title: Assays for precise quantification of total (including short) and elongated HIV-1 transcripts.
abstract: 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.
authors.affiliation: San Francisco Veterans Affairs (VA) Medical Center and University of California, San Francisco (UCSF), 4150 Clement Street, 111W, San Francisco, CA, 94121, USA; Current address: Department of Infectious Diseases, Luzerner Kantonsspital, 6000 Lucerne 16, Switzerland.
authors.forename: Philipp
authors.initials: P
authors.lastname: Kaiser
authors.affiliation: San Francisco Veterans Affairs (VA) Medical Center and University of California, San Francisco (UCSF), 4150 Clement Street, 111W, San Francisco, CA, 94121, USA.
authors.forename: Sunil K
authors.initials: SK
authors.lastname: Joshi
authors.affiliation: San Francisco Veterans Affairs (VA) Medical Center and University of California, San Francisco (UCSF), 4150 Clement Street, 111W, San Francisco, CA, 94121, USA.
authors.forename: Peggy
authors.initials: P
authors.lastname: Kim
authors.affiliation: San Francisco Veterans Affairs (VA) Medical Center and University of California, San Francisco (UCSF), 4150 Clement Street, 111W, San Francisco, CA, 94121, USA.
authors.forename: Peilin
authors.initials: P
authors.lastname: Li
authors.affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, MS BCM385, Houston, TX, 77030, USA.
authors.forename: Hongbing
authors.initials: H
authors.lastname: Liu
authors.affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, MS BCM385, Houston, TX, 77030, USA.
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Rice
authors.affiliation: San Francisco Veterans Affairs (VA) Medical Center and University of California, San Francisco (UCSF), 4150 Clement Street, 111W, San Francisco, CA, 94121, USA.
authors.forename: Joseph K
authors.initials: JK
authors.lastname: Wong
authors.affiliation: San Francisco Veterans Affairs (VA) Medical Center and University of California, San Francisco (UCSF), 4150 Clement Street, 111W, San Francisco, CA, 94121, USA. Electronic address: Steven.yukl@ucsf.edu.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Yukl
mesh_terms: D015967:Gene Expression Regulation, Viral; D015658:HIV Infections / Q000097:blood / Q000175:diagnosis / Q000188:drug therapy / Q000821:virology*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008297:Male; D026723:Polyadenylation; D012367:RNA, Viral / Q000097:blood* / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D048348:Reverse Transcription; D014158:Transcription, Genetic; D017735:Virus Latency
chemical_list: D012367:RNA, Viral
keywords: Elongation; HIV-1; Latency; Short or abortive transcript; TAR; Transcription