Identification of a human immunodeficiency virus type 1 TAR binding protein in human hepatoblastoma HepG2 cells that trans-activates HIV-1 LTR-directed gene expression.
Recently, we have shown that the human immunodeficiency virus (HIV-1) long terminal repeat (LTR) directed chloramphenicol acetyltransferase (CAT) gene is efficiently expressed in human hepatoblastoma HepG2 cells and these cells can support productive HIV-1 replication. In this study we show that HepG2 cells contain a nuclear factor that binds to the HIV-1 trans-activating region (TAR), which we named HepG2-derived TAR binding protein (HTBP). Gel retardation assays using synthetic oligonucleotide probes carrying different mutations in the TAR region and competition DNA mobility-shift experiments using these oligonucleotides revealed the binding site encompassing between +7 and +13 nucleotides (5'-TCTGGTT-3') in the HIV-1 LTR. An in vivo CAT competition assay using -65HIV-1 LTR CAT as a reporter plasmid and various competitor plasmids containing these mutated oligonucleotides also demonstrated that HTBP can influence the HIV-1 LTR-directed CAT gene expression in HepG2 cells by interaction with a specific sequence in the TAR region.
Pizzella
T
T
Department of Biochemistry, Mount Sinai School of Medicine, New York, NY 10029.
Banerjee
R
R
D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000382:microbiology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000382:microbiology; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D015534:Trans-Activators; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D015534:Trans-Activators