title: Characterization of a novel type of HIV-1 particle assembly inhibitor using a quantitative luciferase-Vpr packaging-based assay.
abstract: The HIV-1 auxiliary protein Vpr and Vpr-fusion proteins can be copackaged with Gag precursor (Pr55Gag) into virions or membrane-enveloped virus-like particles (VLP). Taking advantage of this property, we developed a simple and sensitive method to evaluate potential inhibitors of HIV-1 assembly in a living cell system. Two proteins were coexpressed in recombinant baculovirus-infected Sf9 cells, Pr55Gag, which formed the VLP backbone, and luciferase fused to the N-terminus of Vpr (LucVpr). VLP-encapsidated LucVpr retained the enzymatic activity of free luciferase. The levels of luciferase activity present in the pelletable fraction recovered from the culture medium correlated with the amounts of extracellular VLP released by Sf9 cells assayed by conventional immunological methods. Our luciferase-based assay was then applied to the characterization of betulinic acid (BA) derivatives that differed from the leader compound PA-457 (or DSB) by their substituant on carbon-28. The beta-alanine-conjugated and lysine-conjugated DSB could not be evaluated for their antiviral potentials due to their high cytotoxicity, whereas two other compounds with a lesser cytotoxicity, glycine-conjugated and ε-NH-Boc-lysine-conjugated DSB, exerted a dose-dependent negative effect on VLP assembly and budding. A fifth compound with a low cytotoxicity, EP-39 (ethylene diamine-conjugated DSB), showed a novel type of antiviral effect. EP-39 provoked an aberrant assembly of VLP, resulting in nonenveloped, morula-like particles of 100-nm in diameter. Each morula was composed of nanoparticle subunits of 20-nm in diameter, which possibly mimicked transient intermediates of the HIV-1 Gag assembly process. Chemical cross-linking in situ suggested that EP-39 favored the formation or/and persistence of Pr55Gag trimers over other oligomeric species. EP-39 showed a novel type of negative effect on HIV-1 assembly, targeting the Pr55Gag oligomerisation. The biological effect of EP-39 underlined the critical role of the nature of the side chain at position 28 of BA derivatives in their anti-HIV-1 activity.
authors.affiliation: Université Lyon I & INRA UMR-754, Retrovirus & Comparative Pathology, Lyon, France.
authors.forename: Gaëlle
authors.initials: G
authors.lastname: Gonzalez
authors.forename: Sandrina
authors.initials: S
authors.lastname: DaFonseca
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Errazuriz
authors.forename: Pascale
authors.initials: P
authors.lastname: Coric
authors.forename: Florence
authors.initials: F
authors.lastname: Souquet
authors.forename: Serge
authors.initials: S
authors.lastname: Turcaud
authors.forename: Pierre
authors.initials: P
authors.lastname: Boulanger
authors.forename: Serge
authors.initials: S
authors.lastname: Bouaziz
authors.forename: Saw See
authors.initials: SS
authors.lastname: Hong
mesh_terms: D000818:Animals; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D015497:HIV-1 / Q000502:physiology*; D008854:Microscopy, Electron; D018411:Spodoptera; D014771:Virion / Q000502:physiology* / Q000648:ultrastructure; D054325:vpr Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
chemical_list: D054325:vpr Gene Products, Human Immunodeficiency Virus; C514912:vpr protein, Human immunodeficiency virus 1
references.citation: J Virol. 1995 Nov;69(11):7032-44
references.pmid: 7474123
references.citation: J Virol. 1993 Nov;67(11):6542-50
references.pmid: 8411357
references.citation: Virology. 1999 Mar 1;255(1):20-5
references.pmid: 10049817
references.citation: J Gen Virol. 1998 May;79 ( Pt 5):1069-81
references.pmid: 9603321
references.citation: Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3794-8
references.pmid: 7731985
references.citation: Virology. 1991 Sep;184(1):417-22
references.pmid: 1871977
references.citation: PLoS Pathog. 2010 Nov 24;6(11):e1001215
references.pmid: 21151640
references.citation: Rev Med Virol. 2004 Mar-Apr;14(2):107-21
references.pmid: 15027003
references.citation: Adv Pharmacol. 2007;55:347-87
references.pmid: 17586320
references.citation: J Virol. 1993 Dec;67(12):7229-37
references.pmid: 8230445
references.citation: J Virol. 1995 Feb;69(2):1093-8
references.pmid: 7815488
references.citation: J Virol. 2003 Apr;77(8):4960-4
references.pmid: 12663801
references.citation: Antiviral Res. 2008 Dec;80(3):251-65
references.pmid: 18789977
references.citation: J Biol Chem. 1991 Feb 15;266(5):2795-804
references.pmid: 1847135
references.citation: Curr Pharm Des. 2004;10(30):3725-39
references.pmid: 15579067
references.citation: J Virol. 1994 Mar;68(3):1926-34
references.pmid: 8107252
references.citation: Trends Mol Med. 2005 Jan;11(1):31-6
references.pmid: 15649820
references.citation: J Med Chem. 1996 Mar 1;39(5):1069-83
references.pmid: 8676342
references.citation: Antiviral Res. 2010 Jan;85(1):119-41
references.pmid: 19782103
references.citation: Bioorg Med Chem Lett. 1999 Apr 19;9(8):1201-4
references.pmid: 10328313
references.citation: J Virol. 2001 Nov;75(21):10537-42
references.pmid: 11581428
references.citation: Virology. 2006 Dec 5-20;356(1-2):217-24
references.pmid: 16930665
references.citation: Retrovirology. 2005 Feb 22;2:11
references.pmid: 15725353
references.citation: J Virol. 2006 Nov;80(22):10957-71
references.pmid: 16956950
references.citation: Virol J. 2008 Dec 23;5:162
references.pmid: 19105849
references.citation: J Biol Chem. 1999 Jan 15;274(3):1635-45
references.pmid: 9880543
references.citation: Proc Natl Acad Sci U S A. 2005 Oct 25;102(43):15453-8
references.pmid: 16230638
references.citation: J Virol. 1998 Mar;72(3):2072-8
references.pmid: 9499062
references.citation: J Biol Chem. 1999 Mar 26;274(13):9083-91
references.pmid: 10085158
references.citation: Nat Struct Mol Biol. 2005 Aug;12(8):671-7
references.pmid: 16041387
references.citation: J Virol. 1995 Nov;69(11):6873-9
references.pmid: 7474102
references.citation: J Virol. 1990 Jun;64(6):3097-9
references.pmid: 2139896
references.citation: Protein Sci. 2005 Feb;14(2):375-86
references.pmid: 15659370
references.citation: Virology. 1994 May 1;200(2):524-34
references.pmid: 8178440
references.citation: J Immunol Methods. 1983 Dec 16;65(1-2):55-63
references.pmid: 6606682
references.citation: Bioorg Med Chem Lett. 2011 Jan 1;21(1):542-5
references.pmid: 21084190
references.citation: J Virol. 1998 Jul;72(7):5441-8
references.pmid: 9620999
references.citation: J Virol. 2000 Oct;74(20):9727-31
references.pmid: 11000245
references.citation: J Virol. 1993 May;67(5):2787-98
references.pmid: 8474175
references.citation: J Mol Biol. 2006 Jan 6;355(1):157-68
references.pmid: 16289202
references.citation: J Virol. 2011 Jan;85(2):753-64
references.pmid: 21068235
references.citation: EMBO J. 1997 May 1;16(9):2294-306
references.pmid: 9171344
references.citation: Cell. 1989 Oct 6;59(1):103-12
references.pmid: 2676191
references.citation: J Virol. 2006 Jun;80(12):5716-22
references.pmid: 16731910
references.citation: J Virol. 1999 Jan;73(1):592-600
references.pmid: 9847364
references.citation: J Virol. 2000 Nov;74(22):10650-7
references.pmid: 11044109
references.citation: J Virol. 1992 May;66(5):3230-5
references.pmid: 1560545
references.citation: Nat Biotechnol. 1997 Jan;15(1):52-6
references.pmid: 9035106
references.citation: J Virol. 1995 Jun;69(6):3389-98
references.pmid: 7745685
references.citation: Antimicrob Agents Chemother. 2001 Apr;45(4):1225-30
references.pmid: 11257038
references.citation: J Virol. 2009 May;83(10):4884-94
references.pmid: 19279107
references.citation: Virology. 1998 Aug 1;247(2):160-9
references.pmid: 9705909
references.citation: J Biol Chem. 2008 Feb 1;283(5):2518-25
references.pmid: 18057006
references.citation: J Virol. 1994 Jan;68(1):111-22
references.pmid: 8254720
references.citation: Antiviral Res. 2009 Jan;81(1):1-5
references.pmid: 18948140
references.citation: Microbes Infect. 2006 Jun;8(7):1875-81
references.pmid: 16807037
references.citation: J Virol. 2001 Jan;75(2):759-71
references.pmid: 11134289
references.citation: J Med Chem. 1996 Mar 1;39(5):1016-7
references.pmid: 8676334
references.citation: Nat Struct Mol Biol. 2004 Jul;11(7):672-5
references.pmid: 15208690
references.citation: J Virol. 2007 Sep;81(17):9572-6
references.pmid: 17553868
references.citation: J Virol. 2011 Feb;85(4):1420-8
references.pmid: 21106735
references.citation: Virology. 2004 Jul 1;324(2):525-30
references.pmid: 15207637
references.citation: Gene. 1989 Jun 30;79(1):71-81
references.pmid: 2673935
references.citation: J Mol Biol. 2003 Apr 11;327(5):1013-20
references.pmid: 12662926
references.citation: J Virol. 2005 Dec;79(23):14498-506
references.pmid: 16282449
references.citation: J Gen Virol. 1997 Jan;78 ( Pt 1):131-42
references.pmid: 9010296
references.citation: J Virol. 1999 Dec;73(12):9992-9
references.pmid: 10559313
references.citation: J Virol. 2010 Feb;84(3):1585-96
references.pmid: 19923179
references.citation: J Virol. 1995 Apr;69(4):2366-77
references.pmid: 7884882
references.citation: Nat Biotechnol. 2002 Nov;20(11):1151-4
references.pmid: 12355096
references.citation: Virology. 2008 Feb 5;371(1):155-64
references.pmid: 17950396
references.citation: Cell Mol Life Sci. 2002 Jul;59(7):1166-84
references.pmid: 12222963
references.citation: J Virol. 2004 Jan;78(2):922-9
references.pmid: 14694123
references.citation: Antivir Ther. 2007;12(8):1185-203
references.pmid: 18240859
references.citation: Retrovirology. 2004 Jun 29;1:15
references.pmid: 15225375
references.citation: Nat Struct Mol Biol. 2005 Aug;12(8):678-82
references.pmid: 16041386
references.citation: Retrovirology. 2004 Aug 16;1:21
references.pmid: 15312229
references.citation: J Virol. 1997 Nov;71(11):8230-6
references.pmid: 9343174
references.citation: J Virol. 1995 Jan;69(1):365-75
references.pmid: 7983731
references.citation: Biochimie. 2009 Jul;91(7):916-23
references.pmid: 19401213
references.citation: EMBO J. 2004 Jul 7;23(13):2632-40
references.pmid: 15201866
references.citation: Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3564-8
references.pmid: 8170948
references.citation: Gene Ther. 1999 Sep;6(9):1590-9
references.pmid: 10490769
references.citation: Proc Natl Acad Sci U S A. 2003 Nov 11;100(23):13555-60
references.pmid: 14573704
references.citation: J Virol. 1994 Apr;68(4):2556-69
references.pmid: 8139035
references.citation: Proc Natl Acad Sci U S A. 2001 Apr 24;98(9):5246-51
references.pmid: 11320254
references.citation: J Mol Biol. 2003 Mar 14;327(1):215-27
references.pmid: 12614620