Human papillomavirus type 16 E7 protein inhibits DNA binding by the retinoblastoma gene product.

Abstract

The human papillomavirus E7 gene can transform murine fibroblasts and cooperate with other viral oncogenes in transforming primary cell cultures. One biochemical property associated with the E7 protein is binding to the retinoblastoma tumor suppressor gene product (pRB). Biochemical properties associated with pRB include binding to viral transforming proteins (ElA, large T, and E7), binding to cellular proteins (E2F and Myc), and binding to DNA. The mechanism by which E7 stimulates cell growth is uncertain. However, E7 binding to pRB inhibits binding of cellular proteins to pRB and appears to block the growth-suppressive activity of pRB. We have found that E7 also inhibits binding of pRB to DNA. A 60-kDa version of pRB (pRB60) produced in reticulocyte translation reactions or in bacteria bound quantitatively to DNA-cellulose. Recombinant E7 protein used at a 1:1 or 10:1 molar ratio with pRB60 blocked 50 or >95% of pRB60 DNA-binding activity, respectively. A mutant E7 protein (E7-Ala-24) with reduced pRB60-binding activity exhibited a parallel reduction in its blocking of pRB60 binding to DNA. An E7(20-29) peptide that blocks binding of E7 protein to pRB60 restored the DNA-binding activity of pRB60 in the presence of E7. Peptide E7(2-32) did not block pRB60 binding to DNA, while peptide E7(20-57) and an E7 fragment containing residues 1 to 60 partially blocked DNA binding. E7 species containing residues 3 to 75 were fully effective at blocking pRB60 binding to DNA. These studies indicate that E7 protein specifically blocks pRB60 binding to DNA and suggest that the E7 region responsible for this property lies between residues 32 and 75. The functional significance of these observations is unclear. However, we have found that a point mutation in pRB60 that impairs DNA-binding activity also blocks the ability of pRB60 to inhibit cell growth. This correlation suggests that the DNA-binding activity of retinoblastoma proteins contributes to their biological properties.Human papillomaviruses (HPV) are the etiologic agents responsible for warts and are believed to contribute to the pathogenesis of cervical cancer (16,55). The most commonly studied isolates of HPV associated with cervical cancer include 54). The HPV E6 and E7 genes are believed to participate in cell growth regulation. These genes are generally found in cervical cancer cells and cell lines associated with HPV infections, while the remainder of the HPV genome is commonly absent (3,41,43). Moreover, the E6 and E7 genes together are capable of immortalizing primary human keratinocytes (37). The E7 gene by itself is capable of transforming NIH 3T3 cells and can cooperate with ras in the transformation of primary baby rat kidney cells (39,46). E6 alone does not transform murine fibroblasts or cooperate with ras in transforming rat cells (5, 46). Since the E7 gene appears to be the most potent transforming activity in the HPV-16 genome, our laboratory has focused on the role of the E7 gene product in the pathogenesis of HPV-induced disease.

Stabilization of the Retinoblastoma Protein by A-Type Nuclear Lamins Is Required for INK4A-Mediated Cell Cycle Arrest

“…Similar results were found when GFP-pRB was expressed in littermate Lmna ϩ/ϩ cells, as well as the siLmna and siGFP cells. This finding is consistent with experiments in NIH 3T3 cells, where enforced pRB expression confers reduced cell proliferation (24,53,64), and indicates that increased expression of pRB in cells with reduced lamin A/C expression can still confer G 1 arrest. Cotransfection of GFP-p16 ink4a and pRB led to a further reduction in the percentage of S-phase cells relative to controls.…”

The activation domain of transcription factor PU.1 binds the retinoblastoma (RB) protein and the transcription factor TFIID in vitro: RB shows sequence similarity to TFIID and TFIIB.

“…6). It is also worth noting that, like TFIID (24), RB contains an imperfect repeat in this region (residues 399-462 and 505-563) and displays DNA-binding activity (26,27).…”

Protein domains governing interactions between E2F, the retinoblastoma gene product, and human papillomavirus type 16 E7 protein.

“…This observation suggests that the E7-and E2F-binding sites on pRB are not identical. Interestingly, this same C-terminal region has previously been shown to contribute to pRB's binding to doublestranded DNA (39,40). It is not yet apparent whether these two activities are related.…”

“…The role of the metal-binding region on E7's biochemical activity is quite dramatic. Affinity for pRB60, ability to block pRB's E2F-binding activity, and ability to block pRB's DNA-binding activity are all affected by the presence of at least one Cys-X-X-Cys motif, with both domains required for optimal activity (39). Together with the observation that optimal E2F binding requires the C-terminal tail of pRB (amino acids 777 to 909), these results suggest that the metal-binding domain of E7 occludes an interaction of E2F with the C-terminal region of pRB (Fig.…”