HIV-1-based lentiviral vectors are a promising tool for gene therapy. However, integration of a lentiviral vector into host cell genes may lead to the development of cancer. Therefore, control of integration site selection is critical to the successful outcome of gene therapy approaches that use these vectors. The discovery that integration site selection by HIV-1 and HIV-1-based vectors is controlled by the LEDGF/p75 protein has presented new opportunities to control integration site selection. In this study, we tested the hypothesis that a fusion protein containing the C-terminal HIV integrase-binding portion of LEDGF/p75, and the N-terminal chromodomain of heterochromatin protein-1α (HP1α), can target HIV-1 vector DNA outside of genes. We show that this fusion protein, termed TIHPLE, associates with the heterochromatin hallmark trimethylated Lys-9 of histone H3 (H3K9me3). Transient overexpression of TIHPLE alters integration site selection by an HIV-1-based vector and decreases the number of integration events that occur in genes. This change in integration site selection was achieved without a reduction in overall integration efficiency. Furthermore, we show that TIHPLE increases integration in the vicinity of H3K9me3 and in repetitive DNA sequences. These data provide a novel approach to address the problem of the tendency of retroviral vectors to integrate at undesirable sites of the human genome.
Get full access to this article
View all access options for this article.
References
1.
BarsovE.V., HughesS.H.1996. Gene transfer into mammalian cells by a Rous sarcoma virus-based retroviral vector with the host range of the amphotropic murine leukemia virus. J. Virol., 70:3922–3929.
2.
BukrinskyM.I., SharovaN., DempseyM.P., StanwickT.L., BukrinskayaA.G., HaggertyS., StevensonM.1992. Active nuclear import of human immunodeficiency virus type 1 preintegration complexes. Proc. Natl. Acad. Sci. U.S.A., 89:6580–6584.
3.
BukrinskyM.I., HaggertyS., DempseyM.P., SharovaN., AdzhubelA., SpitzL., LewisP., GoldfarbD., EmermanM., StevensonM.1993. A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells. Nature, 365:666–669.
4.
BushmanF.D., MillerM.D.1997. Tethering human immunodeficiency virus type 1 preintegration complexes to target DNA promotes integration at nearby sites. J. Virol., 71:458–464.
5.
BusschotsK., VercammenJ., EmilianiS., BenarousR., EngelborghsY., ChristF., DebyserZ.2005. The interaction of LEDGF/p75 with integrase is lentivirus-specific and promotes DNA binding. J. Biol. Chem., 280:17841–17847.
6.
CherepanovP., MaertensG., ProostP., DevreeseB., Van BeeumenJ., EngelborghsY., De ClercqE., DebyserZ.2003. HIV-1 integrase forms stable tetramers and associates with LEDGF/p75 protein in human cells. J. Biol. Chem., 278:372–381.
DanielR.2006. DNA repair in HIV-1 infection: A case for inhibitors of cellular co-factors?Curr. HIV Res., 4:411–421.
9.
DanielR., KatzR.A., SkalkaA.M.1999. A role for DNA-PK in retroviral DNA integration. Science, 284:644–647.
10.
DanielR., MyersC.B., KulkoskyJ., TaganovK., GregerJ.G., MerkelG., WeberI.T., HarrisonR.W., SkalkaA.M.2004. Characterization of a naphthalene derivative inhibitor of retroviral integrases. AIDS Res. Hum. Retroviruses, 20:135–144.
11.
DimitriP., CorradiniN., RossiF., VerniF.2005. The paradox of functional heterochromatin. Bioessays, 27:29–41.
12.
FlintS.J., EnquistL.W., KrugA.M., RacanielloV.R., SkalkaA.M.2000. Principles of virology. Molecular Biology, Pathogenesis, and Control. ASM Press: New York.
13.
GrandgenettD.P., GoodarziG.1994. Folding of the multidomain human immunodeficiency virus type-I integrase. Protein Sci., 3:888–897.
14.
GrewalS.I., MoazedD.2003. Heterochromatin and epigenetic control of gene expression. Science, 301:798–802.
15.
Hacein-Bey-AbinaS., Von KalleC., SchmidtM., McCormackM.P., WulffraatN., LeboulchP., LimA., OsborneC.S., PawliukR., MorillonE., SorensenR., ForsterA., FraserP., CohenJ.I., De Saint BasileG., AlexanderI., WintergerstU., FrebourgT., AuriasA., Stoppa-LyonnetD., RomanaS., Radford-WeissI., GrossF., ValensiF., DelabesseE., MacintyreE., SigauxF., SoulierJ., LeivaL.E., WisslerM., PrinzC., RabbittsT.H., Le DeistF., FischerA., Cavazzana-CalvoM.2003. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science, 302:415–419.
16.
Holmes-SonM.L., ChowS.A.2000. Integrase–LexA fusion proteins incorporated into human immunodeficiency virus type 1 that contains a catalytically inactive integrase gene are functional to mediate integration. J. Virol., 74:11548–11556.
IwaseS., LanF., BaylissP., De La Torre-UbietaL., HuarteM., QiH.H., WhetstineJ.R., BonniA., RobertsT.M., ShiY.2007. The X-linked mental retardation gene SMCX/JARID1C defines a family of histone H3 lysine 4 demethylases. Cell, 128:1077–1088.
19.
JonesD.O., CowellI.G., SinghP.B.2000. Mammalian chromodomain proteins: Their role in genome organisation and expression. Bioessays, 22:124–137.
20.
KatzR.A., MerkelG., SkalkaA.M.1996. Targeting of retroviral integrase by fusion to a heterologous DNA binding domain: In vitro activities and incorporation of a fusion protein into viral particles. Virology, 217:178–190.
21.
KatzR.A., GregerJ.G., DarbyK., BoimelP., RallG.F., SkalkaA.M.2002. Transduction of interphase cells by avian sarcoma virus. J. Virol., 76:5422–5434.
LewisP., HenselM., EmermanM.1992. Human immunodeficiency virus infection of cells arrested in the cell cycle. EMBO J., 11:3053–3058.
24.
LlanoM., SaenzD.T., MeehanA., WongthidaP., PeretzM., WalkerW.H., TeoW., PoeschlaE.M.2006a. An essential role for LEDGF/p75 in HIV integration. Science, 314:461–464.
25.
LlanoM., VanegasM., HutchinsN., ThompsonD., DelgadoS., PoeschlaE.M.2006b. Identification and characterization of the chromatin-binding domains of the HIV-1 integrase interactor LEDGF/p75. J. Mol. Biol., 360:760–773.
26.
LombardoA., GenoveseP., BeausejourC.M., ColleoniS., LeeY.L., KimK.A., AndoD., UrnovF.D., GalliC., GregoryP.D., HolmesM.C., NaldiniL.2007. Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat. Biotechnol., 25:1298–1306.
27.
MaisonC., AlmouzniG.2004. HP1 and the dynamics of heterochromatin maintenance. Nat. Rev. Mol. Cell. Biol., 5:296–304.
28.
MeehanA.M., SaenzD.T., MorrisonJ.H., Garcia-RiveraJ.A., PeretzM., LlanoM., PoeschlaE.M.2009. LEDGF/p75 proteins with alternative chromatin tethers are functional HIV-1 cofactors. PLoS Pathog., 5:e1000522.
29.
MitchellR.S., BeitzelB.F., SchroderA.R., ShinnP., ChenH., BerryC.C., EckerJ.R., BushmanF.D.2004. Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences. PLoS Biol., 2:E234.
30.
NaldiniL., BlomerU., GallayP., OryD., MulliganR., GageF.H., VermaI.M., TronoD.1996. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science, 272:263–267.
31.
NarezkinaA., TaganovK.D., LitwinS., StoyanovaR., HayashiJ., SeegerC., SkalkaA.M., KatzR.A.2004. Genome-wide analyses of avian sarcoma virus integration sites. J. Virol., 78:11656–11663.
32.
NielsenA.L., Oulad-AbdelghaniM., OrtizJ.A., RemboutsikaE., ChambonP., LossonR.2001. Heterochromatin formation in mammalian cells: Interaction between histones and HP1 proteins. Mol. Cell, 7:729–739.
33.
RoeT., ReynoldsT.C., YuG., BrownP.O.1993. Integration of murine leukemia virus DNA depends on mitosis. EMBO J., 12:2099–2108.
34.
SchroderA.R., ShinnP., ChenH., BerryC., EckerJ.R., BushmanF.2002. HIV-1 integration in the human genome favors active genes and local hotspots. Cell, 110:521–529.
35.
SchubelerD., MacalpineD.M., ScalzoD., WirbelauerC., KooperbergC., Van LeeuwenF., GottschlingD.E., O'NeillL.P., TurnerB.M., DelrowJ., BellS.P., GroudineM.2004. The histone modification pattern of active genes revealed through genome-wide chromatin analysis of a higher eukaryote. Genes Dev., 18:1263–1271.
SinghD.P., KimuraA., ChylackL.T.Jr., ShinoharaT.2000. Lens epithelium-derived growth factor (LEDGF/p75) and p52 are derived from a single gene by alternative splicing. Gene, 242:265–273.
38.
SkalkaA.M.1999. Retroviral DNA Integration. Academic Press: New York.
39.
SmallwoodA., EsteveP.O., PradhanS., CareyM.2007. Functional cooperation between HP1 and DNMT1 mediates gene silencing. Genes Dev., 21:1169–1178.
40.
SmitA.F.1999. Interspersed repeats and other mementos of transposable elements in mammalian genomes. Curr. Opin. Genet. Dev., 9:657–663.
41.
SmithJ.A., WangF.X., ZhangH., WuK.J., WilliamsK.J., DanielR.2008. Evidence that the Nijmegen breakage syndrome protein, an early sensor of double-strand DNA breaks (DSB), is involved in HIV-1 post-integration repair by recruiting the ataxia telangiectasia-mutated kinase in a process similar to, but distinct from, cellular DSB repair. Virol. J., 5:11.
42.
StaunstrupN.H., MoldtB., MatesL., VillesenP., JakobsenM., IvicsZ., IzsvakZ., MikkelsenJ.G.2009. Hybrid lentivirustransposon vectors with a random integration profile in human cells. Mol. Ther., 17:1205–1214.
43.
SutherlandH.G., NewtonK., BrownsteinD.G., HolmesM.C., KressC., SempleC.A., BickmoreW.A.2006. Disruption of Ledgf/Psip1 results in perinatal mortality and homeotic skeletal transformations. Mol. Cell. Biol., 26:7201–7210.
44.
TanW., ZhuK., SegalD.J., BarbasC.F.III, ChowS.A.2004. Fusion proteins consisting of human immunodeficiency virus type 1 integrase and the designed polydactyl zinc finger protein E2C direct integration of viral DNA into specific sites. J. Virol., 78:1301–1313.
45.
TanW., DongZ., WilkinsonT.A., BarbasC.F.III, ChowS.A.2006. Human immunodeficiency virus type 1 incorporated with fusion proteins consisting of integrase and the designed polydactyl zinc finger protein E2C can bias integration of viral DNA into a predetermined chromosomal region in human cells. J. Virol., 80:1939–1948.
46.
VanegasM., LlanoM., DelgadoS., ThompsonD., PeretzM., PoeschlaE.2005. Identification of the LEDGF/p75 HIV-1 integraseinteraction domain and NLS reveals NLS-independent chromatin tethering. J. Cell Sci., 118:1733–1743.
47.
WangG.P., CiuffiA., LeipzigJ., BerryC.C., BushmanF.D.2007. HIV integration site selection: Analysis by massively parallel pyrosequencing reveals association with epigenetic modifications. Genome Res., 17:1186–1194.
48.
WeinbergJ.B., MatthewsT.J., CullenB.R., MalimM.H.1991. Productive human immunodeficiency virus type 1 (HIV-1) infection of nonproliferating human monocytes. J. Exp. Med., 174:1477–1482.
49.
WuX., LiY., CriseB., BurgessS.M.2003. Transcription start regions in the human genome are favored targets for MLV integration. Science, 300:1749–1751.
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
