The relation between HIV- 1 integration and latency

The relation between HIV- 1 integration and latency Linos Vandekerckhove HIV translational research, Ghent Department of General Internal Medicine, Infectious Diseases and Psychosomatic Medicine University Hospital Ghent

Agenda Mechanism Of HIV integration Of HIV latency Quantification of HIV Integration Quantification of HIV Reservoir The relation between residual viremia and integration events The relation between integration and latency Can we interfere with integration to diminish latency and residual viremia?

HIV-1 virion www.icb.ufmg.br/~lbcd/ grupo3/hiv.jpg

Genomic organization and structure of the HIV-1 virion 5 CAP R U5 gag pol vpr vif vpu tat rev env nef U3 R AAA 3 gp41(tm) gp120(su) p32(in) p66/p51(rt) p10(pr) p17(ma) p7(nc) p24(ca) Lipid bilayer (can contain cellular proteins, e.g. HLA) Genomic RNA

HIV-1 replication cycle and HAART (Highly Active Antiretroviral Therapy) gp 120 CD4 Entry Inhibitors CXCR4 CCR5 Protease inhibitors LTR HIV-1 LTR Reverse transcriptase inhibitors Integrase inhibitors

HIV integration occurs in several steps P. Cherepanov et al. Current opinion in structural Biology 2011

Hypothetical decay curve for plasma virus levels in a patient treated with HAART Blips

Barriers for Eradication

HIV-1 latent reservoirs are not eradicated by HAART HAART Host immune response Productively infected cells most commun - cell death (days) Uninfected CD4+ T lymphocytes Infected Latently infected cells rare event - long duration (months) HIV-1 RESERVOIRS POST-INTEGRATION or PROVIRAL LATENCY

The multifactorial nature of HIV post-integration latency 1. The site of integration into the host-cell genome, the cellular chromatin environment at this site and mechanisms of transcriptional interference 2. The absence of crucial inducible host transcription factors, such as NF- B, NFAT and STAT5, that are excluded from the nuclei of resting cells and transiently activated by various stimuli 3. The chromatin structure of the HIV-1 promoter and the presence of a repressive nucleosome (nuc-1) 4. The epigenetic control of the HIV-1 promoter (histone post-translational modifications [such acetylation and methylation] and DNA methylation) 5. The absence of the viral transactivator Tat, which promotes transcription by mediating the recruitment to the HIV-1 promoter of the kinase complex P-TEFb (CyclinT1/CDK9), of histone-modifying enzymes and of ATP-dependent chromatin-remodeling complexes required for nucleosomal disruption and transcriptional processivity 6. The sequestration of P-TEFb in a HEXIM/7SK snrna-bound inactive form

Eukaryotic DNA is packaged into chromatin nuc Heterochromatin : highly condensed chromatin structure (inactive genes) Euchromatin :a less compacted structure where genes are typically expressed

The HIV-1 integration sites and heterochromatic silencing Initial findings on the integration sites in a model cell line (J-Lat) indicated that latent provirus favoured heterochromatic regions, especially in or close to satellite repeat elements. However, an in vivo study of purified resting CD4+ T cells from HIV+ individuals on HAART revealed a strong preference for proviral integration in coding gene regions. This finding as been supported by several recent studies both in in vitro infected PBMCs and in several cell lines that confirmed preferential integration into transcriptional units.

Mechanisms of transcriptional interference Transcriptional interference due to integration into a higly transcribed gene can result in proviral latency. Transcriptional interference can occur through several different mechanisms including: - enhancer trapping - promoter occlusion - steric hindrance

Enhancer trapping: depending on the site of viral integration, the enhancer activity of the viral LTR can be hijacked by nearby host gene promoters Promoter 5 LTR Promoter occlusion: read-through from the cellular upstream promoter prevents assembly of the pre-initiation complex on the viral promoter Promoter 5 LTR Steric hindrance: RNA polymerases driven by opposing promoters collide, resulting in reduced transcription from the weaker promoter Promoter 3 LTR 5 LTR

The multifactorial nature of HIV post-integration latency 1. The site of integration into the host-cell genome, the cellular chromatin environment at this site and mechanisms of transcriptional interference 2. The absence of crucial inducible host transcription factors, such as NF- B, NFAT and STAT5, that are excluded from the nuclei of resting cells and transiently activated by various stimuli 3. The chromatin structure of the HIV-1 promoter and the presence of a repressive nucleosome (nuc-1) 4. The epigenetic control of the HIV-1 promoter (histone post-translational modifications [such acetylation and methylation] and DNA methylation) 5. The absence of the viral transactivator Tat, which promotes transcription by mediating the recruitment to the HIV-1 promoter of the kinase complex P-TEFb (CyclinT1/CDK9), of histone-modifying enzymes and of ATP-dependent chromatin-remodeling complexes required for nucleosomal disruption and transcriptional processivity 6. The sequestration of P-TEFb in a HEXIM/7SK snrna-bound inactive form

The multifactorial nature of HIV post-integration latency 1. The site of integration into the host-cell genome, the cellular chromatin environment at this site and mechanisms of transcriptional interference 2. The absence of crucial inducible host transcription factors, such as NF- B, NFAT and STAT5, that are excluded from the nuclei of resting cells and transiently activated by various stimuli 3. The chromatin structure of the HIV-1 promoter and the presence of a repressive nucleosome (nuc-1) 4. The epigenetic control of the HIV-1 promoter (histone post-translational modifications [such acetylation and methylation] and DNA methylation) 5. The absence of the viral transactivator Tat, which promotes transcription by mediating the recruitment to the HIV-1 promoter of the kinase complex P-TEFb (CyclinT1/CDK9), of histone-modifying enzymes and of ATP-dependent chromatin-remodeling complexes required for nucleosomal disruption and transcriptional processivity 6. The sequestration of P-TEFb in a HEXIM/7SK snrna-bound inactive form

Nucleosome disruption during transcriptional activation of the HIV-1 promoter nuc nuc-0 sites B nuc-1 nuc-2 1 789 activation (TNF, PMA)

Nucleosomal structure

Covalent post-translational histone modifications p = phosphorylation, m = methylation, a = acetylation, Ub = ubiquitination, r = poly-adp-ribosylation epigenetic modifications: dynamic, rapidly changing depending on the signaling conditions within the cell, function sequentially or in combination to form the «histone code», which is read by effector proteins to produce distinct

The multifactorial nature of HIV post-integration latency 1. The site of integration into the host-cell genome, the cellular chromatin environment at this site and mechanisms of transcriptional interference 2. The absence of crucial inducible host transcription factors, such as NF- B, NFAT and STAT5, that are excluded from the nuclei of resting cells and transiently activated by various stimuli 3. The chromatin structure of the HIV-1 promoter and the presence of a repressive nucleosome (nuc-1) 4. The epigenetic control of the HIV-1 promoter (histone post-translational modifications [such acetylation and methylation] and DNA methylation) 5. The absence of the viral transactivator Tat, which promotes transcription by mediating the recruitment to the HIV-1 promoter of the kinase complex P-TEFb (CyclinT1/CDK9), of histone-modifying enzymes and of ATP-dependent chromatin-remodeling complexes required for nucleosomal disruption and transcriptional processivity 6. The sequestration of P-TEFb in a HEXIM/7SK snrna-bound inactive form

Agenda Mechanism Of HIV integration Of HIV latency Quantification of HIV Integration Quantification of HIV Reservoir The relation between residual viremia and integration events The relation between integration and latency Can we interfere with integration to diminish latency

Effect of Intensification on Persistent Viremia in all Patients Conclusion: No individual patient shows evidence of a decline in persistent viremia during intensification n

Quantification of HIV Integration: Alu-PCR to measure integrated HIV copies LTR LTR ALU ALU ALU PCR qpcr standardisation with cell culture NL4.3 on HeLaP4 cells: mix of different fragments to mimic in vivo situation

Drawbacks of Alu-HIV qpcr Variable sizes of Alu-HIV PCR amplicons in the first PCR the amount of Alu hybridization sites the nested PCR itself the background of unintegrated viral DNA the low amount of HIV DNA copies in HIV infected patients on HAART

Co-culture for quantification of latent reservoir 1. Isolation and purification of CD4+ CD25- HLA-DR- PBMC from HIV+ patients: latent memory CD4+ T lymfocytes 2. Uniform activation by addition of IL-2, PHA and irradiated PBMC in 10-fold from donors (macrophages, dendritic cells) 3. Amplify signal for p24 ELISA detection by addition d2/d7 of CD4+ CD8- lymfoblasts from donor

Co-culture for quantification of latent reservoir 1. Isolation and purification of CD4+ CD25- HLA-DR- PBMC from HIV+ patients: latent memory CD4+ T lymfocytes 2. Uniform activation by addition of IL-2, PHA and irradiated PBMC in 10-fold from donors (macrophages, dendritic cells) 3. Amplify signal for p24 ELISA detection by addition d2/d7 of CD4+ CD8- lymfoblasts from donor

Co-culture for quantification of latent reservoir 1. Isolation and purification of CD4+ CD25- HLA-DR- PBMC from HIV+ patients: latent memory CD4+ T lymfocytes 2. Uniform activation by addition of IL-2, PHA and irradiated PBMC in 10-fold from donors (macrophages, dendritic cells) 3. Amplify signal for p24 ELISA detection by addition d2/d7 of CD4+ CD8- lymfoblasts from donor

Co-culture for quantification of latent reservoir Patient: +-300-350ml Hep blood to obtain 60mio memory CD4 lymfocytes PBMC from healthy donors: 2 buffy coats from blood bank per experiment Selection of CD4+ CD25- HLA-DR- T-lymfo s + Selection of CD8- PBMC (lymfoblast addition d2/d7/d15): MACS Separation columns with magnetic micro beads (Miltenyi Biotec) Lymphoprep (Ficoll gradient) 250ml per experiment Biochrome QG RPMI 1640 medium: 2L per experiment IL-2 (220 stock) + PHA Innogenetics Innotest HIV Ag mab (625 per 96well plate) Labor-intensive technique + costly!

The relation between number integration events and latency

Agenda Mechanism Of HIV integration Of HIV latency Quantification of HIV Integration Quantification of HIV Reservoir The relation between residual viremia and integration events The relation between integration and latency Can we interfere with integration to diminish latency and residual viremia?

MONOI ANRS 136 225 patients A S48 A S96 HIV RNA < 50 cp ART duration 7.8 vs 8.7 CD4 Baseline : 582/ mm VL < 1 c/ml at BL VL > 50 c/ml at BL OR (IC 95 %) 0,24 (0,05-0,86) 7,84 (1,22-52,2) p OR (IC 95 %) 0,042-0,025 - p Nadir 220/mm 3 HIV RNA <1 cp (40%) HIV DNA 4 log 10 6 PBMC Poor Compliance (< 100 %) Prior duration ART (Increase by 5 years) HIV-1 DNA at D0 (log10 cells C/10 6) * - - - 3,84 (1,29-12,49) 2,93 (1,43-6,66) 2,66 (1,11-7,48) 0,02 0,00 6 0,04 HIV DNA is predictive of maintaining an HIV RNA <50 cp / ml on mono DRV at W96

HIV DNA Log copies/10 6 PBMC ANRS Cohorts : Cell-associated HIV-DNA Quantification inpbmc 6,00 Lewin and Rouzioux, Review, AIDS 2011 5,00 4,93 4,00 4,07 4,2 3,00 3,68 3,35 2,95 3,22 2,87 2,94 2,99 3,07 2,00 1,00 1,09 2,47 2,27 1,7 2,10 1,78 1,44 1,11 0,70 0,00 0,3 0,36 0,3 PRIMO inclusion SEROCO inclusion ALT HIC Visconti Primary infection Ghosn, JAC 2010 Seroco Rouzioux, JID 2005 ALT Martinez,JID 2008 Elite controllers Lambotte 2008 ART/PI Hocqueloux 2010

Visconti Patients 12 patients treated at Primary infection then stop ART Duration (med ): 35 months Duration Off ART : 5 years CD4 -pre ART 489 ( 371-955) -at ART stop: 931 (354-1639 -last value : 837( 388-1598 HIV RNA - preart : 5.0 log ( 3-7.3) - last value : 1.7 log ( 1.7-2.4) A. Saez-Cirion et al., # F-126 CROI 2011 (Boston)

127 ART treated patients Med Duration : 6.5 y CD4 med : 580 /mm3 HIV RNA ( taqman)< 50 cp : 100% CD4/CD8 : 0.8 HIV RNA us < 1 cp/ml: 37% CV median: 2.6 cp/ml Correlation between residual HIV RNA and CD4/CD8 and DNA No association between residual HIV RNA and activation markers (CRP, IL6, stnf, CD38) J Infect Dis. 2011 Jul;204(1):135-8. 448 1027

Agenda Mechanism Of HIV integration Of HIV latency Quantification of HIV Integration Quantification of HIV Reservoir The relation between residual viremia and integration events The relation between integration and latency Can we interfere with integration to diminish latency and residual viremia?

The relation between integration and latency

The relation between integration and latency

The relation between site of integration and latency

The relation between site of integration and latency Integration sites were enriched within active transcription units in both cell types

Agenda Mechanism Of HIV integration Of HIV latency Quantification of HIV Integration Quantification of HIV Reservoir The relation between residual viremia and integration events The relation between integration and latency Can we interfere with integration to diminish latency and residual viremia?

Can we interfere with integration to diminish latency (and residual viremia)? R.T Gandghi, Plos Med 2010

Can we interfere with integration to diminish latency and residual viremia?

Shortcommings of intensification studies

Can we interfere with the integration site selection

Different patients respond different to antilatency cocktail S.Reuze, Plos One 2009

Can we interfere with the integration site selection

Can we interfere with the integration site selection H.M. Marshall, Plos One 2007

Can we interfere with the integration site selection H.M. Marshall, Plos One 2007

Agenda Mechanism Of HIV integration Of HIV latency Quantification of HIV Integration Quantification of HIV Reservoir The relation between integration and latency The relation between residual viremia and integration events Can we interfere with integration to diminish latency and residual viremia?

Take home message The relation between integration and latency Latency can be established by several molecular mechanisms of which some of them are influenced by the site of integration The relation between residual viremia and integration events The number of integration events seems to be related to a phenotype of disease progression and therefore this could serve as an important tool in a diagnostic setting

Take home message Can we interfere with integration to diminish latency and residual viremia? ART compounds that interfere with integration/integration site selection are the preferred compounds that might directly influence latency, however integration events might also be diminsdhed by antilatency compounds that affect eg epigenetic regulation, NF- B, NFAT and STAT... A combination of new diagnostic tools to assess the extend of the reservoir will help evaluating this new drugs