Argonaute 1 contributes to the transcriptional silencing of HIV-1.

HIV-1 latency remains a major barrier to viral eradication, and the mechanisms underlying the maintenance of proviral transcriptional silencing are not yet fully understood. Argonaute (Ago) proteins are well known for their roles in post-transcriptional gene silencing through microRNA-mediated pathways, but their involvement in transcriptional regulation, particularly in the context of HIV-1 infection, remains poorly characterized. Here, we demonstrate that Ago1 represses HIV-1 promoter activity across diverse latency models, independently of microRNA biogenesis pathways. Knockdown of Ago1, but not Ago2 or Dicer, significantly increased expression of latent proviruses in J-Lat cells, correlating with elevated nascent HIV-1 transcripts and reduced deposition of the repressive H3K9me3 mark at the LTR promoter. Single-molecule RNA FISH coupled with immunofluorescence revealed that Ago1 can accumulate at HIV-1 transcription sites upon activation, and biochemical analyses showed that Ago1 associates with chromatin and interacts with both initiating and elongating forms of RNA polymerase II. Notably, RNA was required for Ago1 chromatin recruitment but was dispensable for its interaction with RNA polymerase II. Using targeted HIV-1 promoter mutants, we further showed that Ago1-mediated repression is independent of key cis-regulatory elements, including the TAR stem-loop, the splice donor site SD1, and the polyadenylation signal. Collectively, our findings uncover a noncanonical role of Ago1 in HIV-1 transcriptional repression, further expanding its functional repertoire beyond RNA interference and providing new insights into the mechanisms maintaining HIV-1 latency, with possible implications for strategies aimed at reversing proviral silencing.