Fig 2: Nonimmune controls for the TIAR-G3BP1 and DDX6-XRN1 proximity ligation assays. (A) NI controls for the TIAR-G3BP1 PLA. Above each PLA image is the schematic corresponding to the PLA approach in that panel. Untreated 293T cells chronically infected with HIV-1 (293T-HIV) were analyzed by PLA. For the positive control (image and schematic on the right), PLA was performed by incubating with primary antibodies, mouse anti-TIAR and rabbit anti-G3BP1, followed by PLA secondary antibodies and other reagents as described in Fig. 6. In the two negative controls (images and schematics at left and center), one primary antibody was replaced with an NI control antibody from the same species, as indicated. Red spots indicating colocalization of the TIAR with G3BP should be absent when either primary antibody is replaced by an NI antibody. After PLA, TIAR or G3BP IF was performed to mark host protein-expressing cells with low-level green fluorescence. Images show a representative field for each of the three antibody pairs. Fields are shown as a merge of three color channels: the red channel shows TIAR-G3BP1 PLA, TIAR-NI PLA, or NI-G3BP1 PLA, as indicated by red labeling above images; the green channel shows TIAR IF or G3BP1 IF as indicated by green labeling above images; and the blue channel shows DAPI-stained nuclei. Scale bars, 10 μm. Graph below shows the average number of PLA spots per cell for each antibody pair. Five fields were analyzed for each group (containing a total of 45 to 62 cells per group), with error bars showing the SEM. (B) NI controls for the DDX6-XRN1 PLA. Above each PLA image is the schematic corresponding to the to the PLA approach in that panel. Untreated 293T cells chronically infected with HIV-1 (293T-HIV) were analyzed by PLA. For the positive control (image and schematic on the right), PLA was performed by incubating with primary antibodies, mouse anti-DDX6 and rabbit anti-XRN1, followed by PLA secondary antibodies and other reagents as described in Fig. 6. In the two negative controls (images and schematics at left and center), one primary antibody was replaced with an NI control antibody from the same species, as indicated. Red spots indicating colocalization of the DDX6 with XRN1 should be absent when either primary antibody is replaced by a NI antibody. Following PLA, DDX6 or XRN1 IF was performed to mark host protein-expressing cells with low-level green fluorescence. Images show a representative field for each of the three antibody pairs. Fields are shown as a merge of three color channels: the red channel shows DDX6-XRN1 PLA, DDX6-NI PLA, or NI-XRN1 PLA, as indicated by red labeling above images; the green channel shows DDX6 IF or XRN1 IF, as indicated by green labeling above images; and the blue channel shows DAPI-stained nuclei. Scale bars, 10 μm. Graph below shows the average number of PLA spots per cell for each antibody pair. Five fields were analyzed for each group (containing a total of 46 to 73 cells per group), with error bars showing the SEM.

Fig 3: Nonimmune controls for the biotin-Gag, biotin-ABCE1, and biotin-DDX6 proximity ligation assays. (A) NI controls for the biotin-Gag PLA. Above each PLA image is shown the schematic corresponding to the PLA approach in that panel. 293T cells chronically infected with HIV-1 (293T-HIV) were treated with 10 μM PAV-818 (the biotinylated active compound) for 16 h. For the positive control (image and schematic on the right), PLA was performed by incubation with primary antibodies, mouse anti-Gag and rabbit anti-biotin, followed by PLA secondary antibodies and other reagents as described in Fig. 6. In the two negative controls (image and schematic at left and center), one primary antibody was replaced with a NI control antibody from the same species, as indicated. Red spots indicating colocalization of the biotinylated compound with Gag should be absent when either primary antibody is replaced by a NI antibody. After PLA, IF was performed to mark either Gag-expressing or biotin-containing cells with low-level green fluorescence. Images show a representative field for each of the three antibody pairs. Fields are shown as a merge of three color channels: the red channel shows biotin-NI PLA, NI-Gag PLA, or biotin-Gag PLA, as indicated by red labeling above images; the green channel shows biotin IF or Gag IF, as indicated by green labeling above images; and the blue channel shows DAPI-stained nuclei. Scale bars, 10 μm. Graph below shows the average number of PLA spots per cell for each antibody pair. A total of 10 to 20 fields were analyzed for each group (containing 118 to 213 cells per group), with error bars showing the SEM. (B) NI controls for the biotin-ABCE1 PLA (top row of images) and biotin-DDX6 PLA (bottom row of images). Figure organization and 293T-HIV treatments are as in panel A above. For the positive control (images and schematic on the right), PLA was performed by incubation with primary antibodies, rabbit anti-host protein (ABCE1 in top row; DDX6 in bottom row) and mouse anti-biotin, followed by PLA secondary antibodies and other reagents as described in Fig. 6. In the two negative controls (images and schematics at left and center), one primary antibody was replaced with a NI control antibody from the same species, as indicated. Red spots indicating colocalization of the biotinylated compound with the host proteins ABCE1 and DDX6 should be absent when either primary antibody is replaced by a NI antibody. After PLA, IF was performed with the indicated antibody (green fluorescence). Images show a representative field for each of the three antibody pairs. Fields are shown as a merge of three color channels: the red channel shows biotin-NI PLA, NI-host protein PLA, or biotin-host protein PLA, as indicated by red labeling above images; the green channel shows biotin IF or host protein IF, as indicated by green labeling above images; and the blue channel shows DAPI-stained nuclei. Scale bars, 10 μm. Graphs below shows the average number of PLA spots per cell for each antibody pair (ABCE1 on the left; DDX6 on the right). Five fields were analyzed for each group (containing a total of 43 to 77 cells per group), with error bars showing the SEM.

Fig 4: The biotinylated antiretroviral PAV-206 analog colocalizes in situ with DDX6, an RNA granule protein in assembly intermediates that colocalizes with ABCE1. (A) Schematic of the PLA approach for detecting colocalization of compound with DDX6. 293T cells chronically infected with HIV-1 (293T-HIV) or uninfected 293T cells were treated with indicated amounts of PAV-818 (the biotinylated active compound), PAV-543 (the biotinylated inactive compound), or DMSO for 16 h. PLA was performed by incubation with primary antibodies (mouse anti-biotin and rabbit anti-DDX6), followed by PLA secondary antibodies and other reagents as described in Fig. 6. Red spots indicate sites where biotinylated compound and DDX6 are colocalized in situ. After PLA, IF was performed (green star) to mark intracellular DDX6 with low-level green fluorescence. (B) The graph shows the average number of biotin-DDX6 PLA spots per cell for each condition, with “+” indicating HIV-1-infected cells and “–” indicating uninfected cells. Five fields were analyzed for each group (containing a total of 39 to 73 cells per group), with error bars showing the SEM. *, Significant difference in the number of biotin-DDX6 PLA spots per cell when comparing treatment with PAV-818 versus PAV-543, both at 10 μM (P < 0.001). (C) A representative field for each group quantified in panel B is shown, except for DMSO treatment. Fields on the left show biotin-DDX6 PLA spots alone in grayscale. To the right are the same fields shown as a merge of three color channels: biotin-DDX6 PLA (red), DDX6 IF (green), and DAPI-stained nuclei (blue). Scale bars, 10 μm.

Fig 5: Model for the action of PAV-206, a possible first-in-class selective inhibitor of HIV-1 assembly. Upper diagram shows the intracellular pathway for HIV-1 immature capsid assembly, in the presence of an inactive compound, starting with synthesis of HIV-1 Gag and formation of the early ∼10S assembly intermediate. The ∼80S assembly intermediate is formed when HIV-1 Gag coopts a host RNP complex related to an RNA granule to form the ∼80S cytosolic intermediate. This and subsequent intermediates contain Gag associated with ABCE1, DDX6, other host factors, and HIV-1 gRNA. After targeting of the cytosolic ∼80S assembly intermediate to the host plasma membrane, Gag multimerization continues leading to the formation of the ∼500S assembly intermediate. The host proteins are released upon formation of the ∼750S completed immature capsid, and budding results in release of the completed immature capsid. The lower diagram shows one model for PAV-117/PAV-206 activity based on the findings in this study. Our colocalization studies suggest that this chemotype is associated with both the HIV-1 assembly intermediates and the host RNA-granule-related complex from which they are derived, since the compound colocalizes with two host components of this complex, ABCE1 and DDX6, in the presence and absence of Gag. The compound does not colocalize with G3BP1 or XRN1, proteins found in stress granules and P-bodies, indicating that this chemotype acts with some selectivity for the RNP complex that is coopted to form assembly intermediates. In addition, we observed a reduction of virus production and the amount of ∼500S assembly intermediate in PAV-117-treated infected cells. Thus, we hypothesize that PAV-117/PAV-206 inhibits virus production by acting during the HIV-1 immature capsid assembly pathway to inhibit progression of assembly past the ∼80S/150S assembly intermediate.