6337–6348. doi: 10.1128/JVI.02576-07 18417593
61. Hobiger K, Utesch T, Mroginski MA, Friedrich T, (2012) Coupling of Ci-VSP modules requires a combination of structure and electrostatics within the linker. Biophys J 102: 1313–1322. doi: 10.1016/j.bpj.2012.02.027 22455914
62. Seo JY, Yaneva R, Hinson ER, Cresswell P, (2011) Human cytomegalovirus directly induces the antiviral protein viperin to enhance infectivity. Science 332: 1093–1097. doi: 10.1126/science.1202007 21527675
63. Heaton NS, Randall G, (2010) Dengue virus-induced autophagy regulates lipid metabolism. Cell Host Microbe 8: 422–432. doi: 10.1016/j.chom.2010.10.006 21075353
64. Eslam M, Khattab MA, Harrison SA, (2011) Peroxisome proliferator-activated receptors and hepatitis C virus. Therap Adv Gastroenterol 4: 419–431. doi: 10.1177/1756283X11405251 22043232
65. Seo JY, Cresswell P, (2013) Viperin regulates cellular lipid metabolism during human cytomegalovirus infection. PLoS Pathog 9: e1003497. doi: 10.1371/journal.ppat.1003497 23935494
66. Chan YL, Chang TH, Liao CL, Lin YL, (2008) The cellular antiviral protein viperin is attenuated by proteasome-mediated protein degradation in Japanese encephalitis virus-infected cells. J Virol 82: 10455–10464. doi: 10.1128/JVI.00438-08 18768981
67. Moser TS, Schieffer D, Cherry S, (2012) AMP-activated kinase restricts Rift Valley fever virus infection by inhibiting fatty acid synthesis. PLoS Pathog 8: e1002661. doi: 10.1371/journal.ppat.1002661 22532801
68. Tien CF, Cheng SC, Ho YP, Chen YS, Hsu JH, et al. (2014) Inhibition of aldolase A blocks biogenesis of ATP and attenuates Japanese encephalitis virus production. Biochem Biophys Res Commun 443: 464–469. doi: 10.1016/j.bbrc.2013.11.128 24321549
69. Yu Y, Clippinger AJ, Alwine JC, (2011) Viral effects on metabolism: changes in glucose and glutamine utilization during human cytomegalovirus infection. Trends Microbiol 19: 360–367. doi: 10.1016/j.tim.2011.04.002 21570293
70. Vander Heiden MG, Cantley LC, Thompson CB, (2009) Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324: 1029–1033. doi: 10.1126/science.1160809 19460998
71. Marie SK, Shinjo SM, (2011) Metabolism and brain cancer. Clinics (Sao Paulo) 66 Suppl 1: 33–43.
72. Vastag L, Koyuncu E, Grady SL, Shenk TE, Rabinowitz JD, (2011) Divergent effects of human cytomegalovirus and herpes simplex virus-1 on cellular metabolism. PLoS Pathog 7: e1002124. doi: 10.1371/journal.ppat.1002124 21779165
73. Ramiere C, Rodriguez J, Enache LS, Lotteau V, Andre P, et al. (2014) Activity of hexokinase is increased by its interaction with hepatitis C virus protein NS5A. J Virol 88: 3246–3254. doi: 10.1128/JVI.02862-13 24390321
74. Sengupta N, Ghosh S, Vasaikar SV, Gomes J, Basu A, (2014) Modulation of neuronal proteome profile in response to Japanese encephalitis virus infection. PLoS One 9: e90211. doi: 10.1371/journal.pone.0090211 24599148
75. Gatenby RA, Gillies RJ, (2004) Why do cancers have high aerobic glycolysis? Nat Rev Cancer 4: 891–899. 15516961
76. Ventura FV, Ruiter JP, L IJ, de Almeida IT, Wanders RJ, (1998) Lactic acidosis in long-chain fatty acid beta-oxidation disorders. J Inherit Metab Dis 21: 645–654. 9762600
77. Rakheja D, Bennett MJ, Rogers BB, (2002) Long-chain L-3-hydroxyacyl-coenzyme a dehydrogenase deficiency: a molecular and biochemical review. Lab Invest 82: 815–824. 12118083
78. Solomon T, Dung NM, Kneen R, Thao le TT, Gainsborough M, et al. (2002) Seizures and raised intracranial pressure in Vietnamese patients with Japanese encephalitis. Brain 125: 1084–1093. 11960897
79. Schonfeld P, Wojtczak L, (2008) Fatty acids as modulators of the cellular production of reactive oxygen species. Free Radic Biol Med 45: 231–241. doi: 10.1016/j.freeradbiomed.2008.04.029 18482593
80. Cho HK, Kim SY, Yoo SK, Choi YH, Cheong J, (2014) Fatty acids increase hepatitis B virus X protein stabilization and HBx-induced inflammatory gene expression. FEBS J 281: 2228–2239. doi: 10.1111/febs.12776 24612645
81. Chen LK, Lin YL, Liao CL, Lin CG, Huang YL, et al. (1996) Generation and characterization of organ-tropism mutants of Japanese encephalitis virus in vivo and in vitro. Virology 223: 79–88. 8806542
82. Makarova O, Kamberov E, Margolis B, (2000) Generation of deletion and point mutations with one primer in a single cloning step. Biotechniques 29: 970–972. 11084856
83. Yoshizumi T, Ichinohe T, Sasaki O, Otera H, Kawabata S, et al. (2014) Influenza A virus protein PB1-F2 translocates into mitochondria via Tom40 channels and impairs innate immunity. Nat Commun 5: 4713. doi: 10.1038/ncomms5713 25140902
84. Bononi A, Pinton P, (2014) Study of PTEN subcellular localization. Methods. In press.
85. Hase T, Dubois DR, Summers PL, (1990) Comparative study of mouse brains infected with Japanese encephalitis virus by intracerebral or intraperitoneal inoculation. Int J Exp Pathol 71: 857–869. 2177623
86. Tu YC, Yu CY, Liang JJ, Lin E, Liao CL, et al. (2012) Blocking double-stranded RNA-activated protein kinase PKR by Japanese encephalitis virus nonstructural protein 2A. J Virol 86: 10347–10358. doi: 10.1128/JVI.00525-12 22787234