Background
Antiretroviral therapy (ART) reduces but does not eliminate intracellular HIV-1 transcription in people with HIV (PWH). While proximal virus transcripts can be detected in blood and tissue, the presence of unspliced distal transcripts is less well characterized. Qualitatively, the sequences of HIV-1 caRNA that are induced by latency reversal agents (LRA) are less well studied than their quantitative levels.
Methods
To understand if similar parts of the HIV-1 proviral landscape are transcribed during LRA exposure, we used cryopreserved PBMC from three PWH on ART to amplify near-full length (NFL) and env-specific HIV-1 proviral DNA and caRNA single genome amplicons (SGA) before and after a 24-hour exposure to PMA-ionomycin or the LRA boosting combination of low-dose bryostatin (1nM) and romidepsin 20nM. Amplicons were sequenced, assessed for hypermutation, trimmed, and aligned. Neighbor-joining trees with the Tamura-Nei model using HXB2 as an outgroup, 1,000 bootstrapped replicates, and a support threshold of 70% were generated. A convenience sample size of 20 SGAs each for HIV-1 NFL DNA, env-specific DNA, and env caRNA for each condition was used.
Results
An average of 100 SGA were generated per participant. HIV-1 DNA contamination was not detected in caRNA. HIV-1 env-containing caRNA were amplifiable from all three participants’ PBMC before LRA exposure; based on our primer locations, these SGA represent unspliced HIV-1 caRNA. These unspliced caRNAs were spread across the phylogenies and included hypermutated sequences. The choice of primers, NFL vs env-specific, affected the detection of hypermutated proviral DNA. We found that LRA boosting and PMAi can activate HIV-1 transcription from similar parts of the virus reservoir. LRA boosting stimulated hypermutated HIV-1 caRNA transcription in some participants, whereas PMAi did not. We found that LRA boosting and PMAi can induce identical HIV-1 mRNA transcripts as occurs when ART is interrupted.
Conclusions
Unspliced HIV-1 caRNA of 8-9kb in length, some of which were hypermutated, were detected during ART and were most likely from >1 transcriptionally active cellular clones. HIV-1 latency during treated infection is best described a period of inefficient, but not absent, virus mRNA production in blood. Hypermutants may contribute to HIV-1 caRNA quantifications. Treatment with HIV-1 LRA can induce virus RNA transcription identical in sequence to that produced during treatment interruptions.
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