Potent latency reversal enables in-depth transcriptomic analyses of the HIV reservoir


BACKGROUND: Extensive characterization of the translation-competent reservoir has been hampered by the limited capacity of current latency reversing agents (LRAs) at inducing HIV reactivation in vitro. Here, we describe a new LRA combination (JNJ877+PNB) that induces potent latency reversal without inducing global T cell activation, and we took advantage of these unique properties to study transcriptomic features of the inducible reservoir.
METHODS: CD4 T cells from 22 ART-treated individuals were stimulated for 24H with PMA/ionomycin (PMA/i) or with JNJ877 in combination with panobinostat (PNB). The frequency of cells expressing p24 and viral release in the supernatant were assessed by HIV-Flow and p24-SIMOA, respectively. HIV clones reactivated by JNJ877+PNB were compared to those reactivated with PMA/i using the STIP-seq assay, which allows for the simultaneous assessment of the integration site and proviral sequence from p24+ cells. Single-cell RNA-seq on sorted p24-/p24+ cells from 7 ART-treated individuals was used to study cellular and viral transcripts following stimulation with JNJ877 or JNJ877+PNB.
RESULTS: JNJ877+PNB induced HIV reactivation in a larger fraction of CD4 T cells than PMA/i (n=22, p<0.00001, fold increase=5X). Similar results were obtained with SIMOA (n=4), confirming an effective release of viral particles in the supernatant. Clones reactivated with JNJ877+PNB were mostly shared with the ones induced by PMA/i, although some were represented in different proportions. Single-cell RNA-seq analyses showed that JNJ877 does not modify the cellular transcriptome of CD4 T cells. Following JNJ877 treatment, p24+ cells significantly expressed higher levels of a novel long non-coding RNA, SOD1P3, CCL5 and GZMA, while expressing lower levels of ATG10 and IL7R when compared to p24- cells. Finally, transcriptomic analyses on 321 p24+ cells revealed that proviruses with a defective major splice donor (MSD) site use alternative splice sites up- and/or downstream of the MSD, suggesting an underestimated role of these proviruses in HIV pathogenesis.
CONCLUSIONS: We report a combination of LRAs that induces latency reversal in a higher proportion of latently infected cells compared to PMA/i, without inducing global T cell activation. Therefore, JNJ877+PNB appears as a promising LRA combination to reactivate HIV in vitro and in vivo, paving the way to an HIV cure.