Despite antiretroviral therapy (ART) effectively suppresses HIV-1 replication, it does not eliminate the latent reservoir of transcriptionally silent, replication-competent proviruses that can reactivate infection upon treatment interruption. “Shock and kill” strategies aimed at reversing latency have not yet achieved durable remission in patients. In contrast, the “block and lock” approach induce long-term transcriptional silencing by epigenetically locking the HIV-1 promoter into a deep latent state, resistant to reactivation.
In this study, we investigate a promoter-targeted short hairpin RNA, shPromA, to recruit Argonaute 1 (AGO1), a core RNA-induced transcriptional silencing complex (RITS) component, to the HIV-1 5′LTR. We investigated its ability to establish repressive chromatin, suppress transcription, and maintain latency even under potent latency reversing agent (LRA) challenge in a J-Lat cell model.
J-Lat A72 cells were transduced with shPromA or scrambled shRNA (shScramb) and treated with TNF-α (0.1-100 ng/mL), SAHA (0.39–100 µM), or both (1.56-25 ng/mL TNF-α + 0.39-6.25 µM SAHA) for up to 21 days. Chromatin immunoprecipitation (ChIP-qPCR) quantified Ago1, repressive histone marker H3K27me3, and activating marker H3K27ac at the HIV-1 promoter. Gag mRNA was measured by RT-qPCR. One-way ANOVA with Tukey’s post hoc test was used (n = 4; p < 0.05).
Without stimulation, shPromA enriched AGO1 (4.8 ± 0.6-fold) and H3K27me3 (3.9 ± 0.4-fold), while reducing H3K27ac to 32 ± 5% of control (p < 0.001), corresponding to a 93 ± 2%
reduction in Gag mRNA. Upon TNF-α challenge, shPromA maintained suppression at 0.09 ± 0.02-fold (p < 0.0001) compared to control. SAHA induced 28 ± 4-fold Gag upregulation in controls, versus only 0.14 ± 0.03-fold in shPromA cells (p < 0.0001). Combined TNF-α + SAHA triggered a 175 ± 20-fold increase in controls but was limited to 0.17 ± 0.04-fold in shPromA cells, with repression persisting for 21 days. ChIP confirmed sustained AGO1 enrichment (~25-fold), elevated H3K27me3 and H3K9me3, and depleted H3K27ac (~120-fold; p < 0.0001). Under combined TNF + SAHA, Ago1 enriched 25-fold and H3K9me3 increased 3.5-fold (p < 0.0001), while H3K27Ac remained suppressed, supporting the establishment of a heterochromatin state resistant to reactivation.
shPromA induces durable epigenetic silencing at the HIV-1 promoter by guiding Ago1 and repressive histone modifications, effectively locking the virus in a latent state. This RNA-guided, promoter-targeted “block and lock” strategy confers robust resistance to potent LRA-induced reactivation and represents a promising avenue toward sustained HIV-1 remission without lifelong ART.
HIV-1 latency, shPromA, Ago1, Block and Lock, epigenetic silencing, RNA therapeutics