Identification of a novel cellular transcriptional repressor interacting with the latent nuclear antigen of Kaposi's sarcoma-associated herpesvirus.
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.
Pan
Hong-Yi
HY
Tumor Virology Program, Children's Cancer Research Institute, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA.
Zhang
Yan-Jin
YJ
Wang
Xin-Ping
XP
Deng
Jian-Hong
JH
Zhou
Fu-Chun
FC
Gao
Shou-Jiang
SJ
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D001483:Base Sequence; D019556:COS Cells; D004247:DNA / Q000378:metabolism; D015723:Gene Library; D019288:Herpesvirus 8, Human / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000502:physiology*; D010750:Phosphoproteins / Q000502:physiology*; D012097:Repressor Proteins / Q000502:physiology*; D020798:Two-Hybrid System Techniques
D000914:Antibodies, Viral; D009687:Nuclear Proteins; D010750:Phosphoproteins; D012097:Repressor Proteins; C118485:latent nuclear antigen (LNA); D004247:DNA