Fig. 1: Screen identifies a role for SMC5/6 in silencing unintegrated HIV-1 DNA.
From: Epigenetic silencing by the SMC5/6 complex mediates HIV-1 latency
a, Volcano plot of the mean fold change of sgRNAs specific for each gene and their false discovery rate (FDR)-corrected P values. Genes with fold change >16 and P value <0.0005 (delineated by the red lines) are labelled. Briefly, P values for individual sgRNAs were calculated using a negative binomial (NB) model, then sorted sgRNA P values were used to calculate FDR-corrected P values for individual genes using the robust rank aggregation (αRRA) algorithm in MAGeCK-VISPR50,51. Members of the SMC5/6 complex are in green (FDR-corrected P values: SMC5 = 7.8 × 10−7, SMC6 = 0.00033, NSMCE3 = 0.00010, SLF1 = 0.00017). b, Flow cytometry of WT CEM-SS cells and the indicated clonal knockout cell lines at 2 dpi with an IN− NL-GFPΔEnv reporter virus at an MOI of ~0.3. A representative experiment from 3 biological replicates is shown. c,d, Flow cytometry of WT (c) or ΔSMC5 CEM-SS (d) cells at 2 dpi with IN+ NL-GFPΔEnv reporter virus at an MOI of ~0.3. Representative experiments from 3 biological replicates are shown. e–h, Time course of the infection of the parental CEM-SS Cas9 cells, or the ∆SMC5 and ∆SLF2 CEM-SS clones with IN+ or IN− NL-NLuc. e, Live cells. f, Virally encoded NLuc expression. g, Total HIV-1 DNA. h, Total HIV-1 RNA expression quantified at the indicated dpi. All IN+ HIV-1-infected cultures died from viral cytopathicity by 7 dpi. DNA and RNA levels were quantified by qPCR and normalized to IN+ HIV-1-infected CEM-SS Cas9 cells at 1 dpi, which was set to 1. Mean ± s.d. of 3 biological replicates.
