CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

or romidepsin (RMD). Dose-ranging studies were performed and, after treatment with a standardized panel of death peptides, BH3 profiles were generated. To determine the selectivity of priming for latently HIV-infected cells, we used the J89 cell line in a series of dual HDACi and small-molecule Bcl-2 inhibitor experiments. Results: Marked changes in the magnitude and phenotype of CD4 + T cell mitochondrial priming were observed after immune activation or exposure to PNB and RMD, compared to untreated HIV-infected PBMC. BH3 profiles varied as a function of drug type, dose, and duration of exposure. Increased priming in response to the peptide BAD and decreased permeabilization to the peptide NOXAA was observed with both transcriptional and immune activating stimuli, signifying an increased dependence on the anti-apoptotic Bcl-2 protein. The rank order of increased mitochondrial priming was RMD > PNB > a-CD3/a-CD28 beads >> SAHA. Similar magnitudes of priming occurred in central and transitional memory CD4 + T cell subsets. Increasing concentrations of the Bcl-2 inhibitors ABT-263 or -199 in the presence of RMD 20nM led to progressive losses of HIV-expressing J89 cells. Conclusions: Histone deacetylase inhibition increased primary CD4 + T cell mitochondrial priming and cellular reliance on the anti-apoptotic protein Bcl-2. The mitochondrial phenotypes that we observed were specific to the immune activation or transcriptional re-activation stimulus being used. Combination HDACi and Bcl-2-inhibition selectively eliminated HIV-expressing cells in a latency cell line. Small molecule Bcl-2 inhibition merits further investigation as a targeted HIV eradication strategy.

TUESDAY, FEBRUARY 24, 2015 Session P-F7 Poster Session

Poster Hall

2:30 pm– 4:00 pm Pharmacologic Latency-Reactivation Agents 412 Reactivation of HIV Latently Infected T Cells by Targeting Tat IRES Translation Georges Khoury 1 ; Sri Ramarathinam 2 ; Charlene Mackenzie 1 ; DavidYurick 1 ; Con Sonza 1 ;Tony Purcell 2 ; Damian F. Purcell 1 1 University of Melbourne At the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia; 2 Monash University/Alfred Hospital, Melbourne, Australia

Background: In virologically suppressed patients, residual latent HIV provirus is predominantly integrated into introns of transcriptionally active genes of memory CD4+ T cells. Much of this provirus is subjected to transcriptional interference, where read-through transcription and splicing incorporate HIV RNA into mature cellular RNAs. We identified the inclusion of Tat first coding exon (Tat-exon2) within a cellular mRNA in latently infected cells, and low level expression of Tat mediated by an Internal Ribosome Entry Site (IRES) present in Tat-exon2. To understand the function of Tat IRES requires a detailed knowledge of the RNA structure and the cellular factors that binds to these chimeric RNAs. Methods: The folded structure of IRES-active tat mRNA was determined by chemical probing experiments. Cellular proteins binding Tat IRES were purified by fusing 3 binding sites for the MS2 coat protein to the 3’end of tat mRNAs with and without native IRES. The RNP complexes were formed by incubation with a protein extract from a T cell line model of latent infection (J-Lat clone 6.3). The proteins were then identified by 1D SDS-PAGE followed by mass spectrometry analysis. Results: 2D structure analysis of tat mRNAs by SHAPE revealed that Tat-exon2 harbours an IRES element that folds independently of the 5’UTR region. Sequence alignment of Tat-exon2 from 2233 HIV strains showed highly conserved sequences near the Tat start-codon. We confirmed the role of one conserved element on the IRES-mediated Tat translation by RNA transfection in TZM-bL reporter cells. Silent point mutations within this conserved element that disrupted tat mRNA structure induced a significant reduction in virus production measured by quantifying the p24 viral capsid protein by ELISA. Several splicing and translation factors interacting with this cis -acting element were identified by mass spectrometry analysis. An important role for these cell proteins in virus protein translation and the reactivation of virion production from latency was determined by over- expression and shRNA knockdown analysis. Conclusions: Our study revealed an important role for a conserved HIV RNA sequence-structure and several RNA binding proteins in Tat IRES translation. The low level of Tat expression from the IRES element within readthrough transcripts that incorporate latent HIV may participate in residual pathogenesis during treated HIV infection, and constitute potential drug target. 413 Targeting HIV-1 Latency With a Potent Tat Inhibitor Background: Despite the immense success of HIV anti-retroviral therapy (ART) to reduce replication to very low levels, it fails to eradicate the virus. HIV persists in latently and productively infected CD4 + T cells in infected subjects undergoing ART. Thus, we need novel classes of therapeutic agents that target different stages of the virus life cycle to limit latent HIV disease. Methods: The HIV Tat protein, binds the 5’ terminal region of HIV mRNAs, and potently activates transcription. Tat is a very attractive drug target because 1) is expressed early during virus replication, 2) has no cellular homologs and 3) direct inhibition of Tat blocks the feedback loop that drives viral exponential production. Compounds that block Tat have been highly sought after; however, none is yet in the clinic. We have shown that didehydro-Cortistatin A (dCA), an analog of a natural steroidal alkaloid, selectively inhibits Tat-activity with no cellular associated toxicity. dCA binds specifically to the RNA-binding domain of Tat reducing HIV-1 RNA production in infected cultured and primary cells with an EC 50 as low as 0.7 pM (1). Results: Here we show that dCA abrogates antigenic virus reactivation from latently infected CD4 + T primary cells explanted from patients receiving suppressive ART. dCA can reduce cell-associated HIV-1 RNA production from primary cells and cell-line models of latency by reducing RNA Polymerase II recruitment to the HIV promoter and as a result, cells become refractory to viral reactivation by several anti-latency agents (cytokines, HDAC inhibitors, PKC activators). Furthermore, arrest of dCA treatment does not result in viral rebound, as the promoter is transcriptionally shut-off. As expected, latent cell lines containing virus mutated in either TAR or Tat are insensitive to dCA. Conclusions: dCA treatment combined with ART may inhibit and persistently abrogate residual HIV production from cellular reservoirs in blood and tissues from virally suppressed subjects, block viral reactivation, reduce reservoir replenishment, and may ultimately decrease the size of the latent reservoir. Our experiments provide a proof-of-concept for the use of transcriptional suppressors in therapeutic approaches for a functional HIV cure. 1. Mousseau et al., Cell Host Microbe (2012). 12(1): 97-108 Guillaume Mousseau 1 ; Remi Fromentin 2 ; Cari Kessing 2 ; LydieTrautmann 2 ; Nicolas Chomont 2 ; Susana T. Valente 1 1 The Scripps Reasearch institute, Jupiter, FL, US; 2 The Vaccine and Gene Therapy Institute, Port Saint Lucie, FL, US

Poster Abstracts

295

CROI 2015