Tracking HIV-1-Infected Cell Clones Using Integration Site-Specific qPCR

Abstract

Efforts to cure HIV-1 infection require better quantification of the HIV-1 reservoir, particularly the clones of cells harboring replication-competent (intact) proviruses, termed repliclones. The digital droplet PCR assays commonly used to quantify intact proviruses do not differentiate among specific repliclones, thus the dynamics of repliclones are not well defined. The major challenge in tracking repliclones is the relative rarity of the cells carrying specific intact proviruses. To date, detection and accurate quantification of repliclones requires in-depth integration site sequencing. Here, we describe a simplified workflow using integration site-specific qPCR (IS-qPCR) to determine the frequencies of the proviruses integrated in individual repliclones. We designed IS-qPCR to determine the frequencies of repliclones and clones of cells that carry defective proviruses in samples from three donors. Comparing the results of IS-qPCR with deep integration site sequencing data showed that the two methods yielded concordant estimates of clone frequencies (r = 0.838). IS-qPCR is a potentially valuable tool that can be applied to multiple samples and cell types over time to measure the dynamics of individual repliclones and the efficacy of treatments designed to eliminate them.

Keywords: HIV-1 reservoir; HIV-1-infected cell clones; proviral integration sites; repliclones.

Figure 1

IS-qPCR workflow. (a) Proviruses whose sequences matched gag-pro-pol RNA sequences from plasma and quantitative viral outgrowth assays (QVOA) were analyzed as described previously [11,30]. (b) Genomic DNA (gDNA) was diluted to a proviral endpoint, amplified by MDA and (c) screened (e.g., near-full-length or gag-pro-pol PCR) for the proviruses of interest. (d) The corresponding MDA product was analyzed to determine the integration site and orientation of the provirus. (e) The sequence and integration sites of proviruses of interest were confirmed by PCR amplification and sequencing of the full-length proviruses as described [11]. (f) Host and HIV-1 primers flanking the integration site were designed to amplify a ~150 bp amplicon and a probe spanning the host–virus junction was prepared. A within-run quantification standard for each provirus was generated by amplifying a sequence (<700 bp) spanning the host–virus junction from genomic DNA. (g) IS-qPCR analysis using gDNA from sample(s) of interest.

Figure 2

Alignments of the sequences of the HIV-1 proviruses in clonally expanded cells that were used to validate the IS-qPCR assay. Sequences were aligned using MUSCLE pairwise alignment. Black lines represent the sequences obtained for each of the proviruses and blank spaces represent deletions. Sequences are labeled with a letter and number designation to indicate the donor who was the source of the clone followed by the name of the gene in which the provirus was integrated. Intact infectious proviruses are denoted by ^†.

Figure 3

Strong positive correlation between clone frequencies (%) estimated by IS-qPCR and ISA for (a) all proviruses of interest, and (b) excluding provirus R-09 ABCA11P (apparent outlier). The Pearson correlation (r) and two-tailed p value were computed using the clones frequencies reported in Table 2.

Figure 4

Numbers of proviruses in specific clones, calculated from IS-qPCR, relative to total provirus in all the infected cells. Values are reported as mean copies adjusted per million total CD4+ T cells.