Number of and distance between response elements in Kaposi's sarcoma-associated herpesvirus ORF57 promoter influence its activation by replication and transcription activator and its repression by interferon regulatory factor 7

Abstract

Kaposi's sarcoma-associated herpesvirus ORF57 expression is highly responsive to replication and transcription activator (RTA) and interferon regulatory factor 7 (IRF-7). Three RTA response elements (RREs) have been identified in the ORF57 promoter. Here, we show evidence of another functional RRE located between nt 82003 and 82081, which can complement the loss of RTA activation resulting from RRE1 deletion. Repeats of a recombination signal-binding protein Jkappa (RBP-Jkappa) site enhanced RTA activation, which could not be suppressed by IRF-7. Alteration of the distance between the RBP-Jkappa site and RRE2 modulated responsiveness to RTA and IRF-7. These results will help to elucidate the precise regulation of viral gene expression.

Fig. 1

Characterization of RREs in the ORF57 promoter and the responsiveness to RTA transactivation and IRF-7 suppression. a Schematic representation of deletion constructs used in transient transfection analysis. The locations of various putative promoter regulatory elements are indicated on the promoter segment, and the transcription start sites (TSS) 1 and 2 are indicated by arrows. The sequence of the 40-bp region is boxed, nucleotides of RRE1 and RRE2 are shaded and the RBP-Jκ site italicized. Solid lines represent insertions of different deletion reporters, with the encompassed regions of the ORF57 promoter indicated by nt numbers. b Analysis of the responsiveness of reporters S1–S5 to RTA activation and IRF-7 repression. 293 T-cells were co-transfected with each of the reporter plasmids S1–S5, RTA and IRF-7 expression plasmids. Luciferase activities were measured 48 h post-transfection, and transfection efficiency was normalized by using the pCMV-β expression plasmid as an internal control. The activation fold was normalized to one in the absence of RTA and IRF-7 (white columns). The degree of activation when the reporters were co-transfected with RTA expression plasmid (grey columns) or with both RTA and IRF-7 expression plasmids (black columns) is shown. Results are averages of three independent experiments, and the standard deviations are shown. In parallel, the expression of RTA, IRF-7, and β-tubulin was detected by western blotting using anti-RTA, anti-Flag, and anti-β-tubulin antibodies. c Analysis of the responsiveness of reporters L1–L5 to RTA activation and IRF-7 repression

Fig. 1

Characterization of RREs in the ORF57 promoter and the responsiveness to RTA transactivation and IRF-7 suppression. a Schematic representation of deletion constructs used in transient transfection analysis. The locations of various putative promoter regulatory elements are indicated on the promoter segment, and the transcription start sites (TSS) 1 and 2 are indicated by arrows. The sequence of the 40-bp region is boxed, nucleotides of RRE1 and RRE2 are shaded and the RBP-Jκ site italicized. Solid lines represent insertions of different deletion reporters, with the encompassed regions of the ORF57 promoter indicated by nt numbers. b Analysis of the responsiveness of reporters S1–S5 to RTA activation and IRF-7 repression. 293 T-cells were co-transfected with each of the reporter plasmids S1–S5, RTA and IRF-7 expression plasmids. Luciferase activities were measured 48 h post-transfection, and transfection efficiency was normalized by using the pCMV-β expression plasmid as an internal control. The activation fold was normalized to one in the absence of RTA and IRF-7 (white columns). The degree of activation when the reporters were co-transfected with RTA expression plasmid (grey columns) or with both RTA and IRF-7 expression plasmids (black columns) is shown. Results are averages of three independent experiments, and the standard deviations are shown. In parallel, the expression of RTA, IRF-7, and β-tubulin was detected by western blotting using anti-RTA, anti-Flag, and anti-β-tubulin antibodies. c Analysis of the responsiveness of reporters L1–L5 to RTA activation and IRF-7 repression

Fig. 2

Effect of the number of and the distance between response elements in mediating RTA transactivation and IRF-7 suppression. a Schematic representation of constructs used in transient transfection assays. Nucleotide sequences replacing the DNA fragment from nt 81904 to 81943 in reporter plasmid S2 are shown, with RRE1, RBP-Jκ site and RRE2 indicated. b Analysis of the responsiveness of reporters C1–C8 to RTA activation and IRF-7 repression