title: Viral membrane fusion and nucleocapsid delivery into the cytoplasm are distinct events in some flaviviruses.
abstract: Flaviviruses deliver their genome into the cell by fusing the viral lipid membrane to an endosomal membrane. The sequence and kinetics of the steps required for nucleocapsid delivery into the cytoplasm remain unclear. Here we dissect the cell entry pathway of virions and virus-like particles from two flaviviruses using single-particle tracking in live cells, a biochemical membrane fusion assay and virus infectivity assays. We show that the virus particles fuse with a small endosomal compartment in which the nucleocapsid remains trapped for several minutes. Endosomal maturation inhibitors inhibit infectivity but not membrane fusion. We propose a flavivirus cell entry mechanism in which the virus particles fuse preferentially with small endosomal carrier vesicles and depend on back-fusion of the vesicles with the late endosomal membrane to deliver the nucleocapsid into the cytoplasm. Virus entry modulates intracellular calcium release and phosphatidylinositol-3-phosphate kinase signaling. Moreover, the broadly cross-reactive therapeutic antibody scFv11 binds to virus-like particles and inhibits fusion.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, United States of America.
authors.forename: Adel M
authors.initials: AM
authors.lastname: Nour
authors.forename: Yue
authors.initials: Y
authors.lastname: Li
authors.forename: Joseph
authors.initials: J
authors.lastname: Wolenski
authors.forename: Yorgo
authors.initials: Y
authors.lastname: Modis
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000235:genetics; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000821:virology*; D011992:Endosomes / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000821:virology; D005416:Flavivirus / Q000235:genetics / Q000378:metabolism*; D018177:Flavivirus Infections / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008561:Membrane Fusion; D019251:Nucleocapsid / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000494:pharmacology; D014709:Vero Cells; D053586:Virus Internalization
chemical_list: D000914:Antibodies, Viral; D019869:Phosphatidylinositol 3-Kinases; D057127:Single-Chain Antibodies
references.citation: J Virol. 2008 Jun;82(12):6024-33
references.pmid: 18400851
references.citation: Curr Top Microbiol Immunol. 2005;285:139-73
references.pmid: 15609503
references.citation: Nature. 2005 Sep 29;437(7059):764-9
references.pmid: 16193056
references.citation: J Virol. 2008 Jul;82(13):6470-80
references.pmid: 18448543
references.citation: J Virol. 2007 May;81(9):4881-5
references.pmid: 17301152
references.citation: Annu Rev Microbiol. 1990;44:649-88
references.pmid: 2174669
references.citation: J Virol. 2009 May;83(9):4338-44
references.pmid: 19244332
references.citation: EMBO J. 2002 Mar 15;21(6):1289-300
references.pmid: 11889035
references.citation: Nat Cell Biol. 2005 Jul;7(7):653-64
references.pmid: 15951806
references.citation: J Gen Virol. 2012 Jan;93(Pt 1):61-71
references.pmid: 21940409
references.citation: Virology. 1992 Jun;188(2):714-20
references.pmid: 1585642
references.citation: J Virol. 2011 Jan;85(1):422-31
references.pmid: 20980511
references.citation: Nat Cell Biol. 2005 Aug;7(8):808-16
references.pmid: 16025105
references.citation: Annu Rev Physiol. 2003;65:701-34
references.pmid: 12471163
references.citation: Cell. 1991 Mar 8;64(5):915-25
references.pmid: 1900457
references.citation: J Immunol. 2009 Jul 1;183(1):650-60
references.pmid: 19535627
references.citation: J Virol. 2005 Dec;79(23):14606-13
references.pmid: 16282460
references.citation: Mol Microbiol. 1997 Feb;23(3):445-57
references.pmid: 9044279
references.citation: J Biol Chem. 1996 Jul 19;271(29):17205-10
references.pmid: 8663431
references.citation: Annu Rev Cell Dev Biol. 2004;20:395-425
references.pmid: 15473846
references.citation: J Virol. 2003 Jul;77(14):7856-62
references.pmid: 12829825
references.citation: J Virol. 1997 Dec;71(12):9608-17
references.pmid: 9371625
references.citation: J Virol. 2004 Oct;78(19):10543-55
references.pmid: 15367621
references.citation: Cell Microbiol. 2009 Oct;11(10):1533-49
references.pmid: 19523154
references.citation: PLoS Pathog. 2008 Feb 8;4(2):e17
references.pmid: 18266465
references.citation: J Gastroenterol. 2010 Feb;45(2):195-203
references.pmid: 19760134
references.citation: Nat Protoc. 2008;3(4):619-28
references.pmid: 18388944
references.citation: Biochim Biophys Acta. 2011 Nov;1808(11):2638-45
references.pmid: 21810406
references.citation: J Virol. 2001 May;75(9):4040-7
references.pmid: 11287553
references.citation: Nature. 1998 Mar 12;392(6672):193-7
references.pmid: 9515966
references.citation: Trends Microbiol. 2003 Sep;11(9):438-44
references.pmid: 13678860
references.citation: J Gen Virol. 1990 Aug;71 ( Pt 8):1845-50
references.pmid: 2167941
references.citation: J Gen Virol. 2003 Jul;84(Pt 7):1737-1741
references.pmid: 12810867
references.citation: Annu Rev Biophys. 2010;39:407-27
references.pmid: 20192774
references.citation: Nat Cell Biol. 1999 Jun;1(2):113-8
references.pmid: 10559883
references.citation: PLoS Pathog. 2008 Aug 29;4(8):e1000141
references.pmid: 18769720
references.citation: J Virol. 2011 Nov;85(22):11567-80
references.pmid: 21917960
references.citation: J Cell Biol. 2004 Aug 30;166(5):645-51
references.pmid: 15337774
references.citation: J Biol Chem. 2011 May 20;286(20):17921-33
references.pmid: 21385879
references.citation: J Virol. 2010 Sep;84(17):8721-31
references.pmid: 20538858
references.citation: Virology. 2002 Jan 5;292(1):162-8
references.pmid: 11878919
references.citation: Biochim Biophys Acta. 2009 Apr;1793(4):674-83
references.pmid: 19014978
references.citation: Curr Biol. 1999 Jan 14;9(1):55-8
references.pmid: 9889123
references.citation: Nat Struct Mol Biol. 2008 Mar;15(3):312-7
references.pmid: 18264114
references.citation: Biol Chem. 2001 Mar;382(3):483-5
references.pmid: 11347897
references.citation: PLoS Pathog. 2010 Oct 07;6(10):e1001131
references.pmid: 20949067
references.citation: J Cell Biol. 2008 Oct 20;183(2):353-61
references.pmid: 18936253
references.citation: Nature. 2004 Jan 22;427(6972):313-9
references.pmid: 14737159
references.citation: J Neurochem. 2010 Oct;115(2):537-49
references.pmid: 20722967
references.citation: EMBO J. 2000 Sep 1;19(17):4577-88
references.pmid: 10970851
references.citation: Mol Cell. 2001 Mar;7(3):593-602
references.pmid: 11463384
references.citation: J Biol Chem. 2007 Jan 12;282(2):821-5
references.pmid: 17130120
references.citation: J Virol. 2004 Oct;78(20):10920-6
references.pmid: 15452212
references.citation: Microbes Infect. 2012 Feb;14(2):159-68
references.pmid: 21946213
references.citation: Vaccine. 2007 Nov 14;25(46):7885-92
references.pmid: 17949864
references.citation: J Virol. 2007 Oct;81(20):11526-31
references.pmid: 17670824
references.citation: J Biol Chem. 2000 Aug 11;275(32):24595-600
references.pmid: 10807926
references.citation: J Virol. 2013 Jan;87(1):52-66
references.pmid: 23077306
references.citation: J Gen Virol. 1986 Jan;67 ( Pt 1):157-66
references.pmid: 3944582
references.citation: Curr Opin Cell Biol. 1995 Aug;7(4):552-63
references.pmid: 7495576
references.citation: J Virol. 2010 Mar;84(6):2798-807
references.pmid: 20053738
references.citation: J Virol. 1998 Jun;72(6):4925-30
references.pmid: 9573260
references.citation: PLoS Pathog. 2008 Dec;4(12):e1000244
references.pmid: 19096510
references.citation: Nat Rev Mol Cell Biol. 2001 Oct;2(10):721-30
references.pmid: 11584299
references.citation: Nature. 1995 May 25;375(6529):291-8
references.pmid: 7753193
references.citation: Traffic. 2003 Apr;4(4):222-31
references.pmid: 12694561