To confirm that virion binding induces NF-kB and to examine the kinetics, an NF-kB promoter-driven luciferase construct was transfected into HFF cells and cells were treated with DgH virions (1000 particles per cell) or were mock-treated. Cells were harvested every hour until 9 hr post treatment, lysed and assayed for luciferase activity in triplicate. Figure 1(a) shows the fold-change in luciferase activity of those HFFs inoculated with DgH in comparison to mock-inoculated cells at the same time point. Biological duplicates of the inoculations were performed with the lysates used in triplicate reporter assays. At 1 hpi, there is an average of 2.4 fold change in luciferase activity from the NF-kB reporter, rising to 3.5 at 2 hpi and is maintained through to 3 hpi whereby it drops to 1.8 at 4 hpi, which correlates with the half-life of luciferase [28] . By 5 hpi, and until 9 hpi, luciferase activity drops to baseline levels at, or near, 1-fold change. Error bars represent the standard error across the six readings taken from technical triplicates on biological duplicates. As induction of the NF-kB reporter construct occurred within one hour of inoculation with DgH virions and peaked at around two-and-a-half hours post-inoculation, then the transcripts previ- HFFs were stimulated with 1000 particles/cell of DgB, DgD or DgH HSV-1 for six hours and a cDNA microarray corresponding to targets of 19 signalling pathways was used to detect changes in cellular gene expression when compared to mock-infected. Infections and mock infections were carried out in duplicate. A number of signalling pathways were shown to be stimulated by one or more of the entry-defective mutants. These data were used to independently verify changes in expression using real-time PCR. doi:10.1371/journal.pone.0009560.t001 ously shown in Table 2 to be up-regulated as a result of DgH virions binding were examined for changes in transcript levels at two-and-a-half hours after inoculation with DgH. Figure 1 (b) compares expression levels at two-and-a-half and six hours postinoculation. Transcripts for the NF-kB-responsive chemokine CCL2 were up-regulated to a greater degree at two-and-a-half hours than at six hours post-inoculation. Of the NF-kB-responsive genes, REL transcription was induced to a higher level whereas PECAM1 transcript abundance was reduced in comparison to six hours. Although JAK/STAT-responsive targets were not differentially expressed by two-and-a-half hours, the JAK/Src targets BCL2 and BCL2L1 were up-regulated above the two-fold arbitrary threshold to similar transcript levels that were seen at six hours post-inoculation.