<p>A. Relative promoter strength was assayed by transfection of rabbit skin cells with the constructs employed to generate the viral promoter/reporter mutants and analysis of the amount of b-gal determined with a CPRG assay as detailed in methods. Transfection efficiencies were normalized by including a renilla luciferase plasmid in the assay. Lane 1 = LATpLacZ, 2 = VP16pLacZ, 3 = ICP0pLacZ. The LAT promoter was the weakest and the levels of expression of this construct were set to one. Where indicated, a VP16 expressing plasmid was included in the transfections. Each bar represents the average of 3 transfection experiments of 3 wells each. B. Photomicrographs of TG from mice infected with17LATpLacZ processed for LATp activity and viral protein expression. At 22 hrs pi, expression was restricted to the LATp. A 36 hrs, viral proteins are expressed but restricted to a subset of neurons that are marked by LATp activity. C. At the indicated times pi, eyes and TG were harvested. Unsectioned TG were processed for the histochemical detection of b-gal activity and IHC detection of viral proteins as detailed previously [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005877#ppat.1005877.ref004" target="_blank">4</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005877#ppat.1005877.ref011" target="_blank">11</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005877#ppat.1005877.ref048" target="_blank">48</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005877#ppat.1005877.ref054" target="_blank">54</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005877#ppat.1005877.ref078" target="_blank">78</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005877#ppat.1005877.ref081" target="_blank">81</a>]. Bars represent the number of neurons positive in an individual TG. The number of TG positive over the number tested in each group is indicated. All TG tested from mice infected with 17LATpLacZ contained one or more neurons positive for b-gal activity at 22 hrs pi, whereas no TG infected with the lytic stage promoter reporter viruses were positive (p≤0.0001, ANOVA). At 36 hrs pi a subset of ganglia examined contained neurons positive for both b-gal and viral protein. The number of ganglia positive and the number of positive neurons in the TG was not different between groups (p≥0.5, ANOVA). Importantly, all neurons positive for viral protein were also positive for LAT promoter activity in the 17LATpLacZ group.</p
