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. 2020 Dec 3;224(2):258–268. doi: 10.1093/infdis/jiaa736

Figure 1.

Figure 1.

Analysis of intact proviral frequency and inducibility in paired PB and LN samples. (A) Map of the HIV-1 proviral genome and HIV-1 RNA species indicating positions of the IPDA and QVIA amplicons. For the IPDA, there are 2 amplicons: 1 in the packaging signal (Ψ, blue arrow), and 1 in the conserved Rev response element in the env gene (green arrow). Intact proviruses give amplification at both sites, whereas most defective proviruses (>90%) fail to give amplification at one or the other position [12]. The QVIA amplicon covers the shared 3’ end of all HIV-1 mRNAs (unspliced, singly spliced, and multiply spliced) and partially overlaps the polyA tail [30]. The transcription start site (right arrow and dotted line) and the polyadenylation signal (dashed line) are indicated. (B) Methods of analysis. The PB and LN samples were processed to PB mononuclear cells and LN mononuclear cells, respectively, and then CD4+ T cells were isolated from each preparation. CD4+ T cells from each sample were then analyzed with the IPDA to detect cells with intact proviruses [12] and the QVIA to detect inducible proviruses. In the IPDA, individual proviruses distribute into nanoliter-sized droplets are interrogated with duplex PCRs amplifying the Ψ signal region and a conserved region of the env gene. Most defective proviruses fail to give amplification at one or both sites due to large deletions and/or hypermutation, whereas intact proviruses can be directly counted as double-positive droplets [12]. Separate ddPCR analysis of a cellular gene (RPP30) allows for quantitation of input cell number and for correction for DNA shearing [12]. The QVIA is a limiting dilution RT-PCR assay that detects cells expressing polyadenylated HIV-1 RNA after activation with PMA and ionomycin [30]. The precise quantitation afforded by the use of a single round of PCR distinguishes cells with high and low levels of HIV-1 RNA, typically separated by 1–2 cycle threshold values (Supplementary Figure 1). The RT-PCR analysis of induced RNA indicated that cells expressing only low levels of HIV-1 RNA typically contained defective proviruses (Supplementary Figure 1). These cells were excluded from the analysis. DMSO, dimethyl sulfoxide; LTR, long terminal repeat.