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Transcript
Alternative RNA splicing in latently infected T cells generates chimeric cellular:HIV mRNAs with the potential to generate Tat and reactivate infection Con Sonza, Talia Mota, Jonathan Jacobson, Michelle Lee, Giovana Bernardi, Jane Howard, Damian Purcell The University of Melbourne, Department of Microbiology and Immunology at the Peter Doherty Institute The current need for Latency targeting therapy: CD4+ T cell (weeks) HIV-1 infected cell Interruption of cART cART Early infection Latency Rebound - AIDS cART is able to suppress plasma viraemia below detectable levels, however the reservoir of latently infected cells persists. Interruption or discontinuation of cART is followed by rebound of viraemia and progression to AIDS. HIV gene expression during latency in resting memory T-cells HIV specific host microRNA Cellular factors -Limited transcriptional activators Impaired RNA export from nucleus Viral factors -Site of integration (into active gene) -Transcriptional interference* -Low acetylation -High methylation Chromatin remodelling (epigenetics) RNA transcription RNA splicing m7G-capping Tat microRNA expression Cell-activation / senescence Mechanisms that establish and/or maintain latency: Transcriptional Interference Adapted from Siliciano RF, Greene WC. 2011 CENTRAL HYPOTHESIS: cART selects HIV provirus integrated into the introns of transcriptionally active genes where read-through transcription includes HIV RNA A7 Cap-dependent Tat vs IRES-Tat Transfection of TZM-bl reporter cells pcDNA3.150 100 150 200 250 300 350 400 450 500 550 600 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 Relative Luciferase Activity 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 Cap-Tat (ng) IRES-Tat (ng) Michelle Lee, 2014 Read-through transcription splices HIV tat exon2 onto cell NT5C3 mRNA in the ACH-2 cell line model of HIV-1 latency PCR primers Ex4 Ex5 D1 2605 2595 - pA A3 2601 Ex6 D4 Ex7 Ex8 Ex9 - pA Ex1 introns Ex2 Ex3 - pA AUG ACH2 latent T cell line 2603 2604 32P-probes Odp2605 HIV-U3 Odp2601 gag-5’ Odp2603 tat exon 2 Odp2604 env/vpu 1 2 3 1 2 3 1 2 3 1 2 3 Ex5 U3 2605 1. Uninfected Jurkat cells 2601 A2 A3 D1 2. Unstimulated ACH2 cells 2604 2603 D4 3. PMA-stimulated ACH2 cells Jane Howard, 2013 Latently infected J-Lat6.3 cells produce chimeric cell:tat RNA by read-through transcription and splicing Michelle Lee, 2013 Functional Tat protein is expressed from spliced cell:tat mRNA using an Internal Ribosome Entry Site (IRES) underlying the Tat coding sequence ACH2 J-Lat 25000 Luciferase activity Luciferase activity 10000 8000 6000 4000 2000 20000 15000 10000 5000 0 A3.01 Unstimulated PMA stimulated 0 Jurkat A3.01 Unstimulated TNF stimulated Transduction of the latently infected ACH2 and J-Lat6.3 cell lines with a Tat responsive LTR-Luciferase reporter pseudovirus Pseudovirus (after RT): U3 R U5 ∆Gag 2 5 ∆Env Nef Fluc Nef U3 R U5 Giovana Bernardi, 2013 Latently-infected primary CD4 T cells express chimeric cellular:tat mRNAs Southern blot Donor 1 Donor 2 probed with tat exon 2 probe Uninfected control Alu-tat PCRs of cDNA from CCL19-treated, memory CD4 T cells infected with NL4.3 Donor 2 - :RT Donor 1 + Uninfected control MW Nested tat PCR tat exon 2 MW MW + + + + + - Cloned, sequenced + DNAse RT Talia Mota Distribution of integration sites in five patients. A total of 2410 integration sites were obtained from PBMCs or negatively selected CD4+ cells from the five patients. F Maldarelli et al. Science 2014;345:179-183 Detection of chimeric cell:tat RNA in primary resting CD4 T cell latency model STAT5B Exon 5 DDX6 Exon 8 Exon 2 HORMAD2 Exon 2 Exon 9 Exon 5 Exon - pA - pA PCR of cDNA from CCL19 chemokine-induced resting CD4 T cells infected with HIV-1NL4.3 (Saleh et al, Blood, 2007) using various cellular gene exon forward primers and tat exon2 reverse primer A3 D1 D4 Exon Read-through transcription and splicing generate chimeric cell:tat RNAs in latently infected primary CD4 T cells Forward Primers STAT5B 1 2 3 4 5 HORMAD2 1 2 3 4 5 - pA - pA Reverse primers 1. LTR-U3 2. gag 3. tat exon 2-ls 4. tat exon 2-cs 5. SD1/SA3 A3 Exon Exon D1 D4 Conclusions • During read-through transcription in latently infected T cell lines and primary resting CD4 T cells, chimeric cell:tat RNAs are generated by the usual cellular mechanisms of alternative RNA splicing • An IRES-like element in tat leads to translation of this mRNA in a cap-independent manner and expression of functional Tat protein (POSTER THPE006: G. Khoury) • Because of the central role of Tat in the establishment and maintenance of latency, factors affecting transcription, splicing, cytoplasmic localization or translation of Tat from chimeric RNAs will impact on HIV latency • Such factors could be targeted to develop novel, more specific, strategies to assist in the activation and clearance of the latent reservoir or prevent viral rebound upon cessation of cART (POSTER THPE016: J. Jacobson) Acknowledgments University of Melbourne Damian Purcell Talia Mota Jonathan Jacobson Michelle Lee Giovana Bernardi Jane Howard Leanne Ng Alfred Hospital / Burnet institute / Monash University Sharon Lewin Paul Cameron Fiona Whiteman Suha Saleh Vanessa Evans NHMRC and ACH2 for funding.
