FIG. 6.

ATRX represses the LUNA promoter during infection by binding to the LUNA promoter. (A) Fibroblasts were transfected with a siRNA-ATRX cocktail (ATRX) or a mismatch control (Scr) and analyzed 24 h posttransfection by Western blotting for ATRX and GAPDH expression. (B and C) Fibroblasts were transfected with a siRNA-ATRX cocktail or a mismatch control; 24 h posttransfection, cells were infected (MOI of 5) with Towne or CR208, and RNA (B) and DNA (C) were harvested 12 h postinfection. RT-PCR was performed on the RNA using LUNA-, actin-, UL44-, and ATRX-specific primers. Alternatively, RNA was amplified in the LUNA-specific PCR without prior RT. An IE gene-specific PCR was used to amplify viral DNA to confirm equivalent numbers of genomes in the infected cells. (D) Fibroblasts infected with CR208 or Towne at an MOI of 3 were harvested at 3 and 6 h postinfection and analyzed by ChIP for ATRX binding. Sonicated cell lysates were immunoprecipitated with an anti-ATRX antibody or a goat IgG control serum, and the DNA was amplified in a LUNA promoter-specific PCR. The IgG control was used to correct the ATRX samples, which were then expressed as percentages of the input. Results from 2 ChIP experiments are shown. Error bars indicate standard deviations.