Fig 1: SLFN5 represses HIV-1 transcription by depositing of H3K27me2 and H3K27me3 marks and interacting with histone H3 and modifying complex. (A) Co-IP of RBBP7 and Myc-tagged SLFN5 protein of HEK293T cells followed by immunoblot. HEK293T cells were transfected with SLFN5-Myc, 48h later, half of the cell lysis was immunoprecipitated by α-Myc antibody and the association with RBBP7 was detected by α-RBBP7 antibody. In a reciprocal Co-IP, the other half of the lysis was immunoprecipitated by α-RBBP7 antibody and the association with SLFN5-Myc was detected by α-Myc antibody. (B) Co-IP of histone H3 and Myc-tagged SLFN5 protein followed by immunoblot. HEK293T cells were transfected with SLFN5-Myc, 48h later, the cell lysis was immunoprecipitated by α-Myc antibody and the immunoprecipitates were detected by α-histone H3 antibody (left panel). In a reciprocal Co-IP, the cell lysis was immunoprecipitated by α-histone H3 antibody and the immunoprecipitates were detected by α-Myc antibody (right panel). (C) ChIP with α-H3K27me2 antibodies was performed. H3K27me2 associated with the U3 (left panel) and R-U5 regions (right panel) is shown relative to the background signal in IgG negative control ChIPs. (D) ChIP with α-H3K27me3 antibodies was performed. H3K27me3 associated with the U3 (left panel) and R-U5 (right panel) regions is shown relative to the background signal in IgG negative control ChIPs. (E) HEK293T cells were transfected with the indicated siRNA, NL4-3luc.R-E- and either SLFN5 DNA or a control vector. 48h post-transfection, luciferase activity was measured (upper panel) and siRNA knockdown was analysed by immunoblot (lower panel). (F) HEK293T cells were transfected with NL4-3luc.R-E-, and either SLFN5 DNA or a control vector, followed by treatment of BRD4770 (20 μM), UNC1999 (450 nM) or EPZ004777 (4 nM). 5 h post-transfection. 48 h later, luciferase activity was measured. Levels of G9a were determined by immunoblot. (G) Co-IP of EZH1, G9a, H3K27me2 and H3K27me3 with SLFN5 or dN2 using α-Myc antibody in the presence or absence of BRD4770 (20 μM). Results shown are the average of three independent experiments. n.s. non-significant, ** P < 0.01, * P < 0.05, *** P < 0.001.
Fig 2: SLFN5 inhibits basal and Tat-transactivated HIV-1 transcription by specifically binding to the LTR via its N-terminal domain. (A) HEK293T cells were transfected with 500 ng of either NL4-3 LTR-luc (LTR) or SV40 promoter-Luc (SV40 Pr) reporter DNA, and SLFN5 DNA or a control vector (ctrl). 48h later, luciferase activity was measured. Expression of SLFN5 was detected by immunoblot. (B) HEK293T cells were transfected with 500 ng pZM246F-LTR-Luc (ZM246F) or pZM247F-LTR-Luc (ZM247F) and SLFN5 DNA or a control vector. Luciferase activity was measured after 48 h. Expression of SLFN5 was detected by immunoblot. (C) HEK293T cells were transfected with 0.5 μg NL4-3 LTR-luc and 0.5 μg SLFN5 with increasing doses of Tat DNA (0, 0.05, 0.5, 2.5, 5 and 20 ng). 48 h post-transfection, cells were lysed and luciferase activity was measured. Expression of SLFN5 was detected by immunoblot with α-Myc antibodies. (D) HEK293T cells were transfected with 0.5 μg LTR-luc and 0.5 μg SLFN5 with higher doses of Tat DNA (0, 50 and 100 ng). 48 h post-transfection, cells were lysed and luciferase activity was measured. Expression of SLFN5 were detected by immunoblot with α-Myc antibodies (E) 293T cells were transfected with NL4-3luc.R-E-, HIV-2 LTR or GAPDH promoter DNA, or SLFN5 DNA. The cells were subject to ChIP assay using an α-Myc antibody or mouse IgG as a negative control. Primer sets targeting HIV-1 U5-R, HIV-2 LTR or GAPDH promoter were used for qPCR. (F) HEK293T cells were transfected with NL4-3luc.R-E- and SLFN5-dN1, dN2 or dC1-NLS, and were subject to CHIP assay. Primers targeting U5-R were used for qPCR. Results shown are the average of three independent experiments. n.s. non-significant, **P < 0.01, *P < 0.05, ***P < 0.001.
Fig 3: Endogenous SLFN5 inhibits HIV-1 replication. (A, B) HeLa-Ctrl and HeLa-SLFN5 KD cells were infected with VSV-G pseudotyped NL4-3luc.R-E- reporter viruses. 48 h post-infection, luciferase activity in HeLa cells was measured (A), and the expression of SLFN5 in HeLa-Ctrl (Ctrl) and HeLa-SLFN5 KD (SLFN5 KD) cell lines was determined by immunoblot (B). (C, D) HeLa-Ctrl and HeLa-SLFN5 KD cells were treated with 103 U/ml IFN-α/β and infected with VSV-G pseudotyped NL4-3luc.R-E- reporter viruses. 48 h post-infection, luciferase activity in HeLa cells was measured (C), and expression of SLFN5 was determined by immunoblot (D). (E, F) Control cells (SupT1-sgNT) and SLFN5-deficient (SupT1-sgSLFN5) cells were infected with VSV-G pseudotyped NL4-3luc.R-E- reporter viruses. 48h post-infection, luciferase activity in SupT1 cells was measured (E). Expression of SLFN5 in SupT1-sgNT and SupT1-sgSLFN5 cell lines was determined by immunoblot (F). Data represent the mean ± SD of three independent experiments. P-values were calculated using a standard Student's t-test. *P < 0.05, **P < 0.01.
Fig 4: A model to illustrate how SLFN5 inhibits HIV-1 LTR transcription. SLFN5 binds to two sequences in the R and U5 regions through its N-terminal domain. Binding of SLFN5 to HIV-1 LTR increases H3 repressive marks at lysine 27 which are deposited by G9a and EZH1, thereby preventing the recruitment of RNA Pol II and suppressing HIV-1 transcription. Once latent provirus infection is established, SLFN5 prevents reactivation of the provirus by latency reversal agents.
Fig 5: SLFN5 impairs activation of latent proviruses. (A, C) HIV-1 latently infected 2D10 (A) and E4 cells (C) were stimulated by JQ-1 (1 μM) for 48 h, followed by electroporation of SLFN5-Myc DNA or pcDNA4 as a negative control. EGFP + cells were scored by flow cytometry. SLFN5-Myc expression was analysed by immunoblot. (B, D) HIV-1 latently infected 2D10 (B) and E4 cells (D) were stimulated by JQ-1 (1 μM) for 48 h, followed by electroporation of SLFN5 shRNA or ctrl shRNA as a negative control. Levels of endogenous SLFN5 were examined by immunoblot. Results shown are the average of three independent experiments. ** P < 0.01, * P < 0.05.
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