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. 2009 Jun;90(Pt 6):1450–1454. doi: 10.1099/vir.0.007922-0

Fig. 3.

Fig. 3.

Relevance of serine 186 and cysteine 189 for interaction with methylated egr1ZRE and effect of methylation on Zta activation of promoter. (a) Schematic diagram summarizing the known interactions between serine 186, cysteine 189 and arginine 190 with methylated cytosines (indicated as balls) and between cysteine 189, and with the methyl group of a thymidine residue in RpZRE3. (b) Alignment of RpZRE2, RpZRE3 and the distal egr1ZRE. Nucleotides conserved between the distal egr1ZRE with either RpZRE2 or RpZRE3 are highlighted with boxes. (c) Zta, ZtaC189A and ZtaS186A (Karlsson et al., 2008a) were generated in a wheatgerm translation system, together with trace levels of [35S]methionine, and fractionated by SDS-PAGE. The expression levels were determined and equivalent amounts were used for EMSA analysis. (d) The interaction of the indicated Zta proteins, or unprogrammed lysate as control, with the methylated egr1ZRE probe was determined by EMSA. The experiment was repeated with similar results. (e) HeLa cells were transfected with the indicated plasmids using Effectene (Qiagen). After 48 h cell extracts were prepared and assayed for luciferase activity using the luciferase assay system (Promega). The data for each type of plasmid [luciferase activity (μg protein)−1] are expressed relative to the activity levels seen in the absence of Zta, together with the sd, which was derived from at least two experiments. The reporter plasmid used was egr1 (−504/+9)LUC (Chang et al., 2006). The plasmid was transfected in an untreated form, or it underwent a mock methylation reaction (−M.SssI), or it went through a methylation reaction with the methyl transferase M.SssI (New England Biolabs) (+M.SssI), both overnight, followed by purification on a QIAprep column (Qiagen). Prior to transfection, the extent of methylation was evaluated by a diagnostic digestion with the methylation-sensitive restriction enzyme BstUI. The open bars represent transfections undertaken with pBabe BZLF1 (Hicks et al., 2003), while the filled bars represent transfections undertaken with the ‘empty’ pBabe vector.