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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

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Τίτλος: Adaptation of droplet digital PCR-based HIV transcription profiling to digital PCR and association of HIV transcription and total or intact HIV DNA
Συγγραφείς: 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
Πηγή: Tumpach , C , Rhodes , A , Kim , Y , Ong , J , Liu , H , Chibo , D , Druce , J , Williamson , D , Hoh , R , Deeks , S G , Yukl , S A , Roche , M , Lewin , S R & Telwatte , S 2023 , ' Adaptation of droplet digital PCR-based HIV transcription profiling to digital PCR and association of HIV transcription and total or intact HIV DNA ' , Viruses , vol. 15 , no. 7 , 1606 , pp. 1-17 . https://doi.org/10.3390/v15071606
Έτος έκδοσης: 2023
Θεματικοί όροι: Humans, DNA, Viral/genetics, HIV-1/genetics, Polymerase chain reaction, Proviruses/genetics, CD4-positive T-lymphocytes, HIV infections, RNA, Viral/analysis, Viral load/methods
Περιγραφή: 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.
Τύπος εγγράφου: article in journal/newspaper
Γλώσσα: English
DOI: 10.3390/v15071606
Διαθεσιμότητα: https://research-portal.st-andrews.ac.uk/en/publications/7f19ff52-6c4d-4be5-a5c6-f14875f01f68
https://doi.org/10.3390/v15071606
Rights: info:eu-repo/semantics/openAccess
Αριθμός Καταχώρησης: edsbas.93F90D61
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  Data: Adaptation of droplet digital PCR-based HIV transcription profiling to digital PCR and association of HIV transcription and total or intact HIV DNA
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  Data: <searchLink fieldCode="AR" term="%22Tumpach%2C+Carolin%22">Tumpach, Carolin</searchLink><br /><searchLink fieldCode="AR" term="%22Rhodes%2C+Ajantha%22">Rhodes, Ajantha</searchLink><br /><searchLink fieldCode="AR" term="%22Kim%2C+Youry%22">Kim, Youry</searchLink><br /><searchLink fieldCode="AR" term="%22Ong%2C+Jesslyn%22">Ong, Jesslyn</searchLink><br /><searchLink fieldCode="AR" term="%22Liu%2C+Haoming%22">Liu, Haoming</searchLink><br /><searchLink fieldCode="AR" term="%22Chibo%2C+Doris%22">Chibo, Doris</searchLink><br /><searchLink fieldCode="AR" term="%22Druce%2C+Julian%22">Druce, Julian</searchLink><br /><searchLink fieldCode="AR" term="%22Williamson%2C+Deborah%22">Williamson, Deborah</searchLink><br /><searchLink fieldCode="AR" term="%22Hoh%2C+Rebecca%22">Hoh, Rebecca</searchLink><br /><searchLink fieldCode="AR" term="%22Deeks%2C+Steven+G%22">Deeks, Steven G</searchLink><br /><searchLink fieldCode="AR" term="%22Yukl%2C+Steven+A%22">Yukl, Steven A</searchLink><br /><searchLink fieldCode="AR" term="%22Roche%2C+Michael%22">Roche, Michael</searchLink><br /><searchLink fieldCode="AR" term="%22Lewin%2C+Sharon+R%22">Lewin, Sharon R</searchLink><br /><searchLink fieldCode="AR" term="%22Telwatte%2C+Sushama%22">Telwatte, Sushama</searchLink>
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  Data: Tumpach , C , Rhodes , A , Kim , Y , Ong , J , Liu , H , Chibo , D , Druce , J , Williamson , D , Hoh , R , Deeks , S G , Yukl , S A , Roche , M , Lewin , S R & Telwatte , S 2023 , ' Adaptation of droplet digital PCR-based HIV transcription profiling to digital PCR and association of HIV transcription and total or intact HIV DNA ' , Viruses , vol. 15 , no. 7 , 1606 , pp. 1-17 . https://doi.org/10.3390/v15071606
– Name: DatePubCY
  Label: Publication Year
  Group: Date
  Data: 2023
– Name: Subject
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  Data: <searchLink fieldCode="DE" term="%22Humans%22">Humans</searchLink><br /><searchLink fieldCode="DE" term="%22DNA%22">DNA</searchLink><br /><searchLink fieldCode="DE" term="%22Viral%2Fgenetics%22">Viral/genetics</searchLink><br /><searchLink fieldCode="DE" term="%22HIV-1%2Fgenetics%22">HIV-1/genetics</searchLink><br /><searchLink fieldCode="DE" term="%22Polymerase+chain+reaction%22">Polymerase chain reaction</searchLink><br /><searchLink fieldCode="DE" term="%22Proviruses%2Fgenetics%22">Proviruses/genetics</searchLink><br /><searchLink fieldCode="DE" term="%22CD4-positive+T-lymphocytes%22">CD4-positive T-lymphocytes</searchLink><br /><searchLink fieldCode="DE" term="%22HIV+infections%22">HIV infections</searchLink><br /><searchLink fieldCode="DE" term="%22RNA%22">RNA</searchLink><br /><searchLink fieldCode="DE" term="%22Viral%2Fanalysis%22">Viral/analysis</searchLink><br /><searchLink fieldCode="DE" term="%22Viral+load%2Fmethods%22">Viral load/methods</searchLink>
– Name: Abstract
  Label: Description
  Group: Ab
  Data: 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.
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– Name: DOI
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  Data: 10.3390/v15071606
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  Data: https://research-portal.st-andrews.ac.uk/en/publications/7f19ff52-6c4d-4be5-a5c6-f14875f01f68<br />https://doi.org/10.3390/v15071606
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  Data: info:eu-repo/semantics/openAccess
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  Data: edsbas.93F90D61
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