The current need for Latency targeting therapy:

1. 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, GiovanaBernardi, Jane Howard, Damian Purcell The University of Melbourne, Department of Microbiology and Immunology at the Peter Doherty Institute

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

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

4. Mechanisms that establish and/or maintain latency: Transcriptional Interference Adapted from SilicianoRF, Greene WC. 2011

5. CENTRAL HYPOTHESIS: cART selects HIV provirus integrated into the introns of transcriptionally active genes where read-through transcription includes HIV RNA A7

6. Cap-dependent Tat vs IRES-Tat Cap-Tat (ng) IRES-Tat (ng) Michelle Lee, 2014

7. Read-through transcription splices HIV tat exon2 onto cell NT5C3 mRNA in the ACH-2 cell line model of HIV-1 latency ACH2 latent T cell line 2595 2605 2605 2603 2603 introns AUG - pA - pA - pA 2601 2601 A3 Ex2 Ex3 Ex4 Ex5 Ex6 Ex7 Ex8 Ex9 Ex1 D1 D4 2604 2604 PCR primers 32P-probes Odp2604 env/vpu Odp2601 gag-5’ Odp2603 tat exon 2 Odp2605 HIV-U3 A2 A3 1 2 3 1 2 3 1 2 3 1 2 3 U3 Ex5 D4 D1 1. Uninfected Jurkat cells 2. Unstimulated ACH2 cells 3. PMA-stimulated ACH2 cells Jane Howard, 2013

8. Latently infected J-Lat6.3 cells produce chimeric cell:tat RNA by read-through transcription and splicing Michelle Lee, 2013

9. Functional Tat protein is expressed from spliced cell:tatmRNA using an Internal Ribosome Entry Site (IRES) underlying the Tat coding sequence Jurkat 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 2 5 Nef Fluc Nef ∆Gag ∆Env GiovanaBernardi, 2013 U3 R U5

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

11. 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

12. Detection of chimeric cell:tat RNA in primary resting CD4 T cell latency model STAT5B Exon 5 Exon 8 DDX6 Exon 2 Exon 5 HORMAD2 Exon 2 Exon 9 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 - pA - pA A3 Exon Exon D1 D4

13. Read-through transcription and splicing generate chimeric cell:tat RNAs in latently infected primary CD4 T cells Forward Primers STAT5B HORMAD2 1 2 3 4 5 1 2 3 4 5 Reverse primers LTR-U3 gag tat exon 2-ls tat exon 2-cs SD1/SA3 - pA - pA A3 Exon Exon D1 D4

14. 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)

15. Acknowledgments University of Melbourne Damian Purcell Talia Mota Jonathan Jacobson Michelle Lee GiovanaBernardi 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.