Figure 2

PCR with oligonucleotides that overlap the circle junction demonstrate decreased levels of HIV-1 2-LTR circles when TNPO3 is depleted. (A) Schematic representation of 2-LTR circle qPCR in which the forward primer overlaps with the circle junction. In particular the forward primers anneal with the 3′LTR and either 2 (solid arrow, Junct2 fwd) or 4 (dashed arrow, Junct4 fwd) nucleotides of the 5′LTR. (B) Quantification of 2-LTR circle PCR products with forward primer overlapping 2 nucleotides of the 5′LTR (as indicated in G, Junct2 fwd), 24 hours after infection of WT and A105T CA mutant viruses on control (Ctrl) or TNPO3 KD TZM-bl cells. The PCR products were detected using Sybr green. (C) Env- HIV-1 viruses carrying either WT or A105T mutated CA and encoding GFP-reporter (HIV-1_NL4-3GFP) were used to challenge control (Ctrl) and TNPO3 KD cells treated with 10 μM raltegravir. At 48 hrs the percent GFP⁺ cells was determined by flow cytometry as an indication of GFP expression from unintegrated viruses. (D) 2-LTR circles and autointegration events quantified by high-throughput sequencing of low-molecular weight DNA extracted from control (Ctrl) or TNPO3 KD cells, 24 hrs after transduction with WT or A105T CA mutant viruses. Ratios between the levels in control (Ctrl) KD versus TNPO3 KD are plotted. Data represent one of at least three independent experiments. Error bars represent ± SEM (n = 3).