title: Direct visualization of HIV-1 with correlative live-cell microscopy and cryo-electron tomography.
abstract: Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-native state and therefore has the potential to help elucidate early events of HIV-1 infection in host cells. However, structural details of infecting HIV-1 have not been observed, due to technological challenges in working with rare and dynamic HIV-1 particles in human cells. Here, we report structural analysis of HIV-1 and host-cell interactions by means of a correlative high-speed 3D live-cell-imaging and cryoET method. Using this method, we showed under near-native conditions that intact hyperstable mutant HIV-1 cores are released into the cytoplasm of host cells. We further obtained direct evidence to suggest that a hyperstable mutant capsid, E45A, showed delayed capsid disassembly compared to the wild-type capsid. Together, these results demonstrate the advantages of our correlative live-cell and cryoET approach for imaging dynamic processes, such as viral infection.
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA.
authors.forename: Sangmi
authors.initials: S
authors.lastname: Jun
authors.forename: Danxia
authors.initials: D
authors.lastname: Ke
authors.forename: Karl
authors.initials: K
authors.lastname: Debiec
authors.forename: Gongpu
authors.initials: G
authors.lastname: Zhao
authors.forename: Xin
authors.initials: X
authors.lastname: Meng
authors.forename: Zandrea
authors.initials: Z
authors.lastname: Ambrose
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Gibson
authors.forename: Simon C
authors.initials: SC
authors.lastname: Watkins
authors.forename: Peijun
authors.initials: P
authors.lastname: Zhang
mesh_terms: D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D020285:Cryoelectron Microscopy / Q000295:instrumentation* / Q000379:methods; D055032:Electron Microscope Tomography / Q000295:instrumentation* / Q000379:methods; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006367:HeLa Cells; D054884:Host-Pathogen Interactions; D054298:Human Immunodeficiency Virus Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020125:Mutation, Missense; D059010:Single-Cell Analysis / Q000379:methods; D014771:Virion / Q000378:metabolism
chemical_list: D036022:Capsid Proteins; D054298:Human Immunodeficiency Virus Proteins
references.citation: Proc Natl Acad Sci U S A. 2008 Jul 29;105(30):10559-64
references.pmid: 18653756
references.citation: Science. 2002 Apr 19;296(5567):503-7
references.pmid: 11964472
references.citation: PLoS Pathog. 2010 Dec 09;6(12):e1001220
references.pmid: 21170360
references.citation: EMBO J. 2007 Jun 20;26(12):3025-37
references.pmid: 17557080
references.citation: Curr Opin Struct Biol. 2008 Apr;18(2):203-17
references.pmid: 18406133
references.citation: Nat Cell Biol. 2010 Sep;12(9):909-15
references.pmid: 20694000
references.citation: J Struct Biol. 2007 Nov;160(2):135-45
references.pmid: 17884579
references.citation: J Virol. 1997 Jul;71(7):5382-90
references.pmid: 9188609
references.citation: Science. 2002 Nov 8;298(5596):1209-13
references.pmid: 12424373
references.citation: J Virol. 2004 Jul;78(13):7138-47
references.pmid: 15194790
references.citation: J Struct Biol. 2010 Nov;172(2):169-79
references.pmid: 20178848
references.citation: FASEB J. 2005 Jun;19(8):1039-41
references.pmid: 15788449
references.citation: Proc Natl Acad Sci U S A. 2009 Oct 13;106(41):17407-12
references.pmid: 19805028
references.citation: Proc Natl Acad Sci U S A. 2006 May 9;103(19):7465-70
references.pmid: 16648264
references.citation: Curr Opin Biotechnol. 2009 Feb;20(1):83-9
references.pmid: 19345086
references.citation: Retrovirology. 2010 Nov 17;7:96
references.pmid: 21083892
references.citation: EMBO J. 2003 Apr 1;22(7):1707-15
references.pmid: 12660176
references.citation: Nat Methods. 2007 Mar;4(3):215-7
references.pmid: 17277781
references.citation: Retrovirology. 2009 Sep 18;6:84
references.pmid: 19765276
references.citation: J Virol. 2002 Jun;76(11):5667-77
references.pmid: 11991995
references.citation: Structure. 2011 Feb 9;19(2):147-54
references.pmid: 21300284
references.citation: PLoS Pathog. 2010 Nov 24;6(11):e1001173
references.pmid: 21124872
references.citation: Structure. 2006 Jan;14(1):15-20
references.pmid: 16407061
references.citation: Proc Natl Acad Sci U S A. 2011 Jun 14;108(24):9975-80
references.pmid: 21628558
references.citation: Q Rev Biophys. 1988 May;21(2):129-228
references.pmid: 3043536
references.citation: Cell. 2009 May 1;137(3):433-44
references.pmid: 19410541
references.citation: Proc Natl Acad Sci U S A. 2005 Feb 22;102(8):2772-7
references.pmid: 15699328
references.citation: J Vis Exp. 2011 Jun 05;(52):
references.pmid: 21673645
references.citation: Virology. 2007 May 10;361(2):426-34
references.pmid: 17188319
references.citation: Proc Natl Acad Sci U S A. 2007 Mar 6;104(10):3777-81
references.pmid: 17360429
references.citation: Eur J Cell Biol. 2009 Nov;88(11):669-84
references.pmid: 19726102
references.citation: J Virol. 1997 Aug;71(8):5871-7
references.pmid: 9223476
references.citation: Nature. 2008 Jul 10;454(7201):236-40
references.pmid: 18500329
references.citation: Trends Biochem Sci. 2009 Feb;34(2):60-70
references.pmid: 19101147
references.citation: J Struct Biol. 2007 Oct;160(1):70-82
references.pmid: 17692533
references.citation: Virology. 1998 Nov 10;251(1):1-15
references.pmid: 9813197
references.citation: J Acquir Immune Defic Syndr (1988). 1990;3(10):965-74
references.pmid: 2398460
references.citation: J Cell Biol. 2002 Nov 11;159(3):441-52
references.pmid: 12417576
references.citation: Methods. 1997 Aug;12(4):337-42
references.pmid: 9245614
references.citation: Curr Opin HIV AIDS. 2006 May;1(3):194-9
references.pmid: 19372808
references.citation: PLoS One. 2007 May 09;2(5):e420
references.pmid: 17487274
references.citation: J Microsc. 2004 Oct;216(Pt 1):76-83
references.pmid: 15369487
references.citation: J Virol. 2004 Mar;78(5):2545-52
references.pmid: 14963157
references.citation: Nat Methods. 2006 Oct;3(10):817-24
references.pmid: 16990814
references.citation: EMBO J. 2007 Apr 18;26(8):2218-26
references.pmid: 17396149
references.citation: J Struct Biol. 1996 Jan-Feb;116(1):71-6
references.pmid: 8742726
references.citation: J Microsc. 2007 Aug;227(Pt 2):98-109
references.pmid: 17845705
references.citation: Science. 2003 Nov 21;302(5649):1396-8
references.pmid: 14631040