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. 2017 Oct 13;91(21):e00904-17. doi: 10.1128/JVI.00904-17

FIG 4.

FIG 4

Location of GREs on BoHV-1 and intergenic regions analyzed for stress-induced transcriptional activity. (A) Diagram of the linear BoHV-1 genome (horizontal line) with predicted GREs denoted by vertical lines. Lines above the genome represent GREs on the positive/forward DNA strand, while lines below indicate GREs on the negative strand. The terminal repeats are denoted by gray rectangles. Numbers denote genomic coordinates. Genomic regions that contain at least two putative GREs and potential KLF binding sites and are 400 bp or less are denoted by the gene in which they are located. LRG refers to the latency-related gene (10). The Circ gene encodes an IE transcript from the left end of the genome and spans covalently joined genome ends. These sequences were synthesized (Genescript), cloned into the pGL3-promoter vector, and then used for the studies described below. UL, unique long region. (B) Expanded genomic region that includes bICP0, bICP4, and bICP22. Genomic sequences located between bICP4 and bICP22 contain the origin of replication (ORIs, denoted by a black diamond) and IEtu1 and IEtu2 to the left and right of the ORIs, respectively (not highlighted). Black boxes denote enlarged GREs on positive (above) or negative (below) strands. GRE1 and GRE2 within the IEtu1 promoter are indicated, and the genomic region for the beginning and end of the sequences is given. (C) Neuro-2A cells were cotransfected with a plasmid containing the firefly luciferase gene downstream of the designated plasmid constructs (1 μg of DNA) and a plasmid encoding Renilla luciferase (0.05 μg of DNA) using Lipofectamine 3000. The level of promoter activity in the empty luciferase vector (pGL3-promoter vector) was normalized to a value of 1, and fold activation for other constructs is presented as enhancer activity. An asterisk denotes a significant difference between luciferase activities of the designated construct relative to the activity of the empty vector that contains only the SV40 early promoter (pGL3-promoter vector), using the Student's t test.