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Adaptation of Droplet Digital PCR-Based HIV Transcription Profiling to Digital PCR and Association of HIV Transcription and Total or Intact HIV DNA
In most people living with HIV (PLWH) on effective antiretroviral therapy (ART), cell-associated viral transcripts are readily detectable in CD4+ T cells despite the absence of viremia. Quantification of HIV RNA species provides insights into the transcriptional activity of proviruses that persist i...
Autores principales: | , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10384802/ https://www.ncbi.nlm.nih.gov/pubmed/37515292 http://dx.doi.org/10.3390/v15071606 |
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author | Tumpach, Carolin Rhodes, Ajantha Kim, Youry Ong, Jesslyn Liu, Haoming Chibo, Doris Druce, Julian Williamson, Deborah Hoh, Rebecca Deeks, Steven G. Yukl, Steven A. Roche, Michael Lewin, Sharon R. Telwatte, Sushama |
author_facet | Tumpach, Carolin Rhodes, Ajantha Kim, Youry Ong, Jesslyn Liu, Haoming Chibo, Doris Druce, Julian Williamson, Deborah Hoh, Rebecca Deeks, Steven G. Yukl, Steven A. Roche, Michael Lewin, Sharon R. Telwatte, Sushama |
author_sort | Tumpach, Carolin |
collection | PubMed |
description | In most people living with HIV (PLWH) on effective antiretroviral therapy (ART), cell-associated viral transcripts are readily detectable in CD4+ T cells despite the absence of viremia. Quantification of HIV RNA species provides insights into the transcriptional activity of proviruses that persist in cells and tissues throughout the body during ART (‘HIV reservoir’). One such technique for HIV RNA quantitation, ‘HIV transcription profiling’, developed in the Yukl laboratory, measures a series of HIV RNA species using droplet digital PCR. To take advantage of advances in digital (d)PCR, we adapted the ‘HIV transcription profiling’ technique to Qiagen’s dPCR platform (QIAcuity) and compared its performance to droplet digital (dd)PCR (Bio-Rad QX200 system). Using RNA standards, the two technologies were tested in parallel and assessed for multiple parameters including sensitivity, specificity, linearity, and intra- and inter-assay variability. The newly validated dPCR assays were then applied to samples from PLWH to determine HIV transcriptional activity relative to HIV reservoir size. We report that HIV transcriptional profiling was readily adapted to dPCR and assays performed similarly to ddPCR, with no differences in assay characteristics. We applied these assays in a cohort of 23 PLWH and found that HIV reservoir size, based on genetically intact proviral DNA, does not predict HIV transcriptional activity. In contrast, levels of total DNA correlated with levels of most HIV transcripts (initiated, proximally and distally elongated, unspliced, and completed, but not multiply spliced), suggesting that a considerable proportion of HIV transcripts likely originate from defective proviruses. These findings may have implications for measuring and assessing curative strategies and clinical trial outcomes. |
format | Online Article Text |
id | pubmed-10384802 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-103848022023-07-30 Adaptation of Droplet Digital PCR-Based HIV Transcription Profiling to Digital PCR and Association of HIV Transcription and Total or Intact HIV DNA Tumpach, Carolin Rhodes, Ajantha Kim, Youry Ong, Jesslyn Liu, Haoming Chibo, Doris Druce, Julian Williamson, Deborah Hoh, Rebecca Deeks, Steven G. Yukl, Steven A. Roche, Michael Lewin, Sharon R. Telwatte, Sushama Viruses Article In most people living with HIV (PLWH) on effective antiretroviral therapy (ART), cell-associated viral transcripts are readily detectable in CD4+ T cells despite the absence of viremia. Quantification of HIV RNA species provides insights into the transcriptional activity of proviruses that persist in cells and tissues throughout the body during ART (‘HIV reservoir’). One such technique for HIV RNA quantitation, ‘HIV transcription profiling’, developed in the Yukl laboratory, measures a series of HIV RNA species using droplet digital PCR. To take advantage of advances in digital (d)PCR, we adapted the ‘HIV transcription profiling’ technique to Qiagen’s dPCR platform (QIAcuity) and compared its performance to droplet digital (dd)PCR (Bio-Rad QX200 system). Using RNA standards, the two technologies were tested in parallel and assessed for multiple parameters including sensitivity, specificity, linearity, and intra- and inter-assay variability. The newly validated dPCR assays were then applied to samples from PLWH to determine HIV transcriptional activity relative to HIV reservoir size. We report that HIV transcriptional profiling was readily adapted to dPCR and assays performed similarly to ddPCR, with no differences in assay characteristics. We applied these assays in a cohort of 23 PLWH and found that HIV reservoir size, based on genetically intact proviral DNA, does not predict HIV transcriptional activity. In contrast, levels of total DNA correlated with levels of most HIV transcripts (initiated, proximally and distally elongated, unspliced, and completed, but not multiply spliced), suggesting that a considerable proportion of HIV transcripts likely originate from defective proviruses. These findings may have implications for measuring and assessing curative strategies and clinical trial outcomes. MDPI 2023-07-22 /pmc/articles/PMC10384802/ /pubmed/37515292 http://dx.doi.org/10.3390/v15071606 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Tumpach, Carolin Rhodes, Ajantha Kim, Youry Ong, Jesslyn Liu, Haoming Chibo, Doris Druce, Julian Williamson, Deborah Hoh, Rebecca Deeks, Steven G. Yukl, Steven A. Roche, Michael Lewin, Sharon R. Telwatte, Sushama Adaptation of Droplet Digital PCR-Based HIV Transcription Profiling to Digital PCR and Association of HIV Transcription and Total or Intact HIV DNA |
title | Adaptation of Droplet Digital PCR-Based HIV Transcription Profiling to Digital PCR and Association of HIV Transcription and Total or Intact HIV DNA |
title_full | Adaptation of Droplet Digital PCR-Based HIV Transcription Profiling to Digital PCR and Association of HIV Transcription and Total or Intact HIV DNA |
title_fullStr | Adaptation of Droplet Digital PCR-Based HIV Transcription Profiling to Digital PCR and Association of HIV Transcription and Total or Intact HIV DNA |
title_full_unstemmed | Adaptation of Droplet Digital PCR-Based HIV Transcription Profiling to Digital PCR and Association of HIV Transcription and Total or Intact HIV DNA |
title_short | Adaptation of Droplet Digital PCR-Based HIV Transcription Profiling to Digital PCR and Association of HIV Transcription and Total or Intact HIV DNA |
title_sort | adaptation of droplet digital pcr-based hiv transcription profiling to digital pcr and association of hiv transcription and total or intact hiv dna |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10384802/ https://www.ncbi.nlm.nih.gov/pubmed/37515292 http://dx.doi.org/10.3390/v15071606 |
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