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. 2012 Dec;86(23):12741–12759. doi: 10.1128/JVI.01655-12

Fig 7.

Fig 7

CTa′m and CTRS3 binding motifs in HSV-1 display insulator activity as enhancer blockers. Luciferase reporter constructs were generated to test the ability of the CTCF binding motif to block the LAT enhancer. The enhancer-blocking activity of the CTa′m and CTRS3 motifs of HSV-1 were assayed in transient-transfection assays using rabbit skin cells. All transfections were done in triplicate wells of rabbit skin cells. All transfection assays were repeated 5 times (n = 5). *, P values representing unpaired one-tailed Student's t tests in pairwise comparisons to the pGL3-control construct. **, P values representing comparison to the pGL3-control/LTE construct. The data were collected in relative luciferase units and normalized to an internal Renilla luciferase control. All values are expressed relative to the pGL3-control vector, which is normalized to a value of 1. The error bars represent standard deviations from the mean. (A) (1) pGL3-control vector from Promega (containing an SV40 promoter) was used as a base for all reporter constructs generated. (2) Reporter construct containing the 589-bp fragment LAT enhancer (pGL3-control/LTE). (3) A 1.3-kb reporter construct encompassing CTa′m (pGL3-control/CTa′m). (4) Reporter construct with the CTa′m fragment positioned between the SV40 promoter and the LAT enhancer (pGL3-control/LTE+CTa′m). (5) CTRL2 construct used as a positive control (2). The CTa′m fragment was able to significantly block LAT enhancer activity on the SV40 promoter in transient transfections. (B) (1) pGL3-control vector from Promega (containing an SV40 promoter) was used as a base for reporter constructs generated. (2) Reporter construct containing the 589-bp fragment LAT enhancer (pGL3-control/LTE). (3) A 1.6-kb reporter construct encompassing CTRS3 (pGL3-control/CTRS3). (4) Reporter construct with the CTRS3 fragment positioned between the SV40 promoter and the LAT enhancer (pGL3-control/LTE+CTRS3). (5) CTRL2 construct used as a positive control (2). The CTRS3 fragment was able to block LAT enhancer activity on the SV40 promoter. The CTa′m and CTRS3 sites of HSV-1 displayed enhancer-blocking activity comparable to that of the previously reported CTRL2 enhancer blocker of HSV-1 (2).