A single point mutation in cyclin T1 eliminates binding to Hexim1, Cdk9 and RNA but not to AFF4 and enforces repression of HIV transcription

Kuzmina, Alona; Verstraete, Nina; Galker, Sigal; Maatook, Maayan; Bensaude, Olivier; Taube, Ran

doi:10.1186/1742-4690-11-51

Table 1 Summary of HA-Cyclin T1 point mutations

From: A single point mutation in cyclin T1 eliminates binding to Hexim1, Cdk9 and RNA but not to AFF4 and enforces repression of HIV transcription

Cyclin T1 mutation	Hexim1	Cdk9	AFF4	Tat activity [murine cells¹]	LTR basal [human cells²]	Inhibition of Tat activity [human cells³]
*Group I*
wild type	+++	+++	+++	100	100	-
H239A	+++	+++	+++	64	110	-
T143;149A;155A	+++	+++	+++	33	108	-
T143A;155A	+++	+++	+++	65	107	-
Q46A	+++	+++	+++	73	103	-
Q50A	+++	+++	+++	79	111	-
159 F	+++	+++	+++	17	97	-
F176A	+++	+++	+++	25	54	-
Q46A;50A;F176A	+++	+++	+++	20	81	-
R38S	+++	+++	+++	41	69	-
M71E	+++	+++	+++	72	100	-
H154R	+++	+++	+++	32	123	-
L170W	+++	+++	+++	11	95	-
T179A	+++	+++	+	41	74	-
W221R	+++	+++	+	17	101	-
F241S	+++	+++	+++	37	115	-
*Group II*
Q56R	+	+	+	7	104	-
R68I	+	+	-	39	105	-
P85L	+	+	+++	34	76	-
Q97K	+	+	+++	42	85	-
V104G	+	+	+	80	160	-
C111R	+++	+	+++	16	90	-
K168E	+	+	+++	58	125	-
D169P	+	+	+++	66	89	-
P249L	+	+	+++	9	109	-
*Group III*
N60K	-	+	-	50	114	+
L133R	-	+	+++	69	70	-
Y175S	-	+++	+++	22	107	-
Y175E	-	+++	+++	5	69	-
*Group IV*
V107E	-	-	+++	12	28	+
L203P	-	-	-	17	102	-
U7	-	-	-	4	141	+
T143A;149A;155A;E137D	-	-	-	15	92	-

HA-Cyclin T1 mutations were generated by random mutagenesis as described (Verstraete et.al.). Protein interactions between HA-CycT1 mutants and P-TEFb transcription partners (Hexim1; Cdk9; AFF4) were monitored in human HEK-293T cells, following over expression of the HA-CycT1 mutants. Cells were transfected with HA-CycT1 mutants and 48 hr. later were lysed and subjected to Immuno-Precipitation (IP) with α-HA antibody. Precipitated HA-CycT1 proteins were then analyzed by SDS-PAGE and WB for association with endogenous partners of P-TEFb - Hexim1; Cdk9 and AFF4. Binding efficiencies are presented as (+++) for efficient binding, which is comparable to HA-CycT1-wild type, (+) for weak binding, or (−) for lack of protein interactions respectively.
(1) Rescue of HIV Tat transactivation in murine cells - Table 1 also summarizes the ability of HA-CycT1 mutants to activate Tat-dependent transcription from the HIV-LTR promoter in murine cells and corresponds to Data in Figure 1. Experiments were performed in 3T3 cells that were co-transfected with the HIV-LTR promoter, which drives a luciferase reporter gene, HIV Tat and HA-CycT1. Luciferase readings were normalized to Renila expression. Readings are presented relative to those obtained in cells that expressed HA-CycT1-wild-type - set to 100 (CycT1-wild type was set relatively to HIV-LTR-luciferase and Tat alone). Data is a representative of the mean value of triplicate wells; error bars show ± SEM.Ability of HA-CycT1 mutants to inhibit Tat dependent HIV transcription in human cells was also tested as presented in Figure 2.
(2) Inhibition of Tat-independent HIV transcription in human cells by HA-CycT1 mutants - cells were co-transfected with HIV-LTR-luciferase reporter gene and HA-CycT1 mutant 48 hr. post transfection cells were harvested and their luciferase activities were measured. Luciferase readings were normalized to Renila expression and are presented relative to the readings obtained in cells that expressed HA-CycT1-wild-type alone - set to 100. Results are representative of the mean value of triplicate wells; error bars show ± SEM.
(3) Inhibition of Tat-dependent HIV transcription in human cells by HA-CycT1 mutants - HEK-293T cells were transfected with two concentrations of HA-CycT1 mutants and the LTR-Tat-BFP reporter provirus 48 hr. post transfection, cells were harvested and the percentage of cells that expressed BFP was measured by FACS. Cells were also introduced with a pCDNA-CMV-GFP expression plasmid to control for transfection efficiencies by measuring percentage of GFP expressing cells via FACS. Symbols of inhibition (+); or lack of inhibition of Tat transactivation (−). Data are presented relatively to the effect of human HA-CycT1-wild type.

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ISSN: 1742-4690

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