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. 2021 Mar 10;13(3):451. doi: 10.3390/v13030451

Figure 5.

Figure 5

The biotinylated antiretroviral analog of PAV-206 colocalizes with three components of assembly intermediates but does not colocalize with two host proteins that are not associated with Gag or assembly intermediates [29]. (A) Schematic of the PLA approach for detecting colocalization of PAV-206 with either Gag or the host proteins ABCE1 and DDX6. 293T cells chronically infected with HIV-1 (293T-HIV) were treated with 10 µM PAV-818 (the biotinylated active compound). PLA was performed on treated cells by incubating with primary antibody pairs (either rabbit anti-biotin with mouse anti-Gag; mouse anti-biotin with rabbit anti-ABCE1; or mouse anti-biotin with rabbit anti-ABCE1) followed by incubation with PLA secondary antibodies (anti-rabbit IgG coupled to [+] PLA oligonucleotide and anti-mouse IgG coupled to [−] PLA oligonucleotide). Addition of other PLA reagents results in connector oligonucleotides annealing to the “+” and “–” oligonucleotides only if the primary antibodies are colocalized (within 40 nm); this in turn leads to the PLA amplification reaction. The addition of an oligonucleotide that recognizes a sequence in the amplified regions and is coupled to a red fluorophore (red star) results in intense spots only at sites where the two antibody targets (biotinylated compound and Gag, or biotinylated compound and host protein) are colocalized in situ. After PLA, IF was performed by adding secondary antibody conjugated to a green fluorophore (green star) to detect any unoccupied Gag or host protein antibody, thus marking Gag- or host-protein expressing cells with low-level green fluorescence. (B) This schematic illustrates a scenario in which the biotinylated compound and host protein are more than 40 nm apart. The connector oligonucleotide will not anneal to the antibody-conjugated [+] and [−] PLA oligonucleotides; thus, little to no amplification will occur and few or no red spots will be observed. (C) Data in the three panels on the left show colocalization by PLA of Gag, ABCE1, and DDX6 with PAV-818, respectively. Data in the two panels on the right show lack of colocalization with two host proteins that are found in P-bodies and stress granules, respectively, G3BP1 and XRN1, but are not known to be associated with Gag or found in assembly intermediates. Additional PLA negative controls, PLA dose–response curves, and quantitation are found in [29].