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

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. The current need for Latency targeting therapy: Interruption of cART (weeks) cART Adapted from: Eisele And Siliciano. Immunity (2012) HIV-1 infected cell CD4+ T cell LatencyRebound - AIDS Early infection

Adapted from: Han and Siliciano, Nat Med., 2007 Cellular factors -Limited transcriptional activators Cellular factors -Limited transcriptional activators Viral factors -Site of integration (into active gene) -Transcriptional interference* -Low acetylation -High methylation Viral factors -Site of integration (into active gene) -Transcriptional interference* -Low acetylation -High methylation Impaired RNA export from nucleus HIV specific host microRNA HIV gene expression during latency in resting memory T-cells Cell-activation / senescence RNA splicing RNA transcription Chromatin remodelling (epigenetics) microRNA expression m7G-capping Tat

Adapted from Siliciano RF, Greene WC Mechanisms that establish and/or maintain latency: Transcriptional Interference

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 Cap-Tat (ng)IRES-Tat (ng) Michelle Lee, 2014

ACH2 latent T cell line 1. Uninfected Jurkat cells2. Unstimulated ACH2 cells 3. PMA-stimulated ACH2 cells Ex2 Ex6 Ex1 introns AUG Ex3 Ex4 Ex5 Ex7 Ex8 Ex PCR primers 32 P-probes Odp2603 tat exon A2A Odp2601 gag-5’ 2601 D Odp2604 env/vpu 2604 D Odp2605 HIV-U Ex5 U pA A3 D1D4 Read-through transcription splices HIV tat exon2 onto cell NT5C3 mRNA in the ACH-2 cell line model of HIV-1 latency 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 Transduction of the latently infected ACH2 and J-Lat6.3 cell lines with a Tat responsive LTR-Luciferase reporter pseudovirus Fluc Nef ∆ Gag ∆ Env U3 R R U5 Nef U3 R R U5 Pseudovirus (after RT): Jurkat Giovana Bernardi, 2013

Alu-tat PCRs of cDNA from CCL19-treated, memory CD4 T cells infected with NL4.3 Latently-infected primary CD4 T cells express chimeric cellular:tat mRNAs tat exon 2 MW DNAse MW RT MW + - :RT Nested tat PCR Uninfected control Donor 1Donor 2 Uninfected control Donor 1Donor 2 Southern blot probed with tat exon 2 probe Cloned, sequenced 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:

Detection of chimeric cell:tat RNA in primary resting CD4 T cell latency model PCR of cDNA from CCL19 chemokine-induced resting CD4 T cells infected with HIV-1 NL4.3 (Saleh et al, Blood, 2007) using various cellular gene exon forward primers and tat exon2 reverse primer STAT5B Exon 5 Exon 8 DDX6 Exon 2 Exon 5 HORMAD2 Exon 2 Exon 9 Exon - pA A3 D1 D4

Read-through transcription and splicing generate chimeric cell:tat RNAs in latently infected primary CD4 T cells STAT5BHORMAD2 Reverse primers 1.LTR-U3 2.gag 3.tat exon 2-ls 4.tat exon 2-cs 5.SD1/SA3 Forward Primers Exon - pA A3 D1D4

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 Sharon Lewin Paul Cameron Fiona Whiteman Suha Saleh Vanessa Evans Alfred Hospital / Burnet institute / Monash University Damian Purcell Talia Mota Jonathan Jacobson Michelle Lee Giovana Bernardi Jane Howard Leanne Ng University of Melbourne NHMRC and ACH2 for funding.