Fig. 1: Gain-of-function screen reveals ADAP1 as a latent HIV-1 activating factor with undescribed T cell signaling functions.

a Schematic of immune stimulation-mediated CD4⁺ T cell gene expression coupled to HIV-1 gene expression motivating the screen in b. b Gain-of-function screen approach to discover undescribed T cell signaling-transcriptional regulators relevant for latent HIV-1 reactivation and T cell programs. c Schematic of 3-phase screen approach. Pool 72 is highlighted as example. See “Methods” for screen details. d Volcano plot summarizing results of phase 1 of gain-of-function screen (repeat 1). Each dot is a pool sample (representing 96 cDNAs/pool) mean luciferase activity (n = 3) and statistical significance was determined using a multiple unpaired t-test. Dots in red denote positive pools above the chosen cutoff of Log₂Fc > 0 with an FDR < 2.5% and cross-referenced with the results of screen repeat 2 (Supplementary Fig. 1c) to determine which pools to resolve (see Supplementary Fig. 1d details). e Summary of candidate HIV-1 activating factors first identified in the gain-of-function screen and then validated individually. Data represents mean ± s.d. of three independent experiments (n = 3). f Candidates from e were entered into STRING (Search Tool for the Retrieval of Interacting Genes/Proteins), a search database that identifies known and predicted protein–protein functional and direct associations and mined for predicted functional interactions based on available databases (represented by different colored edges). Colored nodes are inputted list from e and first shell of predicted interactors, while white nodes are secondary interactors. Figure displayed contains 30 additional nodes with the inputted 7 nodes. g Domain mapping analysis. Top: schematic of ADAP1 domains and point mutations used for further analysis. Bottom: western blot of Jkt-HIVLuc cells transduced with lentiviruses expressing empty vector (Mock), wild-type (WT) ADAP1, or point mutants (GAP Mut, PH1 Mut), and probed with the indicated antibodies. h Fold luciferase activity of Jkt-HIVLuc cells transduced in g. Data represents mean ± s.d. of seven independent experiments (n = 3) (one-way ANOVA followed by Dunnett’s test for multiple comparisons to mock). ns not significant. Source data are provided as a Source Data file.