title: Establishment of HIV latency in primary CD4+ cells is due to epigenetic transcriptional silencing and P-TEFb restriction.
abstract: The development of suitable experimental systems for studying HIV latency in primary cells that permit detailed biochemical analyses and the screening of drugs is a critical step in the effort to develop viral eradication strategies. Primary CD4(+) T cells were isolated from peripheral blood and amplified by antibodies to the T-cell receptor (TCR). The cells were then infected by lentiviral vectors carrying fluorescent reporters and either the wild-type Tat gene or the attenuated H13L Tat gene. After sorting for the positive cells and reamplification, the infected cells were allowed to spontaneously enter latency by long-term cultivation on the H80 feeder cell line in the absence of TCR stimulation. By 6 weeks almost all of the cells lost fluorescent protein marker expression; however, more than 95% of these latently infected cells could be reactivated after stimulation of the TCR by alpha-CD3/CD28 antibodies. Chromatin immunoprecipitation assays showed that, analogously to Jurkat T cells, latent proviruses in primary CD4(+) T cells are enriched in heterochromatic markers, including high levels of CBF-1, histone deacetylases, and methylated histones. Upon TCR activation, there was recruitment of NF-kappaB to the promoter and conversion of heterochromatin structures present on the latent provirus to active euchromatin structures containing acetylated histones. Surprisingly, latently infected primary cells cannot be induced by tumor necrosis factor alpha because of a restriction in P-TEFb levels, which can be overcome by activation of the TCR. Thus, a combination of restrictive chromatin structures at the HIV long terminal repeat and limiting P-TEFb levels contribute to transcriptional silencing leading to latency in primary CD4(+) T cells.
authors.affiliation: Department of Molecular Biology and Microbiology, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-4960, USA.
authors.forename: Mudit
authors.initials: M
authors.lastname: Tyagi
authors.forename: Richard John
authors.initials: RJ
authors.lastname: Pearson
authors.forename: Jonathan
authors.initials: J
authors.lastname: Karn
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000821:virology*; D002478:Cells, Cultured; D022041:Euchromatin / Q000737:chemistry / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D020868:Gene Silencing; D006678:HIV / Q000502:physiology*; D006570:Heterochromatin / Q000737:chemistry / Q000378:metabolism; D006655:Histone Deacetylases / Q000032:analysis; D006657:Histones / Q000032:analysis; D054884:Host-Pathogen Interactions; D006801:Humans; D047468:Immunoprecipitation; D016328:NF-kappa B / Q000378:metabolism; D042925:Positive Transcriptional Elongation Factor B / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D017735:Virus Latency
chemical_list: D022041:Euchromatin; D006570:Heterochromatin; D006657:Histones; D016328:NF-kappa B; D011948:Receptors, Antigen, T-Cell; D014409:Tumor Necrosis Factor-alpha; D042925:Positive Transcriptional Elongation Factor B; D006655:Histone Deacetylases
references.citation: J Immunol. 1994 Aug 1;153(3):952-9
references.pmid: 7913116
references.citation: EMBO J. 2007 Jan 24;26(2):412-23
references.pmid: 17245431
references.citation: Nature. 1995 Jan 12;373(6510):123-6
references.pmid: 7816094
references.citation: Nature. 1999 Oct 14;401(6754):708-12
references.pmid: 10537110
references.citation: Retrovirology. 2007 Jul 11;4:47
references.pmid: 17625008
references.citation: Mol Cell. 2009 Jun 26;34(6):696-709
references.pmid: 19560422
references.citation: Annu Rev Immunol. 2000;18:665-708
references.pmid: 10837072
references.citation: J Immunol. 1991 Apr 15;146(8):2488-94
references.pmid: 1707907
references.citation: AIDS. 2004 May 21;18(8):1101-8
references.pmid: 15166525
references.citation: J Virol. 1998 Dec;72(12):9881-8
references.pmid: 9811724
references.citation: J Virol. 2009 Apr;83(7):3078-93
references.pmid: 19144703
references.citation: Trends Mol Med. 2004 Nov;10(11):525-31
references.pmid: 15519278
references.citation: J Virol. 2002 Dec;76(24):13077-82
references.pmid: 12438635
references.citation: J Virol. 2005 Nov;79(22):14179-88
references.pmid: 16254353
references.citation: J Virol. 2002 Sep;76(17):8776-86
references.pmid: 12163598
references.citation: PLoS Pathog. 2009 Jun;5(6):e1000495
references.pmid: 19557157
references.citation: Cell Host Microbe. 2008 Aug 14;4(2):123-33
references.pmid: 18692772
references.citation: J Virol. 2004 Jun;78(12):6122-33
references.pmid: 15163705
references.citation: EMBO J. 2003 Apr 15;22(8):1868-77
references.pmid: 12682019
references.citation: PLoS Pathog. 2007 Oct 12;3(10):1459-69
references.pmid: 17937499
references.citation: J Exp Med. 2001 Dec 17;194(12):1731-41
references.pmid: 11748275
references.citation: Nat Med. 2007 Oct;13(10):1241-7
references.pmid: 17906637
references.citation: J Immunol. 2008 Dec 1;181(11):7713-20
references.pmid: 19017960
references.citation: Nat Med. 2001 Apr;7(4):459-64
references.pmid: 11283673
references.citation: J Virol. 2007 Jul;81(14):7424-34
references.pmid: 17475639
references.citation: Nat Med. 2000 Jan;6(1):82-5
references.pmid: 10613829
references.citation: J Virol. 2000 Aug;74(15):6790-9
references.pmid: 10888618
references.citation: JAMA. 1999 Nov 3;282(17):1627-32
references.pmid: 10553788
references.citation: Int Immunol. 2008 Sep;20(9):1189-99
references.pmid: 18635582
references.citation: Nature. 1995 Jan 12;373(6510):117-22
references.pmid: 7529365
references.citation: Nature. 1987 Apr 16-22;326(6114):711-3
references.pmid: 3031512
references.citation: J Virol. 2004 Feb;78(3):1160-8
references.pmid: 14722271
references.citation: J Virol. 2003 Aug;77(15):8602-6
references.pmid: 12857933
references.citation: Nat Med. 1999 May;5(5):512-7
references.pmid: 10229227
references.citation: EMBO J. 1993 Aug;12(8):3249-59
references.pmid: 8344262
references.citation: Immunity. 1997 Mar;6(3):235-44
references.pmid: 9075924
references.citation: Nature. 1999 Oct 28;401(6756):874-5
references.pmid: 10553903
references.citation: Proc Natl Acad Sci U S A. 2009 Jun 9;106(23):9403-8
references.pmid: 19470482
references.citation: EMBO J. 1996 Mar 1;15(5):1112-20
references.pmid: 8605881
references.citation: EMBO J. 2007 Dec 12;26(24):4985-95
references.pmid: 18007589
references.citation: EMBO J. 2006 Aug 9;25(15):3596-604
references.pmid: 16874302
references.citation: Proc Natl Acad Sci U S A. 2003 Oct 28;100(22):12955-60
references.pmid: 14569007
references.citation: J Clin Invest. 2009 Nov;119(11):3473-86
references.pmid: 19805909
references.citation: EMBO J. 2007 Jan 24;26(2):424-35
references.pmid: 17245432
references.citation: Virology. 2009 Apr 25;387(1):98-108
references.pmid: 19268337
references.citation: PLoS Pathog. 2006 Jul;2(7):e68
references.pmid: 16839202
references.citation: J Virol. 2005 Jun;79(11):6610-9
references.pmid: 15890899
references.citation: Microbiol Mol Biol Rev. 2006 Sep;70(3):646-59
references.pmid: 16959964
references.citation: Nat Immunol. 2000 Nov;1(5):433-40
references.pmid: 11062504
references.citation: J Virol. 2003 Apr;77(7):4025-32
references.pmid: 12634362
references.citation: J Virol. 1998 Feb;72(2):1666-70
references.pmid: 9445075
references.citation: Cell Host Microbe. 2008 Aug 14;4(2):134-46
references.pmid: 18692773
references.citation: J Infect Dis. 2008 Mar 1;197(5):714-20
references.pmid: 18260759
references.citation: J Exp Med. 1997 Nov 3;186(9):1407-18
references.pmid: 9348298
references.citation: Virology. 2006 Nov 25;355(2):127-37
references.pmid: 16919704
references.citation: J Virol. 2008 Dec;82(24):12291-303
references.pmid: 18829756