Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) replication and transcription factor activates the K9 (vIRF) gene through two distinct cis elements by a non-DNA-binding mechanism

Abstract

The replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, a homologue of Epstein-Barr virus BRLF1 or Rta, is a strong transactivator and inducer of lytic replication. RTA acting alone can induce lytic replication of KSHV in infected cell lines that originated from primary effusion lymphomas, leading to virus production. During the lytic replication process, RTA activates many kinds of genes, including polyadenylated nuclear RNA, K8, K9 (vIRF), ORF57, and so on. We focused here on the mechanism of how RTA upregulates the K9 (vIRF) promoter and identified two independent cis-acting elements in the K9 (vIRF) promoter that responded to RTA. These elements were finally confined to the sequence 5'-TCTGGGACAGTC-3' in responsive element (RE) I-2B and the sequence 5'-GTACTTAAAATA-3' in RE IIC-2, both of which did not share sequence homology. Multiple factors bound specifically with these elements, and their binding was correlated with the RTA-responsive activity. Electrophoretic mobility shift assay with nuclear extract from infected cells and the N-terminal part of RTA expressed in Escherichia coli, however, did not show that RTA interacted directly with these elements, in contrast to the RTA responsive elements in the PAN/K12 promoter region, the ORF57/K8 promoter region. Thus, it was likely that RTA could transactivate several kinds of unique cis elements without directly binding to the responsive elements, probably through cooperation with other DNA-binding factors.

FIG. 1.

Schematic representation of the RTA-responsive region of the K9 (vIRF) promoter. The positions of sequence fragments used in the present study with the upstream sequence of K9 (vIRF) are indicated. The numbers above the sequence indicate the positions of the nucleotides marked with a dot and are relative to the K9 mRNA start site in the lytic phase (10). Variously shaded bars such as RE I-1, RE I-2, and so on shows the regions described in the present study. The line under the boxes depicts the shorter fragments of each RE. Nucleotides surrounded by a dotted box show putative AP1 and/or SP1 recognition sites, as shown above the line. Previously identified responsive regions. RE I and RE II lie between deletion mutants D4 and D5 and between deletion mutants D8 and D9, respectively, as described elsewhere (10). The RE III region is shown in the hatched box.

FIG. 2.

Core RE in RE I. (A) Responsiveness of REs I-1, I-2, and I-3 in 293L cells. (B) Responsiveness of REs I-1, I-2, and I-3 in BJAB cells. The corresponding regions were inserted upstream of the E1B minimal TATA box, which was followed by a firefly luciferase gene. The constructs were transfected into 293L cells (A) and BJAB cells (B), respectively. The emitted light was measured in relative light units 2 days after transfection, which was normalized with β-galactosidase activity in the same reaction lysate. The perpendicular axis gives the fold activity calculated as the normalized activity with RTA divided by that obtained without RTA.

FIG. 3.

Determination of the core responsive segments in RE I-2 and RE II. (A) Responsiveness of REs I-2A, I-2B, and I-C of the RE I-2 region. (B) Responsiveness of RE IIA, RE IIB, and RE IIC of the RE II region. (C) Responsiveness of REs IIC-1 and II-C2 of the RE II-C region. The smaller segments of RE I-2, RE II, and the further RE IIC region (see Fig. 1A) were arranged as three to five tandem repeats upstream of the E1B minimal TATA box, which was followed by a firefly luciferase gene. The assay was performed as described in Materials and Methods. The value of perpendicular axis denotes the fold activity calculated as the normalized activity obtained with RTA divided by that obtained without RTA as in Fig. 2.

FIG. 4.

Responsiveness to RTA of the mutants of RE I-2B and RE IIC-2. Results for a mutant series of RE I-2B (A) and RE IIC-2 (B) are shown. Three to five tandem copies of each mutated fragment of RE I-2B and RE IIC-2 were placed upstream of the E1B minimal TATA box, which was followed by a firefly luciferase gene. The assay was done as described above. The value of perpendicular axis denotes the fold activity, calculated as the normalized activity obtained with RTA divided by that obtained without RTA. Mutated nucleotides of each RE are shown under the panel.

FIG. 5.

Chronological expression profile of RTA, responsiveness of monomer RE I-2B and RE IIC-2, and the binding activity of α50A in EMSA. (A) NE was prepared from BCBL1 cells at 0, 2, 4, 8, 12, 24, 48, and 72 h postinduction with TPA. A total of 20 μg of the NE was subjected to Western blotting analyses. RTA was detected with a mouse monoclonal antibody to RTA (α50A) as the first antibody and an anti-mouse IgG Fab fragment conjugated with horseradish peroxidase (see Materials and Methods). (B) Monomer constructs of RE I-2B and RE IIC-2 were transfected into 293L cells with β-galactosidase expression vector (pCMVβ) for normalization of transfection efficiency. The fold activity was calculated as mentioned above (see Materials and Methods). (C) EMSA was performed with PAN RRE as a probe. Cold competitors such as PAN RRE, RE I-2B, and RE IIC-2 were added to the reaction mixture in 50-fold excess for each case. Next, 2 μg of specific antibody to RTA (α50A) and Oct1 (αOct1) was added for supershift and/or binding inhibition analysis. The arrow denotes a supershifted complex with α50A. PI, preimmune serum.

FIG. 6.

EMSA with RE I-2B as a probe. (A) Binding complex of RE I-2B probe and NE. A total of 20 μg of NE from BCBL1 cells induced with TPA or uninduced was incubated with the labeled RE I-2B probe. Each competitor—unlabeled RE I-2B, RE IIC, AP1 (AP1-binding consensus sequence), or Oct (Oct family protein-binding consensus)—shown on the panel was added in a 50-fold molar excess. (B) Supershift analysis with specific antibodies. Next, 20 μg of NE from BCBL1 induced with TPA was incubated with the RE I-2B probe and 2 μg of each of the following antibodies: mouse preimmune serum (PI), α50A (mouse monoclonal anti-RTA antibody), or αOct1 (mouse monoclonal anti-Oct1 antibody). (C) Competition analysis of RE I-2B with its mutants. The labeled RE I-2B probe and each mutant unlabeled probe (in a 50-fold molar excess) were mixed with NE and analyzed. Arrowheads show specifically formed DNA-protein complexes, and the stars indicate nonspecific complexes.

FIG. 7.

EMSA with RE IIC-2 as a probe. (A) Binding complex of RE IIC-2 probe and NE. A total of 20 μg of NE from BCBL1 cells either induced with TPA or uninduced was incubated with the labeled RE IIC-2 probe. Each competitor—unlabeled RE IIC, RE IIC-1, RE IIC-2, AP1 (AP1-binding consensus sequence), or Oct (Oct family protein-binding consensus)—shown on the panel was added in a 50-fold molar excess. (B) Supershift analyses with specific antibodies. A total of 20 μg of NE from BCBL1 induced with TPA was incubated with the RE IIC-2 probe and 2 μg of each of the following antibodies: mouse preimmune serum (PI), α50A (mouse monoclonal anti-ORF50 antibody), or αOct1 (mouse monoclonal anti-Oct1 antibody). (C) Competition analyses of RE IIC-2 with its mutants. The labeled RE IIC-2 probe and each mutant unlabeled probe (at a 50-fold molar excess) were mixed with NE and analyzed. Arrowheads show specifically formed DNA-protein complexes, and the stars indicate nonspecific complexes.

FIG. 8.

EMSA with RTA412. E. coli-derived RTA412 protein (0.1 μg) was incubated with RE I-2B and RE IIC-2 (A), PAN RRE (B), and 57/K8 RRE (C) as probes, respectively. PAN RRE and 57/K8 RRE generated specific shifted bands marked by triangles and diamonds, respectively. A mouse monoclonal anti-T7 tag antibody (αT7; Novagen) caused supershift (shown as filled circles with a line). PI refers to mouse preimmune serum, and stars indicate nonspecific complexes. (D) Competition anaysis with RE I-2B and RE II-C2 was shown. In this case, each cold competitor was added by a 50-fold up to a 200-fold molar excess, as indicated in the panel.

FIG. 9.

Comparison of the identified RTA responsive elements. K9 (vIRF) RRE I-2B and K9 (vIRF) RRE IIC-2 were identified in the present study. Boldface indicates the core sequence for the responsiveness to RTA in K9 (vIRF) RRE I-2B, RRE IIC-2, and Rta-p 2.1 (30). PAN RRE/K12 was described by Song et al. (32) and by Chang et al. (6). 57/K8 RRE was described by Lucac et al. (24). A double underline drawn in K12 RRE shows a core element for its RTA responsiveness (6).