The Membrane Fusion Step of Vaccinia Virus Entry Is Cooperatively Mediated by Multiple Viral Proteins and Host Cell Components
For many viruses, one or two proteins allow cell attachment and entry, which occurs through the plasma membrane or following endocytosis at low pH. In contrast, vaccinia virus (VACV) enters cells by both neutral and low pH routes; four proteins mediate cell attachment and twelve that are associated in a membrane complex and conserved in all poxviruses are dedicated to entry. The aim of the present study was to determine the roles of cellular and viral proteins in initial stages of entry, specifically fusion of the membranes of the mature virion and cell. For analysis of the role of cellular components, we used well characterized inhibitors and measured binding of a recombinant VACV virion containing Gaussia luciferase fused to a core protein; viral and cellular membrane lipid mixing with a self-quenching fluorescent probe in the virion membrane; and core entry with a recombinant VACV expressing firefly luciferase and electron microscopy. We determined that inhibitors of tyrosine protein kinases, dynamin GTPase and actin dynamics had little effect on binding of virions to cells but impaired membrane fusion, whereas partial cholesterol depletion and inhibitors of endosomal acidification and membrane blebbing had a severe effect at the later stage of core entry. To determine the role of viral proteins, virions lacking individual membrane components were purified from cells infected with members of a panel of ten conditional-lethal inducible mutants. Each of the entry protein-deficient virions had severely reduced infectivity and except for A28, L1 and L5 greatly impaired membrane fusion. In addition, a potent neutralizing L1 monoclonal antibody blocked entry at a post-membrane lipid-mixing step. Taken together, these results suggested a 2-step entry model and implicated an unprecedented number of viral proteins and cellular components involved in signaling and actin rearrangement for initiation of virus-cell membrane fusion during poxvirus entry.
Vyšlo v časopise:
The Membrane Fusion Step of Vaccinia Virus Entry Is Cooperatively Mediated by Multiple Viral Proteins and Host Cell Components. PLoS Pathog 7(12): e32767. doi:10.1371/journal.ppat.1002446
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.ppat.1002446
Souhrn
For many viruses, one or two proteins allow cell attachment and entry, which occurs through the plasma membrane or following endocytosis at low pH. In contrast, vaccinia virus (VACV) enters cells by both neutral and low pH routes; four proteins mediate cell attachment and twelve that are associated in a membrane complex and conserved in all poxviruses are dedicated to entry. The aim of the present study was to determine the roles of cellular and viral proteins in initial stages of entry, specifically fusion of the membranes of the mature virion and cell. For analysis of the role of cellular components, we used well characterized inhibitors and measured binding of a recombinant VACV virion containing Gaussia luciferase fused to a core protein; viral and cellular membrane lipid mixing with a self-quenching fluorescent probe in the virion membrane; and core entry with a recombinant VACV expressing firefly luciferase and electron microscopy. We determined that inhibitors of tyrosine protein kinases, dynamin GTPase and actin dynamics had little effect on binding of virions to cells but impaired membrane fusion, whereas partial cholesterol depletion and inhibitors of endosomal acidification and membrane blebbing had a severe effect at the later stage of core entry. To determine the role of viral proteins, virions lacking individual membrane components were purified from cells infected with members of a panel of ten conditional-lethal inducible mutants. Each of the entry protein-deficient virions had severely reduced infectivity and except for A28, L1 and L5 greatly impaired membrane fusion. In addition, a potent neutralizing L1 monoclonal antibody blocked entry at a post-membrane lipid-mixing step. Taken together, these results suggested a 2-step entry model and implicated an unprecedented number of viral proteins and cellular components involved in signaling and actin rearrangement for initiation of virus-cell membrane fusion during poxvirus entry.
Zdroje
1. ChernomordikLVKozlovMM 2005 Membrane hemifusion: crossing a chasm in two leaps. Cell 123 375 382
2. WhiteJMDelosSEBrecherMSchornbergK 2008 Structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme. Crit Rev Biochem Mol Biol 43 189 219
3. HeldweinEEKrummenacherC 2008 Entry of herpesviruses into mammalian cells. Cell Mol Life Sci 65 1653 1668
4. ArmstrongJAMetzDHYoungMR 1973 The mode of entry of vaccinia virus into L cells. J Gen Virol 21 533 537
5. CarterGCLawMHollinsheadMSmithGL 2005 Entry of the vaccinia virus intracellular mature virion and its interactions with glycosaminoglycans. J Gen Virol 86 1279 1290
6. TownsleyACWeisbergASWagenaarTRMossB 2006 Vaccinia virus entry into cells via a low pH-dependent-endosomal pathway. J Virol 80 8899 8908
7. BengaliZTownsleyACMossB 2009 Vaccinia virus strain differences in cell attachment and entry. Virology 389 132 140
8. WhitbeckJCFooC-HPonce de LeonMEisenbergRJCohenGH 2009 Vaccinia virus exhibits cell-type-dependent entry characteristics. Virology 385 383 391
9. BengaliZSatheshkumarPSYangZWeisbergASParanN 2011 Drosophila S2 cells are non-permissive for vaccinia virus DNA replication following entry via low pH-dependent endocytosis and early transcription. PLoS One 6 e17248
10. MossB 2006 Poxvirus entry and membrane fusion. Virology 344 48 54
11. MercerJHeleniusA 2008 Vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells. Science 320 531 535
12. MercerJKnebelSSchmidtFICrouseJBurkardC 2010 Vaccinia virus strains use distinct forms of macropinocytosis for host-cell entry. Proc Natl Acad Sci U S A 107 9346 9351
13. MoserTSJonesRGThompsonCBCoyneCBCherryS 2010 A kinome RNAi screen identified AMPK as promoting poxvirus entry through the control of actin dynamics. PLoS Pathog 6 e1000954
14. SandgrenKJWilkinsonJMiranda-SaksenaMMcInerneyGMByth-WilsonK 2010 A differential role for macropinocytosis in mediating entry of the two forms of vaccinia virus into dendritic cells. PLoS Pathog 6 e1000866
15. VillaNYBarteeEMohamedMRRahmanMMBarrettJW 2010 Myxoma and vaccinia viruses exploit different mechanisms to enter and infect human cancer cells. Virology 401 266 279
16. HuangCYLuTYBairCHChangYSJwoJK 2008 A novel cellular protein, VPEF, facilitates vaccinia virus penetration into HeLa cells through fluid phase endocytosis. J Virol 82 7988 7999
17. LawMCarterGCRobertsKLHollinsheadMSmithGL 2006 Ligand-induced and non-fusogenic dissolution of a viral membrane. Proc Natl Acad Sci USA 103 5989 5994
18. IchihashiY 1996 Extracellular enveloped vaccinia virus escapes neutralization. Virology 217 478 485
19. ChungC-SHsiaoJ-CChangY-SChangW 1998 A27L protein mediates vaccinia virus interaction with cell surface heparin sulfate. J Virol 72 1577 1585
20. HsiaoJCChungCSChangW 1998 Cell surface proteoglycans are necessary for A27L protein- mediated cell fusion: Identification of the N-terminal region of A27L protein as the glycosaminoglycan-binding domain. J Virol 72 8374 8379
21. LinCLChungCSHeineHGChangW 2000 Vaccinia virus envelope H3L protein binds to cell surface heparan sulfate and is important for intracellular mature virion morphogenesis and virus infection in vitro and in vivo. J Virol 74 3353 3365
22. ChiuWLLinCLYangMHTzouDLMChangW 2007 Vaccinia virus 4c (A26L) protein on intracellular mature virus binds to the extracellular cellular matrix laminin. J Virol 81 2149 2157
23. OjedaSSenkevichTGMossB 2006 Entry of vaccinia virus and cell-cell fusion require a highly conserved cysteine-rich membrane protein encoded by the A16L gene. J Virol 80 51 61
24. TownsleyASenkevichTGMossB 2005 Vaccinia virus A21 virion membrane protein is required for cell entry and fusion. J Virol 79 9458 9469
25. SenkevichTGWardBMMossB 2004 Vaccinia virus A28L gene encodes an essential protein component of the virion membrane with intramolecular disulfide bonds formed by the viral cytoplasmic redox pathway. J Virol 78 2348 2356
26. BrownESenkevichTGMossB 2006 Vaccinia virus F9 virion membrane protein is required for entry but not virus assembly, in contrast to the related l1 protein. J Virol 80 9455 9464
27. IzmailyanRAHuangCYMohammadSIsaacsSNChangW 2006 The envelope G3L protein is essential for entry of vaccinia virus into host cells. J Virol 80 8402 8410
28. OjedaSDomiAMossB 2006 Vaccinia virus G9 protein is an essential component of the poxvirus entry-fusion complex. J Virol 80 9822 9830
29. SenkevichTGMossB 2005 Vaccinia virus H2 protein is an essential component of a complex involved in virus entry and cell-cell fusion. J Virol 79 4744 4754
30. NicholsRJStanitsaEUngerBTraktmanP 2008 The vaccinia I2L gene encodes a membrane protein with an essential role in virion entry. J Virol 82 10247 10261
31. SenkevichTGOjedaSTownsleyANelsonGEMossB 2005 Poxvirus multiprotein entry-fusion complex. Proc Natl Acad Sci USA 102 18572 18577
32. BishtHWeisbergASMossB 2008 Vaccinia virus L1 protein is required for cell entry and membrane fusion. J Virol 82 8687 8694
33. SatheshkumarPSMossB 2009 Characterization of a newly Identified 35 amino acid component of the vaccinia virus entry/fusion complex conserved in all chordopoxviruses. J Virol 83 12822 12832
34. TownsleyASenkevichTGMossB 2005 The product of the vaccinia virus L5R gene is a fourth membrane protein encoded by all poxviruses that is requried for cell entry and cell-cell fusion. J Virol 79 10988 10998
35. ConditRCMoussatcheNTraktmanP 2006 In a nutshell: structure and assembly of the vaccinia virion. Adv Virus Res 66 31 124
36. NelsonGEWagenaarTRMossB 2008 A conserved sequence within the H2 subunit of the vaccinia virus entry/fusion complex is Important for interaction with the A28 subunit and infectivity. J Virol 82 6244 6250
37. WolfeCLMossB 2011 Interaction between the G3 and L5 proteins of the vaccinia virus entry-fusion complex. Virology 412 278 283
38. WagenaarTROjedaSMossB 2008 Vaccinia virus A56/K2 fusion regulatory protein interacts with the A16 and G9 subunits of the entry fusion complex. J Virol 82 5153 5160
39. LoyterACitovskyVBlumenthalR 1988 The use of fluorescence dequenching measurements to follow viral membrane fusion events. Methods Biochem Anal 33 129 164
40. DomsRWBlumenthalRMossB 1990 Fusion of intra- and extracellular forms of vaccinia virus with the cell membrane. J Virol 64 4884 4892
41. ChungCSHuangCYChangW 2005 Vaccinia virus penetration requires cholesterol and results in specific viral envelope proteins associated with lipid rafts. J Virol 79 1623 1634
42. LockerJKKuehnASchleichSRutterGHohenbergH 2000 Entry of the two infectious forms of vaccinia virus at the plasma membrane is signaling-dependent for the IMV but not the EEV. Mol Biol Cell 11 2497 2511
43. LimouzeJStraightAFMitchisonTSellersJR 2004 Specificity of blebbistatin, an inhibitor of myosin II. J Muscle Res Cell Motil 25 337 341
44. LehmannMJShererNMMarksCBPypaertMMothesW 2005 Actin- and myosin-driven movement of viruses along filopodia precedes their entry into cells. J Cell Biol 170 317 325
45. MaciaEEhrlichMMassolRBoucrotEBrunnerC 2006 Dynasore, a cell-permeable inhibitor of dynamin. Dev Cell 10 839 850
46. GuCYaddanapudiSWeinsAOsbornTReiserJ 2010 Direct dynamin-actin interactions regulate the actin cytoskeleton. EMBO J 29 3593 3606
47. KruchtenAEMcNivenMA 2006 Dynamin as a mover and pincher during cell migration and invasion. J Cell Sci 119 1683 1690
48. PraefckeGJMcMahonHT 2004 The dynamin superfamily: universal membrane tubulation and fission molecules? Nat Rev Mol Cell Biol 5 133 147
49. BrownSSSpudichJA 1981 Mechanism of action of cytochalasin: evidence that it binds to actin filament ends. J Cell Biol 88 487 491
50. YarmolaEGSomasundaramTBoringTASpectorIBubbMR 2000 Actin-latrunculin A structure and function. Differential modulation of actin-binding protein function by latrunculin A. J Biol Chem 275 28120 28127
51. AkiyamaTIshidaJNakagawaSOgawaraHWatanabeS 1987 Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem 262 5592 5595
52. FryDWKrakerAJMcMichaelAAmbrosoLANelsonJM 1994 A specific inhibitor of the epidermal growth factor receptor tyrosine kinase. Science 265 1093 1095
53. RijkenPJHageWJvan Bergen en HenegouwenPMVerkleijAJBoonstraJ 1991 Epidermal growth factor induces rapid reorganization of the actin microfilament system in human A431 cells. J Cell Sci 100 Pt 3 491 499
54. SoodCLWardJMMossB 2008 Vaccinia virus encodes a small hydrophobic virion membrane protein (I5) that enhances replication and virulence in mice. J Virol 82 10071 10078
55. DalesSKajiokaR 1964 The cycle of multiplication of vaccinia virus in Earle's strain L cells. I. Uptake and penetration. Virology 24 278 294
56. WolffeEJVijayaSMossB 1995 A myristylated membrane protein encoded by the vaccinia virus L1R open reading frame is the target of potent neutralizing monoclonal antibodies. Virology 211 53 63
57. NelsonGESislerJRChandranDMossB 2008 Vaccinia virus entry/fusion complex subunit A28 is a target of neutralizing and protective antibodies. Virology 380 394 401
58. KembleGWDanieliTWhiteJM 1994 Lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion. Cell 76 383 391
59. ArmstrongRTKushnirASWhiteJM 2000 The transmembrane domain of influenza hemagglutinin exhibits a stringent length requirement to support the hemifusion to fusion transition. J Cell Biol 151 425 437
60. ZavorotinskayaTQianZFranksJAlbrittonLM 2004 A point mutation in the binding subunit of a retroviral envelope protein arrests virus entry at hemifusion. J Virol 78 473 481
61. ZaitsevaEMittalAGriffinDEChernomordikLV 2005 Class II fusion protein of alphaviruses drives membrane fusion through the same pathway as class I proteins. J Cell Biol 169 167 177
62. ZhengQAChangDC 1991 Reorganization of cytoplasmic structures during cell fusion. J Cell Sci 100 Pt 3 431 442
63. EitzenG 2003 Actin remodeling to facilitate membrane fusion. Biochim Biophys Acta 1641 175 181
64. MassarwaRCarmonSShiloBZSchejterED 2007 WIP/WASp-based actin-polymerization machinery is essential for myoblast fusion in Drosophila. Dev Cell 12 557 569
65. KallewaardNLBowenALCroweJEJr 2005 Cooperativity of actin and microtubule elements during replication of respiratory syncytial virus. Virology 331 73 81
66. GowerTLPasteyMKPeeplesMECollinsPLMcCurdyLH 2005 RhoA signaling is required for respiratory syncytial virus-induced syncytium formation and filamentous virion morphology. J Virol 79 5326 5336
67. PontowSEHeydenNVWeiSRatnerL 2004 Actin cytoskeletal reorganizations and coreceptor-mediated activation of rac during human immunodeficiency virus-induced cell fusion. J Virol 78 7138 7147
68. SchowalterRMWurthMAAguilarHCLeeBMoncmanCL 2006 Rho GTPase activity modulates paramyxovirus fusion protein-mediated cell-cell fusion. Virology 350 323 334
69. StantchevTSMarkovicITelfordWGClouseKABroderCC 2007 The tyrosine kinase inhibitor genistein blocks HIV-1 infection in primary human macrophages. Virus Res 123 178 189
70. HarmonBRatnerL 2008 Induction of the Galpha(q) signaling cascade by the human immunodeficiency virus envelope is required for virus entry. J Virol 82 9191 9205
71. HarmonBCampbellNRatnerL 2010 Role of Abl kinase and the Wave2 signaling complex in HIV-1 entry at a post-hemifusion step. PLoS Pathog 6 e1000956
72. MiyauchiKKimYLatinovicOMorozovVMelikyanGB 2009 HIV enters cells via endocytosis and dynamin-dependent fusion with endosomes. Cell 137 433 444
73. PhalenTKielianM 1991 Cholesterol is required for infection by Semliki Forest virus. J Cell Biol 112 615 623
74. BiswasSYinSRBlankPSZimmerbergJ 2008 Cholesterol promotes hemifusion and pore widening in membrane fusion induced by influenza hemagglutinin. J Gen Physiol 131 503 513
75. LustigSFoggCWhitbeckJCEisenbergRJCohenGH 2005 Combinations of polyclonal or monoclonal antibodies to proteins of the outer membranes of the two infectious forms of vaccinia virus protect mice against a lethal respiratory challenge. J Virol 79 13454 13462
76. SuHPGarmanSCAllisonTJFoggCMossB 2005 The 1.51-A structure of the poxvirus L1 protein, a target of potent neutralizing antibodies. Proc Natl Acad Sci USA 102 4240 4245
77. SuHPGoldenJWGittisAGHooperJWGarbocziDN 2007 Structural basis for the binding of the neutralizing antibody, 7D11, to the poxvirus L1 protein. Virology 368 331 341
78. EarlPLMossB 1998 Characterization of recombinant vaccinia viruses and their products. AusubelFMBrentRKingstonREMooreDDSeidmanJG Current Protocols in Molecular Biology New York Greene Publishing Associates & Wiley Interscience 16.18.11 16.18.11
79. EarlPLCooperNWyattLSMossBCarrollMW 1998 Preparation of cell cultures and vaccinia virus stocks. AusubelFMBrentRKingstonREMooreDDSeidmanJG Current Protocols in Molecular Biology New York John Wiley and Sons 16.16.11 16.16.13
80. EarlPLMossBWyattLSCarrollMW 1998 Generation of recombinant vaccinia viruses. AusubelFMBrentRKingstonREMooreDDSeidmanJG Current Protocols in Molecular Biology New York Greene Publishing Associates & Wiley Interscience 16.17.11 16.17.19
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