Identification of a novel cellular transcriptional repressor interacting with the latent nuclear antigen of Kaposi's sarcoma-associated herpesvirus

Abstract

The latent nuclear antigen (LNA) of Kaposi's sarcoma-associated herpesvirus (KSHV) has an essential role in viral latent infection. LNA maintains the stability of KSHV episomes and modulates the expression of cellular genes. A novel cellular protein KLIP1 was identified to interact with LNA through yeast two-hybrid screening, and confirmed by a glutathione S-transferase pull down assay. Domain mapping showed that KLIP1 interacted with the N-terminal domain of LNA. Northern blot hybridization with a KLIP1 probe identified a major transcript of 1.8 kb and a minor transcript of 2.8 kb. cDNA library screening and 5'-RACE revealed that the major transcript encoded an open-reading-frame of 1,257 bp and had a 5'-untranslated region of 73 nucleotides. The major KLIP1 transcript was ubiquitously present in different cell types examined. A KLIP1 synthetic peptide antibody detected a doublet of 58-kDa and 63-kDa proteins in a Western blot assay. KLIP1 had two putative nuclear localization signals and showed punctate nuclear localization when expressed as a GFP-fusion protein. KLIP1 interacted with LNA in vivo, as demonstrated by coimmunoprecipitation using KSHV-infected cells and colocalization when they were expressed as GFP- and DsRed-fusion proteins, respectively. Consistent with its interaction with LNA, nuclear localization, and possession of two leucine zipper motifs, KLIP1 behaved like a transcriptional factor and repressed herpes simplex virus thymidine kinase (TK) promoter activity in a mammalian one-hybrid assay. In addition, cotransfection with LNA alleviated the transcriptional repression effect of KLIP1 on TK promoter activity. These results suggest that KLIP1 is a new member of cellular transcriptional repressors, and that LNA is involved in deregulating cellular transcription process.

Figures

FIG. 1.

Identification of LNA-interacting proteins by yeast two-hybrid screening. Full-length LNA (KSHV ORF73) was cloned into pAS2-1 vector (A). Detection of LNA GAL4 DBD fusion protein in Y190 yeast cells transformed with pAS2-1-LNA by Western blot assay (B). The arrowhead indicates the LNA band (lane 3). Y190 alone (lane1) and Y190 transformed with a recombinant pAS2-1 plasmid containing vIRF (KSHV ORF-K9) gene (lane 2) were used as negative controls. (C) The prey DNA was extracted from the positive clones after initial screening and transformed with either LNA bait construct or negative control vIRF construct to confirm the interaction. The novel clone was shown as a representative result.

FIG. 2.

Confirmation of LNA and KLIP1 interaction by GST pull down assay. (A) Purified GST (lane 1), GST-KLIP1C (lane 2) fusion proteins visualized by SDS-PAGE and Coomassie blue staining. (B) [35S]methionine-labeled LNA protein was pulled-down by the GST-KLIP1C fusion protein (lane 3) but not by GST (lane 2). Lane 1 showed the amount of input LNA.

FIG. 3.

Mapping of LNA domain interacting with KLIP1 protein by yeast-two hybrid assay. Schematic diagram of the full-length LNA and different truncated LNA fragments fused to GAL4 DBD, and their abilities to activate reporter after cotransfection with pACT-KLIP1 plasmid isolated through the initial yeast two-hybrid screening.

FIG. 4.

Expression of KLIP1 transcripts in different cell lines detected by Northern blot hybridization. Total RNA from cells was separated in a denaturing agarose gel, transferred to a membrane, and probed with a KLIP1 riboprobe. β-actin probe was used to hybridize the same membrane after striping of the membrane to calibrate the RNA loading.

FIG. 5.

cDNA sequence of the major KLIP1 transcript, deduced amino acid sequence of KLIP1 protein, and its structural features. (A) cDNA sequence of the major KLIP1 transcript and deduced amino acid sequence. There are 73 nt at the 5′ end and 294 nt at the 3′ end of the untranslated regions of KLIP1 transcript, respectively. (B) Sequence alignment of the KLIP1 deduced amino acid sequence with a mouse homologous sequence (AK006479). Identical amino acids in the homologous mouse sequence are indicated. (C) The deduced 418 aa protein contains two putative nuclear localization signals (NLS) and two Leucine zipper motifs.

FIG. 6.

Detection of KLIP1 protein in different cell lines by immunoprecipitation and Western blot assay. (A) GFP vector (lane 1 and 3) or GFP-KLIP1 (lanes 2, 4, and 5) were transfected into 293 cells. The cell lysate was immunoprecipitated with an anti-GFP monoclonal antibody and further immunoblotted with the anti-GFP monoclonal antibody (lanes 1 and 2), anti-KLIP1-N peptide antibody (lanes 3 and 4), or preimmune serum (lane 5). Anti-KLIP1 peptide antibody detected the fusion protein GFP-KLIP1 but not GFP alone, while preimmune serum failed to detect GFP-KLIP1 protein band. (B) In Western blot assay, the anti-KLIP1-N synthetic peptide antibody, but not the preimmune serum detected a doublet of 58 and 63 kDa in all cell lines examined.

FIG. 7.

KLIP1 is a nuclear protein. Speckle fluorescence distribution was observed in the nucleus of COS-7 cells transiently transfected with GFP-KLIP1 plasmid DNA. COS-7 cells transfected with GFP vector control had whole cell fluorescence distribution.

FIG. 8.

KLIP1 interacts with LNA in vivo. (A) Lysate from GFP-KLIP1-transfected BCBL-1 cells was immunoprecipitated with either anti-GFP (lane 3) or anti-LNA (lane 4) antibodies and then subjected to Western blotting with these two antibodies, respectively. The cell lysate was also precipitated with preimmune serum (lane 2, denoted Pre-im.) as a negative control. Lysates that were equal to 35% of those used in immunoprecipitation were run in PAGE and detected by Western blotting with anti-GFP and anti-LNA antibodies (lane 1). (B) Reciprocal coimmunoprecipitation was performed with GFP-transfected BCBL-1 cells to verify the specificity of the interaction between LNA and KLIP1. (C) Speckle green and red fluorescence distribution was observed in the nucleus of COS-7 cells transiently transfected with GFP-KLIP1 and pDsRed-LNA plasmid DNA. The distribution of both KLIP1 and LNA fusion proteins was identical and overlapped with each other, indicating colocalization of both proteins.

FIG. 9.

KLIP1 repressed TK promoter activity in a dose-dependent fashion in a mammalian one-hybrid assay. (A) Luciferase activity in 293 cells either transfected with 1 μg G5Eluc vector alone or cotransfected with 1 μg G5Eluc and 1 μg PM2-KLIP1. GAL4-VP16 was cotransfected with G5Eluc as a positive control. (B) Relative CAT activity in 293 cells cotransfected with 1 μg of GAL4-TK-CAT reporter plasmid and 0, 0.25, 0.5, 075, 1, or 2 μg of PM2-KLIP1. At 2 μg, KLIP1 repressed up to 87% of TK promoter activity. (C) Western blotting assay was carried out with anti-GAL4 DBD antibodies to examine the expression of PM2-KLIP1 fusion protein in 293 cells transfected with different doses of the plasmid DNA cited in (B). α-tubulin expression was used as loading control. (D to F) Relative CAT activity in HeLa (D), BJAB (E) and HUVEC (F) cells cotransfected with 1 μg of GAL4-TK-CAT reporter and 0, 0.5, 1, or 2 μg of PM2-KLIP1 plasmid DNA. Percentage denotes the ratio between the acetylated substrate and the whole amount of substrate used in the assay. Results are the averages and standard deviations from three independent experiments except in panels A and F, which are the averages and the high and low values from two independent experiments.

FIG. 9.

KLIP1 repressed TK promoter activity in a dose-dependent fashion in a mammalian one-hybrid assay. (A) Luciferase activity in 293 cells either transfected with 1 μg G5Eluc vector alone or cotransfected with 1 μg G5Eluc and 1 μg PM2-KLIP1. GAL4-VP16 was cotransfected with G5Eluc as a positive control. (B) Relative CAT activity in 293 cells cotransfected with 1 μg of GAL4-TK-CAT reporter plasmid and 0, 0.25, 0.5, 075, 1, or 2 μg of PM2-KLIP1. At 2 μg, KLIP1 repressed up to 87% of TK promoter activity. (C) Western blotting assay was carried out with anti-GAL4 DBD antibodies to examine the expression of PM2-KLIP1 fusion protein in 293 cells transfected with different doses of the plasmid DNA cited in (B). α-tubulin expression was used as loading control. (D to F) Relative CAT activity in HeLa (D), BJAB (E) and HUVEC (F) cells cotransfected with 1 μg of GAL4-TK-CAT reporter and 0, 0.5, 1, or 2 μg of PM2-KLIP1 plasmid DNA. Percentage denotes the ratio between the acetylated substrate and the whole amount of substrate used in the assay. Results are the averages and standard deviations from three independent experiments except in panels A and F, which are the averages and the high and low values from two independent experiments.

FIG. 10.

LNA alleviates KLIP1's transcriptional repression effect in a mammalian one-hybrid assay. (A) Relative CAT activity in 293 cells transfected with GAL4-TK-CAT reporter (1 μg) either alone, or cotransfected together with PM2-KLIP1 (1 μg) or pcDNA3.1-LNA (1 μg), respectively, or with both PM2-KLIP1 (1 μg) and pcDNA3.1-LNA (1 μg). (B) The expression of LNA and PM2-KLIP1 in 293 cells transfected with pcDNA3.1-LNA was detected by Western blot assay using anti-LNA antibodies and anti-GAL4 DBD antibodies, respectively. α-tubulin expression was used as loading control. (C through E) Relative CAT activity in HeLa (C), BJAB (D) and HUVEC (E) cells, transfected with GAL4-TK-CAT reporter (1 μg) either alone, or cotransfected together with PM2-KLIP1 (1 μg) or both PM2-KLIP1 (1 μg) and pcDNA3.1-LNA (1 μg). Percentage indicates the ratio between the acetylated substrate and the whole amount of substrate used in the assay. Results are the averages and standard deviations from three independent experiments.

FIG. 10.

LNA alleviates KLIP1's transcriptional repression effect in a mammalian one-hybrid assay. (A) Relative CAT activity in 293 cells transfected with GAL4-TK-CAT reporter (1 μg) either alone, or cotransfected together with PM2-KLIP1 (1 μg) or pcDNA3.1-LNA (1 μg), respectively, or with both PM2-KLIP1 (1 μg) and pcDNA3.1-LNA (1 μg). (B) The expression of LNA and PM2-KLIP1 in 293 cells transfected with pcDNA3.1-LNA was detected by Western blot assay using anti-LNA antibodies and anti-GAL4 DBD antibodies, respectively. α-tubulin expression was used as loading control. (C through E) Relative CAT activity in HeLa (C), BJAB (D) and HUVEC (E) cells, transfected with GAL4-TK-CAT reporter (1 μg) either alone, or cotransfected together with PM2-KLIP1 (1 μg) or both PM2-KLIP1 (1 μg) and pcDNA3.1-LNA (1 μg). Percentage indicates the ratio between the acetylated substrate and the whole amount of substrate used in the assay. Results are the averages and standard deviations from three independent experiments.