Human hepatitis B virus X protein is detectable in nuclei of transfected cells, and is active for transactivation.
Subcellular localization and transactivation of human hepatitis B virus X protein (HBx), a plausible causative factor for hepatocellular carcinogenesis, were studied in transiently transfected cells. The transactivation was detected not only by the cis-element driven chloramphenicol acetyltransferase (CAT) assay but also by immunostaining of CAT protein cotransfected into human hepatoma cell line HepG2. Scanning fluorescence microscopy showed the majority of immunological signals of HBx to be at the perinuclear region of transfected cytoplasm. HBx was also clearly detectable in the nucleus, though less intensely expressed. This was confirmed by Western analysis and coimmunoprecipitation of HBx with transcription factor IIB (TFIIB) in subcellular fractionations. The percentage of HBx-positive cells coincided with that of CAT-positive cells, and confocal laser microscopy revealed the coexistence of CAT signals in GFP-HBx positive cells. The SV40 large T antigen nuclear localization signal (NLS) appended HBx, regardless of whether NLS was added to the N- or C-terminus, transactivated all the examined X-responsive elements (XRE) similarly as did wild-type HBx. Similar results were obtained in p53 negative Saos-2 cells. The detected nuclear HBx may be involved in modulating the transcription at the promoter level whereas the HBx in cytoplasm may be working through signal transduction pathways.
Nomura
T
T
Department of Molecular Oncology, Cancer Research Institute, Kanazawa University, Takara-machi 13-1, Kanazawa 920, Japan. nomtaka@kenroku.ipc.kanazawa-u.ac.jp
Lin
Y
Y
Dorjsuren
D
D
Ohno
S
S
Yamashita
T
T
Murakami
S
S
D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism; D005070:Evans Blue; D016650:Fluorescein-5-isothiocyanate; D006511:Hepatitis B Antigens / Q000032:analysis*; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010957:Plasmids; D015534:Trans-Activators / Q000032:analysis* / Q000235:genetics; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000032:analysis; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured; D054334:Viral Regulatory and Accessory Proteins
D006511:Hepatitis B Antigens; D015534:Trans-Activators; D035581:Transcription Factor TFIIB; D014157:Transcription Factors; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D005070:Evans Blue; D015500:Chloramphenicol O-Acetyltransferase; D016650:Fluorescein-5-isothiocyanate