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. 2021 Sep 20;12:5541. doi: 10.1038/s41467-021-25839-2

Fig. 1. Screening of potent repressors of HIV-1 derived from fusion of ZFP362 and combination of repressor domains.

Fig. 1

a Schematic representation of the design of the ZD3A constructs leading to ZD3A protein. The NLS containing the ZFP362 domain can bind HIV-1 LTR promoter specifically and DNMT3A can recruit epigenetic silencing complexes. The binding of ZFP362-DNMT3A (ZD3A) can lead to long-term epigenetic transcriptional repression of the LTR represented by methylated CpG DNA and repressive histone methylation (right). b The effect of transfection of ZFP and ZD3A constructs in chronically infected Jurkat (CHI-Ju) cells was measured via RT-qPCR for Gag mRNA at 3 days post-treatment. c Measurement of Gag mRNA from TNFα activated CHI-Ju cells three days after transfection with ZD3A. d The effect of transfection of constructs ZFP and ZD3A in chronically infected Jurkat (CHI-Ju) cells 10 days post-treatment was measured by RT-qPCR for Gag mRNA. e A schematic is shown here depicting the 12 different constructs developed and assessed consisting of ZFP362 with one or more repressor domains like KRAB(K), PWWP, ADD, Methyltransferase (Mtase), Catalytic Domain (CD) of DNMT3A fusions, and the full-length DNMT3a (ZD3A) and control ZFP-362. f CHI-Ju cells were transfected with those fusion constructs depicted in (e) and RT-qPCR for Gag mRNA 10 days post-transfection revealed FC4 and FC10 to be the most potent repressors of LTR activity. FC4 will henceforth be referred to as ZPAMt after constituent ZFP362, PWWP, ADD, and Mtase domains, and FC10 will be referred to as KMt after constituent ZFP362, KRAB, and Mtase domains. g Western blot for Myc tag confirmed the expression of ZPAMt, ZKMt, ZD3A, and ZFP. Representative blot of n = 3 biological replicates (h) LTR driven GFP mRNA expression level measured after overexpression of ZPAMt and ZKMt in microglial cell line HC69.5 Data are represented as mean ± standard deviation (SD) in b–d, f, and h. p-values by one-way ANOVA followed by Tukey’s multiple comparisons post hoc test. For b–d, f, and h the error bars indicate the standard deviation of triplicate treated samples and experiments were repeated twice. ****p ≤ 0.0001. Source data are provided as a source data file.