title: An intrabody based on a llama single-domain antibody targeting the N-terminal alpha-helical multimerization domain of HIV-1 rev prevents viral production.
abstract: The human immunodeficiency virus, type 1 (HIV-1)-encoded Rev protein is essential for the expression of late viral mRNAs. Rev forms a large organized multimeric protein-protein complex on the Rev response element of these viral mRNA species and transports them from the nucleus to the cytoplasm, exploiting the CRM1-mediated cellular machinery. Here we report the selection of a nanobody, derived from a llama heavy-chain only antibody, that efficiently blocks the assembly of Rev multimers. The nanobody inhibits HIV-1 replication in cells and specifically suppresses the Rev-dependent expression of partially spliced and unspliced HIV-1 RNA. In HIV-susceptible cells, this nanobody thus has potential as an effective anti-HIV agent using genetic immunization strategies. Its binding site was mapped to Rev residues Lys-20 and Tyr-23 located in the N-terminal alpha-helical multimerization domain. In the presence of this nanobody, we observed an accumulation of dimeric Rev species, supporting a head-to-head/tail-to-tail molecular model for Rev assembly. The results indicate that the oligomeric assembly of Rev follows an ordered stepwise process and identify a new epitope within Rev that could guide strategies for the development of novel HIV inhibitors.
authors.affiliation: Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.
authors.forename: Thomas
authors.initials: T
authors.lastname: Vercruysse
authors.forename: Els
authors.initials: E
authors.lastname: Pardon
authors.forename: Els
authors.initials: E
authors.lastname: Vanstreels
authors.forename: Jan
authors.initials: J
authors.lastname: Steyaert
authors.forename: Dirk
authors.initials: D
authors.lastname: Daelemans
mesh_terms: D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D019380:Anti-HIV Agents / Q000494:pharmacology; D000906:Antibodies / Q000737:chemistry; D002161:Camelids, New World; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D015694:Gene Products, rev / Q000737:chemistry* / Q000378:metabolism; D015658:HIV Infections / Q000188:drug therapy*; D015497:HIV-1 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D025941:Protein Interaction Mapping; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014443:Tyrosine / Q000737:chemistry
chemical_list: D019380:Anti-HIV Agents; D000906:Antibodies; D015694:Gene Products, rev; D014443:Tyrosine; D008239:Lysine
references.citation: Biophys J. 2004 Jun;86(6):3993-4003
references.pmid: 15189895
references.citation: Proc Natl Acad Sci U S A. 2009 Feb 3;106(5):1404-8
references.pmid: 19164515
references.citation: Nature. 1985 Jul 18-24;316(6025):262-5
references.pmid: 2410792
references.citation: Trends Biochem Sci. 2001 Apr;26(4):230-5
references.pmid: 11295555
references.citation: Nature. 1993 Jun 3;363(6428):446-8
references.pmid: 8502296
references.citation: Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9870-4
references.pmid: 1409715
references.citation: Mol Cell Biol. 1994 Nov;14(11):7436-44
references.pmid: 7935458
references.citation: Virology. 2004 Dec 20;330(2):471-80
references.pmid: 15567440
references.citation: Cell. 1991 Apr 19;65(2):241-8
references.pmid: 2015625
references.citation: RNA. 2005 Dec;11(12):1848-57
references.pmid: 16314457
references.citation: Science. 2005 Nov 11;310(5750):1022-5
references.pmid: 16284179
references.citation: Science. 1989 Dec 22;246(4937):1625-9
references.pmid: 2688093
references.citation: J Virol. 1989 Mar;63(3):1265-74
references.pmid: 2783738
references.citation: Mol Cell. 2000 Nov;6(5):1067-76
references.pmid: 11106746
references.citation: J Virol. 1998 Apr;72(4):2935-44
references.pmid: 9525614
references.citation: Cell. 1986 May 9;45(3):375-85
references.pmid: 2421920
references.citation: J Mol Biol. 2001 Nov 2;313(4):845-59
references.pmid: 11697908
references.citation: Proc Natl Acad Sci U S A. 1994 May 24;91(11):5075-9
references.pmid: 8197188
references.citation: Mol Cell. 2001 Mar;7(3):603-14
references.pmid: 11463385
references.citation: J Virol. 1989 Sep;63(9):3708-13
references.pmid: 2760980
references.citation: Annu Rev Microbiol. 1998;52:491-532
references.pmid: 9891806
references.citation: Mol Cell Biol. 2005 Jan;25(2):728-39
references.pmid: 15632073
references.citation: J Virol. 1990 Feb;64(2):881-5
references.pmid: 2404140
references.citation: Cell. 1993 Jun 4;73(5):1031-40
references.pmid: 7684657
references.citation: Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1256-60
references.pmid: 8108397
references.citation: Oncogene. 1998 Apr 9;16(14):1851-61
references.pmid: 9583682
references.citation: Mol Cell. 2008 Sep 26;31(6):824-34
references.pmid: 18922466
references.citation: Curr Biol. 1997 Oct 1;7(10):767-75
references.pmid: 9368759
references.citation: J Mol Biol. 1994 Aug 12;241(2):193-207
references.pmid: 8057359
references.citation: Cell. 1995 Aug 11;82(3):475-83
references.pmid: 7543368
references.citation: J Struct Biol. 1998 Jan;121(1):41-52
references.pmid: 9573619
references.citation: Bioorg Med Chem. 2008 Nov 1;16(21):9487-97
references.pmid: 18835718
references.citation: J Virol. 1994 Dec;68(12):7944-52
references.pmid: 7966585
references.citation: J Clin Microbiol. 2008 Jul;46(7):2206-11
references.pmid: 18463204
references.citation: Virology. 1994 Jul;202(1):186-94
references.pmid: 7516596
references.citation: Methods Cell Biol. 2008;85:329-51
references.pmid: 18155469
references.citation: AIDS Res Hum Retroviruses. 1994 Apr;10(4):343-50
references.pmid: 8068414
references.citation: J Biol Chem. 2004 Nov 26;279(48):50167-75
references.pmid: 15294891
references.citation: Cell. 1997 Sep 19;90(6):1051-60
references.pmid: 9323133
references.citation: J Virol. 1990 Nov;64(11):5360-6
references.pmid: 2120472
references.citation: Trends Pharmacol Sci. 2004 Jul;25(7):343-6
references.pmid: 15219971
references.citation: Proc Natl Acad Sci U S A. 2002 Oct 29;99(22):14440-5
references.pmid: 12374846
references.citation: Nature. 1997 Nov 20;390(6657):308-11
references.pmid: 9384386
references.citation: Chem Biol. 1997 Feb;4(2):139-47
references.pmid: 9190288
references.citation: J Virol. 1992 Mar;66(3):1809-13
references.pmid: 1738210
references.citation: Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):758-62
references.pmid: 1731351
references.citation: Cell. 1989 Jul 14;58(1):205-14
references.pmid: 2752419
references.citation: Antimicrob Agents Chemother. 2001 Oct;45(10):2807-12
references.pmid: 11557473
references.citation: Cell. 2004 Oct 29;119(3):381-92
references.pmid: 15507209
references.citation: Biochemistry. 1994 Mar 15;33(10):2988-96
references.pmid: 7510518
references.citation: Proc Natl Acad Sci U S A. 1989 Mar;86(5):1495-9
references.pmid: 2784208
references.citation: Nature. 1989 Dec 14;342(6251):816-9
references.pmid: 2481237
references.citation: Nature. 1989 Mar 16;338(6212):254-7
references.pmid: 2784194
references.citation: J Struct Biol. 2009 Apr;166(1):8-15
references.pmid: 19118632
references.citation: FEBS Lett. 1997 Sep 15;414(3):521-6
references.pmid: 9323027
references.citation: Genes Dev. 1994 Jul 1;8(13):1538-47
references.pmid: 7958838