Quantitation of Integrated HIV Provirus by Pulsed-Field Gel Electrophoresis and Droplet Digital PCR

Abstract

We utilized pulsed-field gel electrophoresis (PFGE) to purify high-molecular-weight DNA from HIV-infected cells. This purification, in combination with our previously described droplet digital PCR (ddPCR) assay, was used to develop a method to quantify proviral integrated HIV DNA free of lower-molecular-weight species of HIV DNA. Episomal 2-long-terminal-repeat (2-LTR) circles were completely cleared from HIV DNA samples. Technical replicates of the complete assay, starting with the same specimens, resulted in no statistical differences in quantification of integrated HIV sequences in cellular DNA from cells from HIV-infected subjects after prolonged treatment with antiretroviral therapy (ART). The PFGE ddPCR assay was compared to the quantitative PCR (qPCR) assay, the most widely used assay to measure proviral integrated HIV DNA. Spearman's rho nonparametric correlation determined PFGE ddPCR results to be positively correlated with qPCR results ( = 0.7052; = 0.0273). In summary, PFGE ddPCR is a sensitive, reproducible, and robust method to measure proviral integrated HIV DNA and is theoretically more accurate than previously described assays, because it is a direct measure of integrated HIV DNA.

Next-Generation Sequencing in a Direct Model of HIV Infection Reveals Important Parallels to and Differences from In Vivo Reservoir Dynamics

“…2. This fraction is expected to be enriched for integrated HIV DNA, as previously shown by others (27). NGS revealed a similar decline in the intact HIV copies/cell over time in both activated and resting T cells.…”

“…A similar argument can be raised in studies utilizing NGS to define the pre-ART composition of the reservoir or the change in the viral landscape in the first months after ART initiation (45). The use of BluePippin was proposed by the Richman laboratory as an approach to measure integrated HIV DNA (27). We further advanced the use of this technique by providing proof of concept of the striking difference that exists between sequences obtained from total versus integrated HIV DNA in vitro.…”

Histone deacetylase inhibitors induce complex host responses that contribute to differential potencies of these compounds in HIV reactivation

“…The high-molecular weight fraction of genomic DNA (ϳ23 kb) was recovered from 0.5% low melt agarose gels using the Zymoclean Large Fragment DNA Recovery Kit (Zymo Research Corp., Irvine, CA). The DNA was then quantified using ddPCR for HIV gag, 2LTR circles, and RPP30 host cell genomic DNA (51). Total cell number was determined by dividing the number of detected RPP30 copies by 2.…”

Measuring the Success of HIV-1 Cure Strategies

“…-Cannot distinguish between intact and defective provirus so overestimates the reservoir -Quantification relative to a standard so prone to bias -Highly specific and prone to error from primer/template mismatches Kostrikis et al, 2002Beloukas et al, 2009van der Sluis et al, 2013Munir et al, 2013Casabianca et al, 2014Rouzioux et al, 2014Vandergeeten et al, 2014 Brussel et al, 2005Yu et al, 2008Liszewski et al, 2009Brady et al, 2013Agosto et al, 2007De Spiegelaere et al, 2014Vandergeeten et al, 2014Lada et al, 2018 Digital Henrich et al, 2012Strain et al, 2013Malatinkova et al, 2015Henrich et al, 2017Lada et al, 2018 Cell associated RNA Measures all or different forms of cell associated RNA with the rationale that it is more likely to measure replication competent provirus than defective -More sensitivity for replication competent provirus -Cannot distinguish transcripts that arise from replication competent cells and defective cells Archin et al, 2012Pasternak et al, 2012Shan et al, 2013Cillo et al, 2014Yucha et al, 2017Massanella et al, 2018Yukl et al, 2018 TILDA Measures multiply spliced tat/rev transcripts following stimulation of CD4 T cells plated in limiting dilution -Higher sensitivity for replication competent provirus -Faster, cheaper and less resources needed than VOA Measured transcripts may arise from defective proviral genomes Procopio et al, 2015Frank et al, 2019Bertoldi et al, 2020 ISH and flow cytometry…”

“…Additionally, only 10% of integrated HIV-1 is detected by this assay because 90% of integrated provirus is too far from an Alu sequence to be exponentially amplified and a correction factor must therefore be applied to the quantification (Agosto et al, 2007;Yu et al, 2008;Liszewski et al, 2009;De Spiegelaere et al, 2014). Accuracy is further limited by linear amplification of unintegrated HIV-1 DNA, though the effect of this can be partially negated by simultaneous pre-amplification with only the HIV-1 specific primer to enable distinction between integrated and unintegrated DNA (O'Doherty et al, 2002;Yu et al, 2008) or by pulsed-field gel electrophoresis (PFGE) prior to amplification to remove low molecular weight DNA (Lada et al, 2018). Despite its limitations and owing to the various improvements made, quantification integrated HIV-1 via Alu PCR is a powerful and high-throughput method to quantify the LR.…”