IDENTIFICATION OF A NOVEL RETINOBLASTOMA GENE-PRODUCT BINDING-SITE ONHUMAN PAPILLOMAVIRUS TYPE-16 E7 PROTEIN

Transformation of mammalian cells by human papil lomavirus type 16 app ears to require binding of the viral E7 protein to the cellular retino blastoma growth suppresser gene product (pRB). Binding of E7 protein t o pRB inhibits several of pRB's biochemical properties, including asso ciation with the transcription factor E2F. Fragments of E7 protein der ived from its conserved region 2 (CR2) domain bind to pRB and are suff icient to inhibit binding of full-length E7 protein to pRB. However, t hese CR2 fragments exhibit reduced affinity for pRB compared to the fu ll-length protein and do not inhibit formation of the pRB E2F complex. These observations suggest the existence of additional contact sites between the E7 protein and pRB. In the current study we have identifie d a region of E7, distinct from the CR2 domain, which is sufficient to bind pRB. This new pRB binding motif encompasses the zinc-binding con served region 3 (CR3) domain of E7. Studies with a series of pRB delet ion mutants suggest that pRB residues between amino acids 803 and 841 are necessary for binding to the E7 CR3 domain. An E7 CR3 peptide inhi bits binding of E2F to pRB, indicating that E2F and E7(31-98) bind to pRB at the same or overlapping sites. These results are consistent wit h a model in which optimal binding of E7 to pRB requires at least two distinct contact sites: the previously identified high affinity intera ction between the E7 CR2 domain and the pRB ''pocket'' region, and a s econd interaction between the E7 CR3 domain and the COOH-terminal regi on of pRB. The latter interaction is sufficient for E7's inhibition of E2F binding to pRB.