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Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Regulates Cell Stress Response and Apoptosis


Severe acute respiratory syndrome virus (SARS-CoV) that lacks the envelope (E) gene (rSARS-CoV-ΔE) is attenuated in vivo. To identify factors that contribute to rSARS-CoV-ΔE attenuation, gene expression in cells infected by SARS-CoV with or without E gene was compared. Twenty-five stress response genes were preferentially upregulated during infection in the absence of the E gene. In addition, genes involved in signal transduction, transcription, cell metabolism, immunoregulation, inflammation, apoptosis and cell cycle and differentiation were differentially regulated in cells infected with rSARS-CoV with or without the E gene. Administration of E protein in trans reduced the stress response in cells infected with rSARS-CoV-ΔE or with respiratory syncytial virus, or treated with drugs, such as tunicamycin and thapsigargin that elicit cell stress by different mechanisms. In addition, SARS-CoV E protein down-regulated the signaling pathway inositol-requiring enzyme 1 (IRE-1) of the unfolded protein response, but not the PKR-like ER kinase (PERK) or activating transcription factor 6 (ATF-6) pathways, and reduced cell apoptosis. Overall, the activation of the IRE-1 pathway was not able to restore cell homeostasis, and apoptosis was induced probably as a measure to protect the host by limiting virus production and dissemination. The expression of proinflammatory cytokines was reduced in rSARS-CoV-ΔE-infected cells compared to rSARS-CoV-infected cells, suggesting that the increase in stress responses and the reduction of inflammation in the absence of the E gene contributed to the attenuation of rSARS-CoV-ΔE.


Vyšlo v časopise: Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Regulates Cell Stress Response and Apoptosis. PLoS Pathog 7(10): e32767. doi:10.1371/journal.ppat.1002315
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1002315

Souhrn

Severe acute respiratory syndrome virus (SARS-CoV) that lacks the envelope (E) gene (rSARS-CoV-ΔE) is attenuated in vivo. To identify factors that contribute to rSARS-CoV-ΔE attenuation, gene expression in cells infected by SARS-CoV with or without E gene was compared. Twenty-five stress response genes were preferentially upregulated during infection in the absence of the E gene. In addition, genes involved in signal transduction, transcription, cell metabolism, immunoregulation, inflammation, apoptosis and cell cycle and differentiation were differentially regulated in cells infected with rSARS-CoV with or without the E gene. Administration of E protein in trans reduced the stress response in cells infected with rSARS-CoV-ΔE or with respiratory syncytial virus, or treated with drugs, such as tunicamycin and thapsigargin that elicit cell stress by different mechanisms. In addition, SARS-CoV E protein down-regulated the signaling pathway inositol-requiring enzyme 1 (IRE-1) of the unfolded protein response, but not the PKR-like ER kinase (PERK) or activating transcription factor 6 (ATF-6) pathways, and reduced cell apoptosis. Overall, the activation of the IRE-1 pathway was not able to restore cell homeostasis, and apoptosis was induced probably as a measure to protect the host by limiting virus production and dissemination. The expression of proinflammatory cytokines was reduced in rSARS-CoV-ΔE-infected cells compared to rSARS-CoV-infected cells, suggesting that the increase in stress responses and the reduction of inflammation in the absence of the E gene contributed to the attenuation of rSARS-CoV-ΔE.


Zdroje

1. DrostenCGuntherSPreiserWvan der WerfSBrodtHR 2003 Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med 348 1967 1976

2. FouchierRAKuikenTSchuttenMvan AmerongenGvan DoornumGJ 2003 Aetiology: Koch's postulates fulfilled for SARS virus. Nature 423 240

3. KsiazekTGErdmanDGoldsmithCZakiSPeretT 2003 A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med 348 1953 1966

4. KuikenTFouchierRAMSchuttenMRimmelzwaanGFvan AmerongenG 2003 Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome. Lancet 362 263 270

5. MarraMAJonesSJMAstellCRHoltRABrooks-WilsonA 2003 The genome sequence of the SARS-associated coronavirus. Science 300 1399 1404

6. PeirisJSMLaiSTPoonLLMGuanYYamLYC 2003 Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 361 1319 1325

7. RotaPAObersteMSMonroeSSNixWACampganoliR 2003 Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 300 1394 1399

8. LauSKWooPCLiKSHuangYTsoiHW 2005 Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proc Natl Acad Sci U S A 102 14040 14045

9. LiWShiZYuMRenWSmithC 2005 Bats are natural reservoirs of SARS-like coronaviruses. Science 310 676 679

10. WooPCLauSKLiKSPoonRWWongBH 2006 Molecular diversity of coronaviruses in bats. Virology 351 180 187

11. ZiebuhrJ 2005 The coronavirus replicase. EnjuanesL Coronavirus replication and reverse genetics Berlin Heidelberg, Germany Springer-Verlag 57 94

12. ZiebuhrJSnijderEJGorbalenyaAE 2000 Virus-encoded proteinases and proteolytic processing in the Nidovirales. J Gen Virol 81 853 879

13. EnjuanesLGorbalenyaAEde GrootRJCowleyJAZiebuhrJ 2008 The Nidovirales. MahyBWJVan RegenmortelMWalkerPMajumder-RussellD Encyclopedia of Virology, Third Edition Oxford Elsevier Ltd 419 430

14. HuangCItoNTsengCTMakinoS 2006 Severe acute respiratory syndrome coronavirus 7a accessory protein is a viral structural protein. J Virol 80 7287 7294

15. HuangCPetersCJMakinoS 2007 Severe acute respiratory syndrome coronavirus accessory protein 6 is a virion-associated protein and is released from 6 protein-expressing cells. J Virol 81 5423 5426

16. ItoNMosselECNarayananKPopovVLHuangC 2005 Severe acute respiratory syndrome coronavirus 3a protein is a viral structural protein. J Virol 79 3182 3186

17. SchaecherSRMackenzieJMPekoszA 2007 The ORF7b protein of SARS-CoV is expressed in virus-infected cells and incorporated into SARS-CoV particles. J Virol 81 718 731

18. ShenSLinPSChaoYCZhangAYangX 2005 The severe acute respiratory syndrome coronavirus 3a is a novel structural protein. Biochem Biophys Res Commun 330 286 292

19. TorresJParthasarathyKLinXSaravananRLiuDX 2006 Model of a putative pore: the pentameric alpha-helical bundle of SARS coronavirus E protein in lipid bilayers. Biophys J 91 938 947

20. TorresJMaheswariUParthasarathyKNgLLiuDX 2007 Conductance and amantadine binding of a pore formed by a lysine-flanked transmembrane domain of SARS coronavirus envelope protein. Protein Sci 16 2065 2071

21. WilsonLGagePEwartG 2006 Hexamethylene amiloride blocks E protein ion channels and inhibits coronavirus replication. Virology 353 294 306

22. WilsonLMcKinlayCGageP 2004 SARS coronavirus E protein forms cation-selective ion channels. Virology 330 322 331

23. CurtisKMYountBBaricRS 2002 Heterologous gene expression from transmissible gastroenteritis virus replicon particles. J Virol 76 1422 1434

24. OrtegoJCerianiJEPatinoCPlanaJEnjuanesL 2007 Absence of E protein arrests transmissible gastroenteritis coronavirus maturation in the secretory pathway. Virology 368 296 308

25. OrtegoJEscorsDLaudeHEnjuanesL 2002 Generation of a replication-competent, propagation-deficient virus vector based on the transmissible gastroenteritis coronavirus genome. J Virol 76 11518 11529

26. DeDiegoMLAlvarezEAlmazanFRejasMTLamirandeE 2007 A severe acute respiratory syndrome coronavirus that lacks the E gene is attenuated in vitro and in vivo. J Virol 81 1701 1713

27. DeDiegoMLPeweLAlvarezERejasMTPerlmanS 2008 Pathogenicity of severe acute respiratory coronavirus deletion mutants in hACE-2 transgenic mice. Virology 376 379 389

28. KuoLMastersPS 2003 The small envelope protein E is not essential for murine coronavirus replication. J Virol 77 4597 4608

29. SullivanCSPipasJM 2001 The virus-chaperone connection. Virology 287 1 8

30. SantoroMG 2000 Heat shock factors and the control of the stress response. Biochem Pharmacol 59 55 63

31. NollenEAMorimotoRI 2002 Chaperoning signaling pathways: molecular chaperones as stress-sensing ‘heat shock’ proteins. J Cell Sci 115 2809 2816

32. MoseleyP 2000 Stress proteins and the immune response. Immunopharmacology 48 299 302

33. RobertJ 2003 Evolution of heat shock protein and immunity. Dev Comp Immunol 27 449 464

34. GosertRKanjanahaluethaiAEggerDBienzKBakerSC 2002 RNA replication of mouse hepatitis virus takes place at double-membrane vesicles. J Virol 76 3697 3708

35. SnijderEJvan der MeerYZevenhoven-DobbeJOnderwaterJJvan der MeulenJ 2006 Ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex. J Virol 80 5927 5940

36. KnoopsKKikkertMWormSHZevenhoven-DobbeJCvan der MeerY 2008 SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum. PLoS Biol 6 e226

37. RonDWalterP 2007 Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 8 519 529

38. WangXZHardingHPZhangYJolicoeurEMKurodaM 1998 Cloning of mammalian Ire1 reveals diversity in the ER stress responses. EMBO J 17 5708 5717

39. HardingHPZhangYRonD 1999 Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 397 271 274

40. ChenXShenJPrywesR 2002 The luminal domain of ATF6 senses endoplasmic reticulum (ER) stress and causes translocation of ATF6 from the ER to the Golgi. J Biol Chem 277 13045 13052

41. BoyceMYuanJ 2006 Cellular response to endoplasmic reticulum stress: a matter of life or death. Cell Death Differ 13 363 373

42. WeiLSunSXuCHZhangJXuY 2007 Pathology of the thyroid in severe acute respiratory syndrome. Hum Pathol 38 95 102

43. ZhangQLDingYQHeLWangWZhangJH 2003 Detection of cell apoptosis in the pathological tissues of patients with SARS and its significance. Di Yi Jun Yi Da Xue Xue Bao 23 770 773

44. KrahlingVSteinDASpiegelMWeberFMuhlbergerE 2009 Severe acute respiratory syndrome coronavirus triggers apoptosis via protein kinase R but is resistant to its antiviral activity. J Virol 83 2298 2309

45. TanYJLimSGHongW 2007 Regulation of cell death during infection by the severe acute respiratory syndrome coronavirus and other coronaviruses. Cell Microbiol 9 2552 2561

46. HuangKJSuIJTheronMWuYCLaiSK 2005 An interferon-gamma-related cytokine storm in SARS patients. J Med Virol 75 185 194

47. WongRSWuAToKFLeeNLamCW 2003 Haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis. BMJ 326 1358 1362

48. TangNLChanPKWongCKToKFWuAK 2005 Early enhanced expression of interferon-inducible protein-10 (CXCL-10) and other chemokines predicts adverse outcome in severe acute respiratory syndrome. Clin Chem 51 2333 2340

49. JiangYXuJZhouCWuZZhongS 2005 Characterization of cytokine/chemokine profiles of severe acute respiratory syndrome. Am J Respir Crit Care Med 171 850 857

50. ZhangYLiJZhanYWuLYuX 2004 Analysis of serum cytokines in patients with severe acute respiratory syndrome. Infect Immun 72 4410 4415

51. ReghunathanRJayapalMHsuLYChngHHTaiD 2005 Expression profile of immune response genes in patients with Severe Acute Respiratory Syndrome. BMC Immunol 6 2

52. Gillim-RossLTaylorJSchollDRRidenourJMastersPS 2004 Discovery of novel human and animal cells infected by the severe acute respiratory syndrome coronavirus by replication-specific multiplex reverse transcription-PCR. J Clin Microbiol 42 3196 3206

53. HattermannKMullerMANitscheAWendtSDonoso MantkeO 2005 Susceptibility of different eukaryotic cell lines to SARS-coronavirus. Arch Virol 150 1023 1031

54. LuWZhengBJXuKSchwarzWDuL 2006 Severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release. Proc Natl Acad Sci U S A 103 12540 12545

55. MosselECHuangCNarayananKMakinoSTeshRB 2005 Exogenous ACE2 expression allows refractory cell lines to support severe acute respiratory syndrome coronavirus replication. J Virol 79 3846 3850

56. NetlandJDeDiegoMLZhaoJFettCAlvarezE 2010 Immunization with an attenuated severe acute respiratory syndrome coronavirus deleted in E protein protects against lethal respiratory disease. Virology 399 120 128

57. EmenyJMMorganMJ 1979 Regulation of the interferon system: evidence that Vero cells have a genetic defect in interferon production. J Gen Virol 43 247 252

58. McKimm-BreschkinJLHolmesIH 1982 Conditions required for induction of interferon by rotaviruses and for their sensitivity to its action. Infect Immun 36 857 863

59. LeongWFTanHCOoiEEKohDRChowVT 2005 Microarray and real-time RT-PCR analyses of differential human gene expression patterns induced by severe acute respiratory syndrome (SARS) coronavirus infection of Vero cells. Microbes Infect 7 248 259

60. ZhangYBlattmanJNKennedyNJDuongJNguyenT 2004 Regulation of innate and adaptive immune responses by MAP kinase phosphatase 5. Nature 430 793 797

61. NarayananKHuangCLokugamageKKamitaniWIkegamiT 2008 Severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type I interferon, in infected cells. J Virol 82 4471 4479

62. FriemanMBChenJMorrisonTEWhitmoreAFunkhouserW 2010 SARS-CoV pathogenesis is regulated by a STAT1 dependent but a type I, II and III interferon receptor independent mechanism. PLoS Pathog 6 e1000849

63. SheahanTMorrisonTEFunkhouserWUematsuSAkiraS 2008 MyD88 is required for protection from lethal infection with a mouse-adapted SARS-CoV. PLoS Pathog 4 e1000240

64. BechillJChenZBrewerJWBakerSC 2008 Coronavirus infection modulates the unfolded protein response and mediates sustained translational repression. J Virol 82 4492 4501

65. SalanuevaIJCarrascosaJLRiscoC 1999 Structural maturation of the transmissible gastroenteritis coronavirus. J Virol 73 7952 7964

66. ChanCPSiuKLChinKTYuenKYZhengB 2006 Modulation of the unfolded protein response by the severe acute respiratory syndrome coronavirus spike protein. J Virol 80 9279 9287

67. HeB 2006 Viruses, endoplasmic reticulum stress, and interferon responses. Cell Death Differ 13 393 403

68. WangYShenJArenzanaNTirasophonWKaufmanRJ 2000 Activation of ATF6 and an ATF6 DNA binding site by the endoplasmic reticulum stress response. J Biol Chem 275 27013 27020

69. ShangJLehrmanMA 2004 Discordance of UPR signaling by ATF6 and Ire1p-XBP1 with levels of target transcripts. Biochem Biophys Res Commun 317 390 396

70. WeissSRNavas-MartinS 2005 Coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. Microbiol Mol Biol Rev 69 635 664

71. ChengVCHungIFTangBSChuCMWongMM 2004 Viral replication in the nasopharynx is associated with diarrhea in patients with severe acute respiratory syndrome. Clin Infect Dis 38 467 475

72. PeirisJSYuenKYOsterhausADStohrK 2003 The severe acute respiratory syndrome. N Engl J Med 349 2431 2441

73. DingYWangHShenHLiZGengJ 2003 The clinical pathology of severe acute respiratory syndrome (SARS): a report from China. J Pathol 200 282 289

74. NichollsJMPoonLLLeeKCNgWFLaiST 2003 Lung pathology of fatal severe acute respiratory syndrome. Lancet 361 1773 1778

75. RockxBBaasTZornetzerGAHaagmansBSheahanT 2009 Early upregulation of acute respiratory distress syndrome-associated cytokines promotes lethal disease in an aged-mouse model of severe acute respiratory syndrome coronavirus infection. J Virol 83 7062 7074

76. SmitsSLde LangAvan den BrandJMLeijtenLMvanIWF 2010 Exacerbated innate host response to SARS-CoV in aged non-human primates. PLoS Pathog 6 e1000756

77. Nieto-TorresJLDeDiegoMLAlvarezEEnjuanesL 2010 Cellular localization and topology of severe acute respiratory syndome coronavirus (SARS-CoV) envelope protein. Virology 415 69 82

78. YeZWongCKLiPXieY 2008 A SARS-CoV protein, ORF-6, induces caspase-3 mediated, ER stress and JNK-dependent apoptosis. Biochim Biophys Acta 1780 1383 1387

79. MinakshiRPadhanKRaniMKhanNAhmadF 2009 The SARS Coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type 1 interferon receptor. PLoS One 4 e8342

80. VersteegGAvan de NesPSBredenbeekPJSpaanWJ 2007 The coronavirus spike protein induces endoplasmic reticulum stress and upregulation of intracellular chemokine mRNA concentrations. J Virol 81 10981 10990

81. SungSCChaoCYJengKSYangJYLaiMM 2009 The 8ab protein of SARS-CoV is a luminal ER membrane-associated protein and induces the activation of ATF6. Virology 387 402 413

82. TangBSChanKHChengVCWooPCLauSK 2005 Comparative host gene transcription by microarray analysis early after infection of the Huh7 cell line by severe acute respiratory syndrome coronavirus and human coronavirus 229E. J Virol 79 6180 6193

83. YuSYHuYWLiuXYXiongWZhouZT 2005 Gene expression profiles in peripheral blood mononuclear cells of SARS patients. World J Gastroenterol 11 5037 5043

84. ShaoHLanDDuanZLiuZMinJ 2006 Upregulation of mitochondrial gene expression in PBMC from convalescent SARS patients. J Clin Immunol 26 546 554

85. ZhangLZhangZPZhangXELinFSGeF 2010 Quantitative proteomics analysis reveals BAG3 as a potential target to suppress severe acute respiratory syndrome coronavirus replication. J Virol 84 6050 6059

86. CallahanMKGargMSrivastavaPK 2008 Heat-shock protein 90 associates with N-terminal extended peptides and is required for direct and indirect antigen presentation. Proc Natl Acad Sci U S A 105 1662 1667

87. YangYXiongZZhangSYanYNguyenJ 2005 Bcl-xL inhibits T-cell apoptosis induced by expression of SARS coronavirus E protein in the absence of growth factors. Biochem J 392 135 143

88. AnSChenCJYuXLeibowitzJLMakinoS 1999 Induction of apoptosis in murine coronavirus-infected cultured cells and demonstration of E protein as an apoptosis inducer. J Virol 73 7853 7859

89. BruneW 2010 Inhibition of programmed cell death by cytomegaloviruses. Virus Res 157 144 150

90. ChenCYPingYHLeeHCChenKHLeeYM 2007 Open reading frame 8a of the human severe acute respiratory syndrome coronavirus not only promotes viral replication but also induces apoptosis. J Infect Dis 196 405 415

91. SchaecherSRTouchetteESchriewerJBullerRMPekoszA 2007 Severe acute respiratory syndrome coronavirus gene 7 products contribute to virus-induced apoptosis. J Virol 81 11054 11068

92. TerhuneSTorigoiEMoormanNSilvaMQianZ 2007 Human cytomegalovirus UL38 protein blocks apoptosis. J Virol 81 3109 3123

93. XuanBQianZTorigoiEYuD 2009 Human cytomegalovirus protein pUL38 induces ATF4 expression, inhibits persistent JNK phosphorylation, and suppresses endoplasmic reticulum stress-induced cell death. J Virol 83 3463 3474

94. ChenYVoegeliTSLiuPPNobleEGCurrieRW 2007 Heat shock paradox and a new role of heat shock proteins and their receptors as anti-inflammation targets. Inflamm Allergy Drug Targets 6 91 100

95. JohnsonBJLeTTDobbinCABanovicTHowardCB 2005 Heat shock protein 10 inhibits lipopolysaccharide-induced inflammatory mediator production. J Biol Chem 280 4037 4047

96. WangXLiuY 2007 Regulation of innate immune response by MAP kinase phosphatase-1. Cell Signal 19 1372 1382

97. HammerMMagesJDietrichHServatiusAHowellsN 2006 Dual specificity phosphatase 1 (DUSP1) regulates a subset of LPS-induced genes and protects mice from lethal endotoxin shock. J Exp Med 203 15 20

98. ZhaoQShepherdEGMansonMENelinLDSorokinA 2005 The role of mitogen-activated protein kinase phosphatase-1 in the response of alveolar macrophages to lipopolysaccharide: attenuation of proinflammatory cytokine biosynthesis via feedback control of p38. J Biol Chem 280 8101 8108

99. SalojinKVOwusuIBMillerchipKAPotterMPlattKA 2006 Essential role of MAPK phosphatase-1 in the negative control of innate immune responses. J Immunol 176 1899 1907

100. CameronMJRanLXuLDaneshABermejo-MartinJF 2007 Interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome. J Virol 81 8692 8706

101. AlmazanFDeDiegoMLGalanCEscorsDAlvarezE 2006 Construction of a SARS-CoV infectious cDNA clone and a replicon to study coronavirus RNA synthesis. J Virol 80 10900 10906

102. WettenhallJMSimpsonKMSatterleyKSmythGK 2006 affylmGUI: a graphical user interface for linear modeling of single channel microarray data. Bioinformatics 22 897 899

103. IrizarryRABolstadBMCollinFCopeLMHobbsB 2003 Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res 31 e15

104. BenjaminiYHochbergY 1995 Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Roy Stat Soc B 57 289 300

105. ReinerAYekutieliDBenjaminiY 2003 Identifying differentially expressed genes using false discovery rate controlling procedures. Bioinformatics 19 368 375

106. SubramanianATamayoPMoothaVKMukherjeeSEbertBL 2005 Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 102 15545 15550

107. LivakKJSchmittgenTD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods 25 402 408

108. MartinezILombardiaLHerranzCGarcia-BarrenoBDominguezO 2009 Cultures of HEp-2 cells persistently infected by human respiratory syncytial virus differ in chemokine expression and resistance to apoptosis as compared to lytic infections of the same cell type. Virology 388 31 41

109. MartinezILombardiaLGarcia-BarrenoBDominguezOMeleroJA 2007 Distinct gene subsets are induced at different time points after human respiratory syncytial virus infection of A549 cells. J Gen Virol 88 570 581

110. van EngelandMRamaekersFCSchutteBReutelingspergerCP 1996 A novel assay to measure loss of plasma membrane asymmetry during apoptosis of adherent cells in culture. Cytometry 24 131 139

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