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. 2015 Dec 17;90(1):368–378. doi: 10.1128/JVI.01192-15

FIG 2.

FIG 2

Schematic of vIL-6 luciferase (LUC) promoter and constructs showing the location of the potential XBP response elements (XRE) and activation of the vIL-6 promoter constructs by spliced XBP-1. (A) The sequence of the vIL-6 promoter contains two TATA boxes. Ten XRE core sequences, ACGT (47), are found within 1.7 kb upstream of the vIL-6 start codon (positions 17871 to 19567 of KSHV-BAC36; GenBank accession number HQ404500.1): XRE1, −163 to −160; XRE2, −245 to −242; XRE3, −447 to −444; XRE4, −769 to −766; XRE5, −991 to −988; XRE6, −1262 to −1259; XRE7, −1331 to −1328; XRE8, −1352 to −1349; XRE9, −1401 to −1398; and XRE10, −1607 to −1604. Each potential XRE sequence is denoted as a square. Consensus XREs are indicated in black, and other (core-only) XREs are shown in gray. The direction of each consensus XRE is indicated with an arrow (core XRE sequence only, 5′-ACGT-3′; the core sequence is underlined in the consensus XRE, 5′-NNGNTGACGTGKNNNWT-3′). Constructs pvIL6-2, pvIL6-3, and pvIL6-4 were made by sequential deletions, as shown. (B) Comparison of the activation of the vIL-6 promoter luciferase reporter by the XBP-1 unspliced (XBP-1u) or spliced (XBP-1s) form. Hep3B cells were cotransfected with 300 ng of a vIL-6 promoter luciferase construct and 50 ng of an internal β-Gal control plasmid (pGL3-basic [pGL3b]) in the presence of 100 ng of an expression plasmid encoding XBP-1u, XBP-1s, or the pcDNA3.1 expression plasmid control. Values are expressed as fold increase over the value for the pGL3-basic reporter transfected with an empty expression vector (pcDNA3.1) and represent the mean of three independent experiments. Error bars denote the standard deviations (*, P ≤ 0.01; **, P ≤ 0.005, for the comparisons shown, normalized in each case for the results with the pGL3-basic control). The comparison between results for pvIL6-1/XBP-1u and pvIL6-1/pcDNA was not significant (P > 0.05). (C) Comparison of the activation of truncated forms of the vIL-6 luciferase reporter by XBP-1s or the pcDNA3.1 plasmid control. Hep3B cells were cotransfected with 300 ng of each vIL-6 promoter and 50 ng of an internal β-Gal control plasmid in the presence of 100 ng of an expression plasmid encoding XBP-1s or the pcDNA3.1 control. Values are expressed as fold increase over the value for pvIL6-1 transfected with an empty expression vector (pcDNA3.1) and represent the means of three independent experiments. Error bars denote the standard deviations.