Fig. 2: SUMOylation of chromatinized unintegrated HIV-1 DNA by NSMCE2.
From: Epigenetic silencing by the SMC5/6 complex mediates HIV-1 latency
a, Flow cytometry at 2 dpi of WT or ∆NSMCE CEM-SS T cells transduced with a lentiviral vector expressing nothing, FLAG-NSMCE2 or the FLAG-NSMCE2∆SUMO mutant and then infected with IN− NL-GFPΔEnv at an MOI of ~0.3. Shown is a representative experiment from 3 biological replicates. b, Quantification of virally encoded NLuc expression in the indicated cells infected with IN+ or IN− NL-NLucΔEnv. NLuc expression was normalized to the IN− HIV-1 infection of WT CEM-SS cells, which was set to 1 (**P = 0.0010, ***P = 0.0009, ****P < 0.0001, 1-way analysis of variance (ANOVA), Dunnett’s test). c, Representative western blot of FLAG-tagged WT FLAG-NSMCE and the NSMCE2∆SUMO expression after transduction of the indicated cell types. d, ChIP–qPCR quantification of the level of bound, ectopically expressed FLAG-tagged NSMCE2 or endogenous SMC5 to unintegrated IN− HIV-1 DNA in WT or ∆NSMCE CEM-SS T cells. SUMO2/3 deposition was also determined by ChIP–qPCR (ΔNSMCE2 + NSMCE2 ***P = 0.0002, ΔNSMCE2 + NSMCE2ΔSUMO ***P = 0.0003; 2-way ANOVA, Dunnett’s test). e, Quantification of the level of SUMO2/3 deposition on the HIV-1 LTR at 2 dpi in WT and ∆SMC5 CEM-SS cells infected with IN− NL-GFPΔEnv. IgG served as the negative control (****P < 0.0001, 2-way ANOVA, Sidak’s test). Data in b, d and e are mean ± s.d. of 3 biological replicates.
