Assays for precise quantification of total (including short) and elongated HIV-1 transcripts

• This manuscript reports RT-ddPCR assays to measure total and elongated HIV RNAs.
• Traditional RT priming strategies detect <4% of short HIV transcripts.
• Short HIV transcripts can be efficiently detected by RT from a polyA linker.
• This approach may be applied to other difficult-to-prime RNA targets.

Despite intensive study, it is unclear which mechanisms are responsible for latent HIV infection in vivo. One potential mechanism is inhibition of HIV transcriptional elongation, which results in short abortive transcripts containing the trans-activation response (TAR) region. Because the relative levels of total (including short) and processive transcripts provide measures of HIV transcriptional initiation and elongation, there is a compelling need for techniques that accurately measure both. Nonetheless, prior assays for total transcripts have been semi-quantitative and have seen limited application to patient samples. This manuscript reports the validation of quantitative reverse transcription (RT) droplet digital PCR assays for measurement of total (TAR) and processive (R-U5/gag) HIV transcripts. Traditional RT priming strategies can efficiently detect the TAR region on long HIV transcripts but detect <4% of true short transcripts. The TAR assay presented here utilizes an initial polyadenylation step, which provides an accessible RT priming site and detects short and long transcripts with approximately equal efficiency (70%). By applying these assays to blood samples from 8 ART-treated HIV+ individuals, total HIV transcripts were detected at levels >10-fold higher than elongated transcripts, implying a substantial block to transcriptional elongation in vivo. This approach may be applied to other difficult-to-prime RNA targets.