FIG. 5.

ICP0 counteracts the repressor activity associated with the amino-terminal region of class II HDACs. (A) HeLa cells were transfected with 400 ng of L8G5-Luc reporter and 100 ng of LexA-VP16 expression vector (46), and a combination of the indicated amount of plasmids expressing ICP0 (in nanograms) and 50 ng (+) of HDAC5 or HDAC3 proteins fused to GAL4 DNA-binding domain (GAL4-DB) (46) (see Materials and Methods). A value of 100% represents the luciferase activity calculated in the absence of HDAC5 coexpression but in the presence of 50 ng of vectors expressing the GAL4-DB; standard deviations are indicated. Lanes 2, 8, and 10 represent the controls with empty pcineo vectors. (B) HeLa cells were transfected with 1 μg of 3XMEF2-Luc reporter plasmid and as indicated (+): 200 ng of pMT2-MEF2A expressing plasmid (46) and/or plasmids expressing ICP0 (25 ng) and/or full-length or truncated HA-tagged HDAC5 (25 ng). A value of 100% represents the mean value of luciferase activity calculated in the absence of HDAC5 coexpression; standard deviations are indicated. Lanes 2, 5, 7, and 9 represent the controls with empty pcineo vectors. (C) Comparison of repression activities of truncated HDAC5(123-673), HDAC4, HDAC5, and MITR on MEF2A transcriptional activity. Experimental conditions were similar to those for panel B. A value of 100% represents the value of luciferase activity calculated in the absence of any repressor coexpression. A Western blot is shown to compare the relative amounts of each protein. (D) Same experiment as in panel C, but with the addition of 25 ng of plasmid expressing ICP0 or its RING finger mutant FXE. The black bar represents the normalized MEF2A activity in the presence of the repressor protein but in the absence of ICP0 or FXE. Western blots are shown to compare the relative amounts of each repressor protein expressed in the presence of ICP0 or FXE (lanes 1 to 4) and to show a representative amounts of ICP0 and FXE expression in all of the samples (lane 5). Western blots were performed by using 12CA5 or 11060 MAbs for the detection of HA-tagged HDACs and MITR or ICP0 and FXE, respectively. A total of 100 ng of β-galactosidase expression vector was used in each transfection for protein amount standardization. pcineo plasmid was used in all assays to keep the amount of transfected plasmid constant as a negative control. The luciferase activity was measured 24 h posttransfection and normalized with respect to that of β-galactosidase. All data resulted from at least three independently repeated assays; standard deviations are indicated.