HEXIM1-Tat chimera inhibits HIV-1 replication
Fig 1
A HEXIM1-Tat fusion peptide inhibits gene expression from the HIV promoter.
A. Structure of the HEXIM1-Tat fusion peptides used in the study. The functional domains used from HEXIM1 and Tat include a HEXIM1 Arginine Rich Motif (ARM, black box, residues 150–162) that binds RNA, a HEXIM1 inhibitory domain (ID, light grey box, residues 200–211) that inhibits CDK9 through a PYNT motif, and Tat transactivation domain (AD, dark grey box, residues 1–48) that binds to P-TEFb. The conceptual schemes are not drawn to scale. B. Transient expression of HT1 inhibits Tat-induced LTR-driven Luc expression. Upper panel: a schema depicts the reporter assay. A luciferase reporter gene (Luc) is under the control of the HIV promoter (P-HIV) and can be activated by ectopically expressed Tat. We use a Luc assay to titrate the inhibitory effect of HT1-3 on P-HIV transactivation by Tat. Middle panel: increasing amounts of m:HT1, m:HT2 or m:HT3 expressing plasmid (pHT) were co-transfected in 293T cells with a plasmid (pTat) expressing f:Tat and another (pLTR-Luc) expressing Luc under the control of the HIV promoter. Luc activity was plotted as % activity relative to control (empty vector used instead of pHT), depending on the transfected pHT : pTat ratio. Error bars in the graph represent standard deviation from triplicate experiments. Lower panel: expression levels of m:HT1 and f:Tat were confirmed by WB using anti-Myc and anti-Flag Abs. Housekeeping protein GAPDH was used as loading control. C. Transient expression of HT1 inhibits Luc expression from HIV-1 NL43ΔenvLuc. Left panel: a schema depicts the reporter assay. Luc is inserted in a replication-defective HIV-1 molecular clone (pNL43ΔenvLuc) in which Luc and Tat are under the control of the HIV promoter (P-HIV). We use a Luc assay to titrate the inhibitory effect of HT1 on P-HIV transactivation by Tat. Right panel: increasing amounts of pHT1 were co-transfected in 293T cells with pNL43ΔenvLuc. Luc activity was plotted as % activity relative to control (empty vector used instead of pHT), depending on the transfected amounts of pHT1. Error bars in the graph represent standard deviation from triplicate experiments.