期刊
JOURNAL OF VIROLOGICAL METHODS
卷 242, 期 -, 页码 1-8出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jviromet.2016.12.017
关键词
HIV-1; Latency; Transcription; Elongation; TAR; Short or abortive transcript
资金
- U.S. Department of Veterans Affairs [IK2 CX000520-01, 101 BX000192]
- National Institute of Diabetes and Digestive and Kidney Diseases (N.I.D.D.K.) at the National Institutes of Health [1R01 DK108349-01]
- National Institute of Allergy and Infectious Diseases at the National Institutes of Health [R56 AI116342, R33 A1116218, U19 AI096109, P30 AI036211, R21 A1116173]
- American Foundation for AIDS Research (amfAR) Institute for HIV Cure Research [109301]
- Swiss National Science Foundation [PBZHP3_147260]
- Swiss National Science Foundation (SNF) [PBZHP3_147260] Funding Source: Swiss National Science Foundation (SNF)
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. Published by Elsevier B.V.
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