THE E6 VARIANT PROTEINS E6I-E6IV OF HUMAN PAPILLOMAVIRUS-16 - EXPRESSION IN CELL-FREE SYSTEMS AND BACTERIA AND STUDY OF THEIR INTERACTION WITH P53

Several species of alternatively spliced mRNAs are transcribed from th e E6 gene region of human papillomavirus (HPV) 16. These have the codi ng capacity for either the full length E6 of 151 amino acids (aa) or f our truncated variants, E6I-E6IV, of 43-64 aa. As the first step to id entify the putative E6 variants and their functions, we generated cDNA s corresponding to the various E6 open reading frames (ORF) and examin ed their expression employing in vitro transcription/translation syste ms and the bacterial pET system. in wheat germ extract, in vitro trans lation resulted in the production of all five proteins, E6 and E6I-E6I V. These proteins were also expressed as stable fusion proteins From t he pETl6b and pET17 x b vectors in Escherichia coli. Mobilites of the E6 variant proteins on SDS-acrylamide gels were consistent with their predicted sizes. The authenticity of the synthesized proteins was conf irmed by immunoprecipitation with specific antibodies directed against epitopes in the N-terminal portion of E6 as well as antibodies raised against the individual variant proteins produced in E. coli. In rabbi t reticulocyte lysate, however, only the full length E6 and the E6IV v ariant were synthesized. This could be due to inefficient translation as well as lower stability of the short variants, E6I-III, in reticulo cyte lysate (RTL). The ability of the E6 variants to associate with p5 3 and target its proteolytic degradation in vitro, was examined in coi mmunoprecipitation assays, using in vitro synthesized proteins and mon oclonal antibodies to p53. Results of these assays indicated that only the full length E6 efficiently binds to and promotes the degradation of p53. The E6 variants E6I-E6IV, although able to associate with p53 at a low efficiency, were unable to target its degradation.