title: A high-throughput cellular assay to quantify the p53-degradation activity of E6 from different human papillomavirus types.
abstract: A subset of human papillomaviruses (HPVs), known as the high-risk types, are the causative agents of cervical cancer and other malignancies of the anogenital region and oral mucosa. The capacity of these viruses to induce cancer and to immortalize cells in culture relies in part on a critical function of their E6 oncoprotein, that of promoting the poly-ubiquitination of the cellular tumor suppressor protein p53 and its subsequent degradation by the proteasome. Here, we describe a cellular assay to measure the p53-degradation activity of E6 from different HPV types. This assay is based on a translational fusion of p53 to Renilla luciferase (Rluc-p53) that remains sensitive to degradation by high-risk E6 and whose steady-state levels can be accurately measured in standard luciferase assays. The p53-degradation activity of any E6 protein can be tested and quantified in transiently transfected cells by determining the amount of E6-expression vector required to reduce by half the levels of RLuc-p53 luciferase activity (50 % effective concentration [EC50]). The high-throughput and quantitative nature of this assay makes it particularly useful to compare the p53-degradation activities of E6 from several HPV types in parallel.
authors.affiliation: Laboratory of Molecular Virology, Institut de Recherches Cliniques de Montréal, 110 Pine Avenue West, Montreal, QC, Canada, H2W 1R7.
authors.forename: David
authors.initials: D
authors.lastname: Gagnon
authors.forename: Jacques
authors.initials: J
authors.lastname: Archambault
mesh_terms: D045744:Cell Line, Tumor; D005822:Genetic Vectors / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D059748:Proteolysis; D012097:Repressor Proteins / Q000378:metabolism*; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
chemical_list: C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D012097:Repressor Proteins; D016159:Tumor Suppressor Protein p53; D008156:Luciferases