Fig 1: Unspliced (US) HIV RNA co-localizes with positive and negative effectors at multiple sites within the cell.(A) Representative images of co-localization phenotypes observed using fluorescence in situ hybridization/immunofluorescence (FISH/IF). For each, a merged image of a cell highlighting a site of co-localization (white square) is shown. Enlarged regions of interest (ROIs) of each fluorescence channel are displayed in the associated small panels to separate overlapping US HIV RNA, host protein, HIV Gag polyprotein, and DAPI signals. Some images were obtained from experimental replicates that did not include Gag IF and therefore do not include images from the corresponding channel. Note: Brightness and contrast settings were adjusted individually for each color channel of the images to effectively show co-localization. These settings may be different for the ROIs. (B) Table showing the frequency of observing a particular co-localization phenotype of US HIV RNA with each of 11 host proteins. Frequencies are displayed as the percentage of cells observed with each co-localization phenotype. (C) ROIs from the Hybridization Purification of RNA-Protein Complexes Followed by Mass Spectrometry (HyPR-MS), hierarchically clustered heatmap (Figure 2A) showing the close relation of HNRNPR, FAM120A, and IGF2BP3 interaction profiles, each preferentially interacted with US and partially spliced (PS) HIV RNA. (D) Data for proteins of interest from the siRNA knockdown (KD) screen (Figure 3—source data 1). HIV gene expression decreases for HNRNPR, FAM120A, and IGF2BP3 upon KD with a greater decrease in late gene expression than in early. For mitochondria-related proteins LRPPRC and MBOAT7, HIV gene expression increases upon KD of the host protein.Figure 4—source data 1.Quantitation of host protein and HIV RNA co-localization phenotypes.