CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

573

BACH2 Controls Seeding and Establishment of Long-Lived HIV-1 Reservoir in Memory CD4+ T Cells Hongbo Gao, Yuhao Li, Liang Shan Washington University in St Louis, St Louis, MO, USA Background: Antiretroviral therapy (ART) is not a cure for HIV-1 infection due to the formation of latent reservoirs. Most long-lived viral reservoir cells are infected by the virus near the time of therapy initiation. A better understanding of the early events in viral reservoir seeding may help prevent latent reservoir formation. Unlike quiescent reservoir cells, CD4+CCR5+ T cells as the primary targets in HIV-1 infection present an activated Th1 and Th17 effector phenotype. This discrepancy suggests phenotypic and functional changes occur in CCR5+effector cells after HIV-1 entry, necessitating further elucidation. Methods: We profiled CD4+CCR5+ T cells and evaluated in-vitro re differentiation of activated CCR5+ cells to explore how terminal effectors reprogram into quiescent memory cells. We revealed that BACH2 is a key transcription factor in memory-precursor (MP) conversion of CCR5+cells which was validated through BACH2-knockout and overexpression assays. To investigate the impact of BACH2 on the HIV-1 latent reservoir, BACH2 knockout humanized mice were infected with R5-tropic virus and treated with ART. The measurement of HIV-1 DNA frequency in tissues and viral rebound analysis were performed. Results: Upon TCR stimulation, CCR5 was upregulated predominantly in cells committed to terminal differentiation. Furthermore, the majority of CCR5+ cells became functionally impaired, characterized by elevated activation or exhaustion markers and increased apoptosis susceptibility. However, a small subset of these cells undergoes re-differentiation into memory precursor (MP) cells, a process regulated by BACH2. BACH2 deficiency prevents MP cell formation and reduces TCF1 expression, weakening recall response and cell proliferation. In humanized mice, BACH2 knockout did not affect plasma viral load or HIV-1 DNA frequency in tissues compared to controls. However, after ART, the knockout group had a lower frequency of HIV-1 DNA. Viral rebound occurred in 90% of control mice (9/10) but only in 29% of BACH2-knockout mice (4/14). Conclusions: Our study shows that BACH2 reverses CCR5+cells from terminal differentiated cells to memory precursors. The absence of BACH2 abrogates the seeding of latent reservoirs, revealing the essential role of BACH2 in the seeding and establishment of long-lived HIV-1 reservoir in memory CD4+ T cells. Our finding identifies a potential avenue for targeted interventions. Blockade of HIV Latency Reversal in CD4+ T Cells From ART-Suppressed PLWH by the Antisense RNA AST Rui Li 1 , Fabio Romerio 1 , Kaveh Daneshvar 2 , Xinjie Ji 1 , Michelle Pleet 3 , Grace Igbinosun 1 , Mohd Shameel Iqbal 1 , Fatah Kashanchi 3 , Alan Mullen 4 1 The Johns Hopkins University School of Medicine, Baltimore, MD, USA, 2 Massachusetts General Hospital, Boston, MA, USA, 3 George Mason University, Fairfax, VA, USA, 4 University of Massachusetts, Worcester, MA, USA Background: Cure strategies that seek to permanently block reversal of HIV-1 latency remain largely unexplored. We previously showed that the HIV-1 antisense transcript AST induces a closed chromatin state at the HIV-1 5’LTR that blocks transcription and promotes latency in cell line models. Here, we assessed the ability of AST to prevent latency reversal in CD4+ T cells from PLWH, and its potential application in “block and lock” cure strategies. We also elucidated the underlying molecular mechanisms. Methods: AST or a control RNA were expressed by nucleofection in CD4+ cells from PLWH under suppressive ART. Cells were then treated with SAHA, Panobinostat, or anti-CD3/CD28, and cell-associated HIV-1 gag RNA levels were evaluated by RT-qPCR. Identification of AST structural domains and sequence motifs was performed in the Jurkat E4 latency model. RIP and ChIRP assays were used to study the interaction of AST with chromatin factors and the 5’LTR, respectively. ChIP assays were used to assess epigenetic modifications at the 5’LTR. Results: We found that ectopic expression of AST in CD4+ T cells from ART suppressed PLWH blocks latency reversal in response to the HDAC inhibitors, SAHA and Panobinostat, and to TCR stimulation, enforcing HIV-1 transcriptional silencing. The ability of AST to promote HIV-1 latency requires a quadruplex forming G-rich motif of AST that interacts with and recruits the suppressive epigenetic complex, PRC2 to the 5’LTR. Replacement of this motif with an unrelated sequence disrupts AST binding to PRC2 and reversed latency. Two pyrimidine-rich motifs in the 5’ terminus of AST facilitate its interaction with the 5’ LTR via sequence homology. Targeted nucleotide substitutions in these

two pyrimidine-rich motifs abolish the interaction of AST with the 5’ LTR and its latency-promoting activity. An unbiased proteomics screen of AST interactors revealed an array of previously known and potential new HIV-1-suppressive factors, including the SWI/SNF complex, TDP-43, PTBP1, MATR3, and hnRNPK. Conclusions: Ectopic expression of AST inhibits HIV-1 transcription in PLWH derived CD4+ T cells and locks proviruses in a state of permanent latency. AST functions as a scaffold for the recruitment and assembly of a biomolecular complex at the 5’ LTR leading to epigenetic silencing of HIV-1 transcription. Overall, our studies identify AST as a first-in-class biological molecule capable of enforcing latency in PLWH-derived cells, showing that AST has potential applications in cure strategies.

575

HIV Reactivation Potential Evaluated by RNA-Seq and DNA Methylation Following LRA Stimulation Giuseppe Rubens Pascucci 1 , Elena Emili 2 , Alessia Neri 2 , Elena Morrocchi 2 , Nicole Colantoni 1 , Arianna Rotili 2 , Chiara Pighi 1 , Giulio Olivieri 1 , Stefania Bernardi 1 , Giovanna Leone 1 , Ann M. Chahroudi 3 , Mathias Lichterfeld 4 , Benjamin Reece Bone 4 , Nicola Cotugno 1 , Paolo Palma 1 1 Bambino Gesu Children's Hospital, Rome, Italy, 2 University of Rome Tor Vergata, Rome, Italy, 3 Emory University, Atlanta, GA, USA, 4 Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA Background: Latency-reversing agents (LRAs) have shown promise in reactivating HIV reservoir within “shock and kill”approaches. However, molecular mechanisms underlying HIV reactivation within CD4+ T cells as well as predictors of LRA efficacy remain partially unknown. Here, we investigated the transcriptomic and whole genome DNA methylation profile of CD4+ T cells cultured in vitro with or w/o two LRAs and assessed for associations with HIV viral reservoir inducibility. Methods: Large-scale samples (2,5 – 9,4x10 9 PBMCs) were collected through leukapheresis from 9 study participants (10 – 27 years old, 7 female and 2 male) with perinatally acquired HIV who initiated ART within the first year of life and maintained viral suppression for several years (IQR: 13-22 years). The HIV reservoir was characterized by Flip-Seq, IPDA and QVOA. HIV inducibility was calculated as infectious units/10 6 cells (by QVOA) divided by the number of intact proviruses/10 6 cells. Purified CD4 + T cells were cultured in-vitro for 20h with two LRAs: fimepinostat (CUD) and Ingenol-3-angelate (PEP), left unstimulated (UNS) or cultured with PMA+ionomycin+IL2 as positive control (CTR). After stimulation, RNA and DNA were extracted and sequenced using the long-reads Oxford Nanopore. DNA methylation was analyzed for both 5-methylcytosine (5mC) and N6-methyladenosine (6mA). An algorithm was developed to identify genes and genomic sites potentially associated with HIV inducibility. Transcriptome and epigenome were integrated using 4 public databases by intersecting the methylated sites with the genomic position of enhancer binding sites. Results: The analysis of 3 distinct OMIC layers revealed 2743 Features Associated with Inducibility (FAI) upon four distinct experimental conditions: UNS, CUD, PEP, and CTR vs UNS. CUD showed the highest number of FAI at RNA and 5mC level (see Fig 1). The intersection of FAI informed by RNA-Seq and 5mC showed that Vacuolar Protein Sorting 8 (VPS8) and Phosphodiesterase 8 (PDE8A) genes were associated with HIV inducibility at both OMIC layers. These proteins are involved in HIV replication mechanisms, potentially representing important in vitro correlates of the potential of HIV reactivation by LRAs. Conclusions: MultiOMIC analysis of CD4 + T cells upon LRA stimulation revealed molecular features associated with HIV inducibility. These features may shed

Poster Abstracts

574

CROI 2025 152