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. 2007 Nov;81(22):12680-4.
doi: 10.1128/JVI.00556-07. Epub 2007 Sep 12.

Identification of the DNA Sequence Interacting With Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 1

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Free PMC article

Identification of the DNA Sequence Interacting With Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 1

Junsoo Park et al. J Virol. .
Free PMC article

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma. The open reading frame (K9) of KSHV encodes viral interferon regulatory factor 1 (vIRF1), which functions as a repressor of interferon-mediated signal transduction. The amino-terminal region of vIRF1 displays significant homology to the DNA-binding domain of cellular interferon regulatory factors, supporting the theory that the protein interacts with specific DNA sequences. Here, we identify the consensus sequence of vIRF1-binding sites from a pool of random oligonucleotides. Moreover, our data show that vIRF1 interacts with the K3:viral dihydrofolate reductase:viral interleukin 6 promoter region in the KSHV genome.

Figures

FIG. 1.
FIG. 1.
Identification of the vIRF1-binding sequence. (A) Gel shift analysis of selected vIRF1-binding sites. Gel shift assays were performed with DNA selected at each round as probes, together with GST-vIRF1 protein. The number of selection cycles is shown above each lane. Shifted bands are indicated with arrows. (B) Selected vIRF1-binding sequences. Each cloned sequence after three rounds of selection is presented. Sequences were aligned for maximum identity. (C) Statistical results of each base match.
FIG. 2.
FIG. 2.
Characterization of vIRF1-binding sites. (A) Gel shift assays with the selected vIRF1-binding sites. Gel shift assays were performed with selected DNA (s1, s2, s4, s5, and s11) sequences and GST-vIRF1 (lanes 1 to 5) or GST protein (lanes 6 to 10). Protein-DNA complexes are specified using arrows. (B) Competition assays were performed by preincubation with selected unlabeled vIRF1-binding sites. Labeled s5 probe was used in each gel shift assay. Unlabeled s4 (lanes 1 to 4), s11 (lanes 5 to 8), and random oligonucleotides (lanes 9 to 12) were used. Twofold (lanes 2, 6, and 11), fivefold (lanes 3, 7, and 12), and 10-fold (lanes 4, 8, and 12) molar excesses of unlabeled probes were used, respectively.
FIG. 3.
FIG. 3.
Effects of the amounts of vIRF1 protein. Gel shift assays were performed with increasing amounts of GST-vIRF1 protein and s5 sequence. Shifted bands and free probe are indicated with arrows.
FIG. 4.
FIG. 4.
vIRF1 binds to the K3:vDHFR:vIL6 promoter region. (A) The KSHV K3:vDHFR:vIL6 promoter sequence is presented. Sequences similar to the vIRF1 consensus sequence are underlined; matched sequences are in boldface type. (B) Schematic diagram of the cluster of K3, vDHFR, and vIL6 genes in the KSHV genome. (C) vIRF1 interacts with the K3:vDHFR:vIL6 promoter. Gel shift assays were performed using the promoter region as a probe, together with no protein, GST, or GST-vIRF1 (s1, s2, s4, s5, and s11 sequences). (D) In vivo association of vIRF1 with the K3:vDHFR:vIL6 promoter. ChIP assays were performed with BCBL-1 cells induced or not induced with TPA. Input DNA (before immunoprecipitation, diluted 1:1,000) and ChIP DNA were amplified by using the K3:vDHFR:vIL6 promoter primer. PCR products were resolved by agarose gel electrophoresis. (E) Activation of the K3:vDHFR:vIL6 promoter by vIRF1. 293T cells were cotransfected with Rous sarcoma β-galactosidase expression plasmid (pRSV-β-gal) (0.5 μg) and increasing amounts of vIRF1 expression plasmid together with the K3:vDHFR:vIL6-Luc reporter plasmid (1 μg) or the blank reporter (pGL3 promoter) (1 μg). Transcriptional changes were measured using the luciferase activity and are presented as severalfold activation. Luciferase activity was derived from three independent experiments and normalized with β-galactosidase expression levels.

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