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. 2018 Apr 13;92(9):e01844-17. doi: 10.1128/JVI.01844-17

FIG 2.

FIG 2

RUNX1b- and -1c-induced suppression of HIV-1 partially contributes to the effect of uc002yug.2 on HIV-1. (A and B) Overexpression of RUNX1b and RUNX1c inhibits HIV-1 replication in HEK293T cells. (A) The pNL4-3 vector and plasmids expressing RUNX1 isoforms, with or without CBF-β, were cotransfected into HEK293T cells as indicated. At 48 h after transfection, the levels of expression of RUNX1 isoforms, CBF-β, p55, and CAp24 in cells were determined by immunoblotting. (B) HIV-1 NL4-3 infectivity in the supernatant was measured by detecting luciferase activity after TZM-bl cells were infected with the supernatant for another 48 h. The infectivity of 293T cells transfected with pNL4-3 plus control vector was set as 100%. (C to E) RUNX1 is involved in the function of uc002yug.2 to regulate HIV-1 replication. (C) siRNA targeting RUNX1b and -1c was cotransfected with pNL4-3 into uc002yug.2 knockdown and control HEK293T cells. After 48 h, cells and supernatants were harvested. The qRT-PCR assay was used to detect the mRNA level of RUNX1b and -1c. (D) p55 levels in cell lysate and CAp24 levels in supernatants were determined by immunoblotting, and the densities of bands were analyzed with ImageJ software to calculate the values relative to that for β-actin. (E) The infectivity of HIV-1 was measured as described for panel B. (F to I) Overexpression of uc002yug.2 increases HIV-1 replication in Jurkat cells. (F) Jurkat cells were transduced with lentiviruses containing uc002yug.2 or a scramble control, and at 48 h postinfection, puromycin (1 μg/ml) was added to the medium for selection. The RNA level of uc002yug.2 was detected by qRT-PCR. (G) The uc002yug.2-overexpressing or control Jurkat cells were infected with HIV-1 pNL4-3-deltaE-EGFP virus, and GFP-positive cells were measured by flow cytometry at 48 h postinfection. (H) The relative ratio of GFP-positive cells in uc002yug.2-overexpressing or control Jurkat cells was calculated. The ratio of GFP-positive cells in control Jurkat cells infected with pNL4-3-deltaE-EGFP virus was set as 100%. (I) mRNA levels of RUNX1a and RUNX1b and -1c in uc002yug.2-overexpressing or control Jurkat cells was detected with qRT-PCR. Results are representative of those from three independent repeats, and the corresponding value of the mock control was set as 1 or 100%. Data are presented as means ± SDs.