Identification of a novel retinoblastoma gene product binding site on human papillomavirus type 16 E7 protein.
Transformation of mammalian cells by human papillomavirus type 16 appears to require binding of the viral E7 protein to the cellular retinoblastoma growth suppressor gene product (pRB). Binding of E7 protein to pRB inhibits several of pRB's biochemical properties, including association with the transcription factor E2F. Fragments of E7 protein derived from its conserved region 2 (CR2) domain bind to pRB and are sufficient to inhibit binding of full-length E7 protein to pRB. However, these CR2 fragments exhibit reduced affinity for pRB compared to the full-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 identified a region of E7, distinct from the CR2 domain, which is sufficient to bind pRB. This new pRB binding motif encompasses the zinc-binding conserved region 3 (CR3) domain of E7. Studies with a series of pRB deletion mutants suggest that pRB residues between amino acids 803 and 841 are necessary for binding to the E7 CR3 domain. An E7 CR3 peptide inhibits 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 with a model in which optimal binding of E7 to pRB requires at least two distinct contact sites: the previously identified high affinity interaction between the E7 CR2 domain and the pRB "pocket" region, and a second interaction between the E7 CR3 domain and the COOH-terminal region of pRB. The latter interaction is sufficient for E7's inhibition of E2F binding to pRB.
Patrick
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DR
Department of Cancer Research, Merck Research Laboratories, West Point, Pennsylvania 19486.
Oliff
A
A
Heimbrook
D C
DC
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D015870:Gene Expression; D016161:Genes, Retinoblastoma; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D027383:Papillomaviridae / Q000378:metabolism*; D050725:Papillomavirus E7 Proteins; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism*
D017931:DNA Primers; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D011994:Recombinant Proteins; D016160:Retinoblastoma Protein; C059731:oncogene protein E7, Human papillomavirus type 16