Fig 1: HIV-CRISPR Screening Identifies HIV Dependency Factors.(A) Negative MAGeCK Gene Scores across both ZAP-KO Screens ranked from most depleted genes on the X-axis. Only the top 25 hits are shown. (B) Left: THP-1 cells were stimulated overnight with IFNa and assayed for cell surface SIGLEC1/CD169 expression by flow cytometry. Right: Control (scrambled - gray) THP-1 cells andTHP-1 cells transduced with a SIGLEC1/CD169-targeting shRNA construct (dotted purple line) were assayed for cell surface SIGLEC1/CD169 expression after overnight IFNa treatment. (C) Infection of control (gray – wild type) and SIGLEC1/CD169 knockdown THP-1 (purple - CD169-KD) with and without IFNa (1000 U/mL u IFNa) and assayed by intracellular p24gag 2 days after infection (D) KO efficiency as determined by ICE analysis (CXCR4) or flow cytometry (TLR2). (E) Infection of control (gray – NTC), CXCR4-KO pools (orange) and TLR2-KO pools (green) were assayed for the % of cells expressing HIV p24gag 2 days post-infection by intracellular staining and flow cytometry. Left: wt HIV-1LAI (n = 3). Right: HIV-1LAI?env + VSV G (n = 3). (F) SEC62 knockdown after transduction with two LKO SEC62 shRNA constructs. Western blot of the sec62-targeting shRNA cell lines is shown together with two control (scrambled in gray) cell lines. Loading control = actin. (G) Infection of SEC62-KD (yellow) and control (scrambled in gray) with wt HIV-1LAI (left panel) or HIV-1LAI?env + VSV G (right panel). The % of cells expressing HIV p24gag 2 days post-infection is shown. (H) The mean fluorescence intensity (MFI) of CD4-APC (left panel) and CXCR4-APC (right panel) cell surface staining of control (scrambled in gray) and SEC62-KD (yellow) THP-1 cell pools.10.7554/eLife.39823.018Figure 5—source data 1.MAGeCK Gene Analysis (Negative Scores) of ZAP-KO THP-1 PIKAHIV HIV-1LAI screens.TargetID. log2FC IFN. ZAPKO11_uIFN-ZAPKO11_THP.gDNA.neg.score. ZAPKO46_uIFN-ZAPKO46_THP.gDNA.neg.score. ZAPKO x2 uIFN NEG. ZAPKO x2 uIFN NEG -log10.