Immunofluorescence assay (IFA) was conducted to determine a possible role for elevated Egr-1 on the expression of KSHVencoded lytic proteins in the above transfected cells (Fig. 4A, B ). KSHV-encoded ORF59, a processivity factor for KSHV DNA polymerase, is expressed in the nucleus of infected cells during early stages of virus reactivation [22] . Target cells transfected with empty vectors showed low levels of ORF59 expression (Fig. 4C) . However, transfection cells with egr-1/pcDNA3.1(+) augmented the level of ORF59 protein expression in KSHV-infected cells (Fig. 4C ); clearly implicating a critical role for Egr-1 in inducing KSHV reactivation. Finally, we analyzed MAPK signaling in the above cells relative to Egr-1 expression levels. Our results suggest that cells transfected with egr-1/pCDNA3.1(+) displayed elevated levels of Egr-1 (Fig. 4D, lane 7) . Transfection of cells with pCDNA3.1(+) (Fig. 4D , lane 5) or mock transfection (Fig. 4D, lane 3) of cells did not significantly alter the expression levels of Egr-1 and phosphorylation state of ERK1/2 compared to untransfected cells (Fig. 4D, lane 1) . Interestingly, treatment of cells with a known inhibitor of MEK1/2 (10 mM of U0126) significantly lowered both the expression levels of Egr-1 and ERK1/2 activity (Fig. 4D, lane  8) . We observed U0126 to dose dependently inhibit ERK1/2 activity and Egr-1 levels in the above cells confirming the specificity of U0126 in targeting MAPK.Egr-1 signaling. The inhibition of ERK1/2 activity and Egr-1 levels was greatest following treatment of infected cells with 10 mM of U0126 (Fig. 4E,  lane 4) . These results suggest Egr-1 to be downstream of the MAPK signaling cascade. [28] and treated with 20 ng/ml of TPA for 8 h. ChIP assays were performed using 2 mg of specific antibodies to Egr-1 or nonspecific IgGs. Primers specifically targeting ORF50P3 or ORF50P8 (see Table 1 ) were used to perform semi-quantitative PCR experiments on 1% of total DNA (input) and IP samples. Respective cDNA at 10, 15, 20, 25, and 30 cycles were removed and resolved on a 2% agarose gel. IP of BCBL-1 DNA using specific antibodies to histone H3 was used as positive controls. (B) A schematic representation of ORF50P used to make the deletions of the luciferase reporter constructs. The nucleotide locations correspond to the old KSHV genome sequence NC_003409 which has since been updated to NC_009333.1. Asterisks refer to the ORF50P3 and ORF50P8 locations, respectively. (C) Overexpression of Egr-1 activates ORF50P via interacting with ORF50P3 and ORF50P8 domains. HEK293 cells were co-transfected with a combination of three vectors, one from the following groups: (i) pcDNA3.1(+) or egr-1/pcDNA3.1(+), (ii) the control vector, pRL-TK, and (iii) empty pGL3 vectors or pGL3 vectors encoding FL ORF50P or one of several deletions (D-2922 to -2044; D-2922 to -1322; D-2922 to -894; and D-2922 to -169). After 48 h post-transfection, the cells were lysed, and relative luciferase activity was monitored. Firefly luciferase was normalized to the corresponding Renilla luciferase activity. The luciferase activation of pGL3 by egr-1/pcDNA3.1(+) was represented as 1-fold. Each point denotes the average 6 SD of three experiments. Columns with different alphabets are statistically significant (P,0.05) by least significant difference (LSD). doi:10.1371/journal.pone.0033364.g002