CROI 2018 Abstract eBook

Abstract eBook

Poster Abstracts

328 IDENTIFICATION OF SMALL MOLECULES THAT INHIBIT REACTIVATION OF LATENT HIV-1 Benni S. Vargas , Feng Han, Nicholas S. Giacobbi, Phalguni Gupta, Nicolas Sluis-Cremer University of Pittsburgh, Pittsburgh, PA, USA Background: A major barrier to curing HIV-1 infection is the persistence of a quiescent but replication-competent latent viral reservoir in resting CD4+ T cells. Theoretically, if one could manipulate the epigenetic state of the HIV-1 provirus, or interfere with the epigenetic control mechanisms involved in viral transcriptional activation, one could silence the latent proviruses for an extended time-period, or possibly for a lifetime, thus enabling a functional cure. In this regard, the primary goal of this study was to identify small molecules that can effectively block the reactivation of latent HIV-1, independent of the stimulus used to reverse latency. Methods: We screened a unique collection of 418 kinase inhibitors (Selleckchem) that target a wide range of cellular signaling pathways using the 24ST1NLESG cell line of HIV-1 latency. The screen was carried out with the kinase inhibitors alone (2μM), or in combination with 3 latency reversing agents: 1μM prostratin, 10nM panobinostat or 1μM JQ-1. Follow-up studies included screening the kinase library for cellular toxicity and detailed dose-response analyses. Results: We identified 21 kinase inhibitors, mostly targeted toward PKC, MEK or ERK, that blocked the activity of prostratin only. Twenty-three kinase inhibitors, targeting mTOR, PI3K or GSK-3, inhibited panobinostat activity only. We identified 4 inhibitors which blocked the activity of JQ-1 only. We found an additional 30 compounds that inhibited the activity of all 3 latency reversing agents. Of these, Danusertib, an Aurora kinase inhibitor; and PF-3758309, a PAK4 inhibitor, were found to be the most potent. The concentration of Danusertib required to inhibit 50% (i.e., IC50) of HIV-1 latency reversal in the 24ST1NLESG cell line by prostratin, panobinostat, JQ-1 and TNF-α was determined to be 40±16, 110±43, 147±21 and 192±23 nM, respectively. The concentration of Danusertib that resulted in 50% cytotoxicity (i.e., CC50) was 29.7±3.4 μM (therapeutic index > 150). The IC50 values determined for PF-3758309 for inhibition of prostratin, panobinostat, JQ-1 and TNF-α activity were 0.07±0.04, 0.4±0.03, 1.2±0.3 and 0.8±0.09 nM, respectively. The CC50 for PF-3758309 was 4.3±1.2 μM (therapeutic index > 3,300). Ongoing studies are evaluating the activity of the inhibitors in cells from HIV-infected individuals. Conclusion: We have identified 2 kinase inhibitors, Danusertib and PF- 3758309, which potently block the reactivation of latent HIV-1, independent of the stimulus used to reverse latency. 329 CONTRIBUTION OF LNCRNAS IN ESTABLISHMENT OF HIV LATENCY IN CENTRAL MEMORY CD4 T CELLS Wim Trypsteen 1 , Cory White 2 , Alberto Bosque 3 , Pieter Mestdagh 1 , Steve Lefever 1 , Celsa A. Spina 4 , Linos Vandekerckhove 1 , Nadejda Beliakova-Bethell 4 1 Ghent University, Ghent, Belgium, 2 University of Southampton, Southampton, UK, 3 The George Washington University, Washington, DC, USA, 4 University of California San Diego, La Jolla, CA, USA Background: HIV cure research has been hampered by the existence of a latent viral reservoir that persists in infected individuals receiving antiretroviral therapy. To date, most of the cure research has focused on protein-coding genes but recently the interest in the study of long non-coding RNA (lncRNA) has risen, as these molecules could provide insight in new therapeutic strategies and further complete insight in the HIV life cycle. Methods: Transcriptome profiling was performed (total RNA-Seq) in two primary HIV latency models of central memory CD4 T cells (TCM) to investigate changes in lncRNA expression. Subsequently, differentially expressed mRNAs and lncRNAs were identified in both models and a guilt-by-association analysis was implemented to infer biological roles for the lncRNAs in HIV latency. Results: In the primary HIV latency models, we respectively identified 826 & 471 mRNAs (87.8% & 76.2%) and 115 & 147 lncRNAs (12.2% & 23.8%) that were significantly differentially expressed (FDR<0.05) between uninfected and latently infected TCM cells. Between models, 10 lncRNAs were overlapping (oa. NEAT1 and PVT1) and many of these lncRNAs were associated with pathways involved in cell cycle regulation and pathways with a link to HIV latency: IL-7, PTEN, CSK and CCR5. In addition, a cluster of 17 lncRNAs was associated with the p53 pathway and corroborate earlier findings in this TCMmodel that illustrated p53-dependent latency establishment. One of these upregulated p53-linked

lncRNAs, 7SLRNA, has a characterized inhibitory role in the p53 pathway and would suit as a possible new therapeutical target. Conclusion: Altogether, this study demonstrates that several lncRNAs play a role in HIV latency and can be linked to biological pathways with importance in HIV latency establishment and maintenance. Some of these lncRNAs, i.e. NEAT1, PVT1 or 7SLRNA, represent possible targets for reversing HIV latency and contribute to a HIV cure.

Poster Abstracts

330 TRANSLATION EFFICIENCY LIMITS HIV-1 LATENCY REVERSAL Phillip Tomezsko 1 , Daniel R. Kuritzkes 2 , Silvi Rouskin 3 , Athe Tsibris 2

1 Harvard University, Cambridge, MA, USA, 2 Brigham and Women’s Hospital, Boston, MA, USA, 3 MIT, Cambridge, MA, USA Background: The synthesis of virus proteins is an absolute requirement for any strategy that leverages humoral or cell-mediated immunity to eradicate HIV. The purpose of this study was to determine if the HIV-1 RNA produced after latency reversal leads to efficiently translated mRNA. Methods: To comprehensively assess LRA-induced HIV-1 translation across the genome, we performed parallel mRNA-seq and ribosome profiling in the ACH-2 and J89 cell lines. We assessed HIV-1 transcription and translation after a 24-hour incubation with PMA/ionomycin (PMAi) or the histone deacetylase inhibitor romidepsin (RMD), relative to an untreated control condition. We developed an analysis pipeline that used Salmon and DESeq2 to quantify cellular and viral transcripts. Transcription and translation were reported in the unit transcripts per million (TPM), a unit of measure that normalizes by gene length and sequence depth. Translational efficiency (TE) was defined as the ratio of ribosome footprint to mRNA TPM for a given HIV-1 gene. Results: Treatment with PMAi and RMD increased virus transcription above basal levels. Averaged across all HIV-1 genes in replicate experiments, approximately 46- and 40-fold more HIV-1 mRNA TPM were observed in PMAi- or RMD-treated ACH-2, respectively, and 238- and 242-fold more HIV-1 mRNA TPM in PMAi- or RMD-treated J89, respectively, relative to untreated cells. For translation, HIV-1 ribosome footprint TPM increased 65- and 11-fold with PMAi and RMD treatment in ACH-2 and 271- and 116-fold in J89, respectively. Using HIV-1 gag, tat, and env as representative examples of unspliced, singly spliced, and multiply spliced mRNA, the TE in PMA/iono-treated ACH-2 was 1.36, 4.83, and 0.2, respectively, whereas TE after RMD was 0.15, 1.21, and 0.04. In PMA/ iono-treated J89 TE was 1.3, 22.6, and 0.9, respectively; TE after RMD was 0.8, 4.8, and 0.3. The TE ratios (PMA/iono:RMD) for gag, tat, and env were 9.4, 4.0, and 4.9 in ACH-2 and 1.8, 4.7, and 2.6 in J89. Conclusion: In both ACH-2 and J89 cells, comparable proportions of HIV-1 mRNA were produced with PMAi or RMD treatment but the translation efficiency was lower with RMD. These data support the premise that a post-transcriptional block may limit the latency reversal potential of HDACi. Evaluation of candidate LRA compounds/combinations should include a rigorous assessment of HIV-1 translation.

331 LRA TREATMENT ALTERS ANTIGEN PROCESSING AND PEPTIDE PRESENTATION IN CD4 T CELLS

Julie Boucau , Alvaro Sanchez Bernabeu, Sylvie Le Gall Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA

Background: HIV persists as a latent infection in memory CD4 T cells. Current approaches for eradication rely on a shock and kill strategy where pharmaceutical agents reactivate the latent reservoir leading to cytopathic

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