Homologous sequences in adenovirus E1A and human papillomavirus E7 proteins mediate interaction with the same set of cellular proteins.

Studies of adenovirus E1A oncoprotein mutants suggest that the association of E1A with the retinoblastoma protein (pRB) is necessary for E1A-mediated transformation. Mutational analysis of E1A indicates that two regions of pRB are required for E1A to form stable complexes with the retinoblastoma protein. In addition to pRB binding, these regions are necessary for E1A association with several other cellular proteins, including p130, p107, cyclin A, and p33cdk2. Here we show that short synthetic peptides containing the pRB-binding sequences of E1A are sufficient for interaction with p107, cyclin A, and p130. The E7 protein of human papillomavirus type 16 contains an element that binds to pRB and appears to be functionally homologous to the E1A sequences. Peptides containing this region of the E7 protein were able to interact with p107, cyclin A, and p130 in addition to pRB. These findings suggest that the common mechanism of transformation used by these viral oncogenes involves their association with a set of polypeptides.     

Identification of HPV-16 E7 peptides that are potent antagonists of E7 binding to the retinoblastoma suppressor protein

Complex formation between the human papilloma virus type-16 E7 protein (HPV-16 E7) and the retinoblastoma suppressor protein (pRB) is believed to be important in the process of cellular transformation that leads to cervical carcinoma. Utilizing an in vitro solution assay as well as a plate binding assay that measures the association between HPV-16 E7 and pRB proteins, we have examined a series of synthetic HPV-16 E7 peptides. HPV-16 E7 peptides which lie between amino acid residues 14 and 32 were found to be potent inhibitors of E7/pRB binding. The minimal peptide structure that possessed full antagonist activity was N-acetyl-E7-(21-29)-peptide amide. This peptide inhibited 100% of E7/pRB binding and exhibited an IC50 of 40 nM in the plate binding assay. A purified beta-galactosidase-E7 fusion protein exhibited an IC50 of 2 nM in the same assay. These results suggest that other regions of the E7 molecule in addition to amino acids 21-29 may contributed to E7/pRB interaction. Analysis of E7-(20-29)-peptides containing single amino acid substitutions suggests that Cys24, Tyr23, Tyr25, Asp21, and Glu26 are important residues for maintaining maximal antagonist activity. This series of peptides should prove useful in analyzing the biological consequences of E7/pRB binding in HPV-infected cells.     

Three regions of the pRB pocket domain affect its inactivation by human papillomavirus E7 proteins

A critical event in papillomavirus transformation of human cells is the inactivation of pRB by the E7 protein. E7, like many other viral oncoproteins, possesses a well-characterized LXCXE peptide motif that interacts with the pocket domain of pRB. Disruption of the LXCXE-binding cleft on pRB renders it resistant to E7 binding and inactivation. Such binding cleft mutants of pRB are capable of inducing a G(1) arrest in the human papillomavirus 18-transformed HeLa cell line. We show here that the efficient inactivation of pRB in HeLa cells does not simply depend on the integrity of the LXCXE-binding cleft. Multiple site-directed mutants that alter conserved surfaces of the pRB pocket domain cause HeLa cells to accumulate in G(1). We divide these mutants into two classes: those that can be bound by E7 and those that cannot. The E7 interacting mutants include changes in conserved residues that lie in a groove between the A and B halves of the pocket. Surprisingly, none of these mutants show a clear defect in any of the known mechanisms for pRB inactivation by E7. Analysis of mutants that are compromised for E7 binding reveals that this interaction depends on both the LXCXE-binding cleft and on a conserved group of lysines adjacent to the cleft. These basic amino acids on pRB define a discrete interaction point with E7. These residues most likely form ionic interactions with conserved acidic amino acids on E7 since a stable pRB/E7 interaction was restored when the lysine residues on pRB and the acidic residues on E7 were interchanged.     

Polyomavirus large T mutants affected in retinoblastoma protein binding are defective in immortalization.

To clarify the relationship between the various activities of the polyomavirus large T antigen and the contribution of this oncogene to neoplastic transformation, we constructed a series of mutants with small deletions or single-amino-acid substitutions in two separate regions of the protein. These sequences were targeted because they showed considerable similarity to conserved regions 1 and 2 of adenovirus E1A which are thought to be binding sites for the retinoblastoma gene product (pRB). The pRB-binding properties of the large T mutants were assessed with an in vitro coimmunoprecipitation assay. pRB binding was readily detected with wild-type large T, but coprecipitation was completely abolished by as little as a single amino acid substitution (Asp-141----Glu or Glu-146----Asp) in region 2 of the polyomavirus large T antigen. Mutants defective in pRB binding were unable to immortalize primary rat embryo fibroblasts, suggesting that association with pRB is an important component of immortalization mediated by polyomavirus large T. The mutations in region 1 affected pRB binding only marginally, yet some of them severely impaired immortalization, indicating that pRB binding may be essential but not sufficient for immortalization.     

Purification and characterization of a functionally homogeneous 60-kDa species of the retinoblastoma gene product.

The retinoblastoma susceptibility gene (RB) encodes a 928-amino acid protein (pRB) that is hypothesized to function in a pathway that restricts cell proliferation. The immortalizing proteins from three distinct DNA tumor viruses (SV40 large T antigen, adenovirus E1a, and human papilloma virus Type 16 E7) have been shown to interact with RB protein through two noncontiguous regions comprised of amino acids 393-572 (domain A) and 646-772 (domain B). We constructed a truncated form of RB (RB p60) that retains these two domains but eliminates the N-terminal 386 amino acids of RB. RB p60 was expressed in Escherichia coli in inclusion bodies. After solubilization, it was refolded in the presence of magnesium chloride, and the active protein was isolated with an E7 peptide affinity column. The protein that elutes from this column is functionally homogenous in its ability to bind immobilized E7 protein. Thermal denaturation studies provide additional evidence for the conformational homogeneity of the isolated protein. This purification scheme allows the isolation of significant amounts of RB p60 protein that is suitable for structural and functional studies.     

Adenovirus E1B 19-kilodalton protein overcomes the cytotoxicity of E1A proteins.

Infection with adenovirus mutants carrying either point mutations or deletions in the coding region for the 19-kDa E1B gene product (19K protein) causes degradation of host cell and viral DNAs (deg phenotype) and enhanced cytopathic effect (cyt phenotype). Therefore, one function of the E1B 19K protein is to protect nuclear DNA integrity and preserve cytoplasmic architecture during productive adenovirus infection. When placed in the background of a virus incapable of expressing a functional E1A gene product, however, E1B 19K gene mutations do not result in the appearance of the cyt and deg phenotypes. This demonstrated that expression of the E1A proteins was responsible for inducing the appearance of the cyt and deg phenotypes. By constructing a panel of viruses possessing E1A mutations spanning each of the three E1A conserved regions in conjunction with E1B 19K gene mutations, we mapped the induction of the cyt and deg phenotypes to the amino-terminal region of E1A. Viruses that fail to express conserved region 3 (amino acids 140 to 185) and/or 2, (amino acids 121 to 185) or nonconserved sequences between conserved regions 2 and 1 of E1A (amino acids 86 to 120) were still capable of inducing cyt and deg. This indicated that activities associated with these regions, such as transactivation and binding to the product of the retinoblastoma susceptibility gene, were dispensable for induction of E1A-dependent cytotoxic effects. In contrast, deletion of sequences in the amino terminus of E1A (amino acids 22 to 107) resulted in extragenic suppression of the cyt and deg phenotypes. Therefore, a function affected by deletion of amino acids 22 to 86 of E1A is responsible for exerting cytotoxic effects in virally infected cells. Furthermore, transient high-level expression of the E1A region using a cytomegalovirus promoter plasmid expression vector was sufficient to induce the cyt and deg phenotypes, demonstrating that E1A expression alone is sufficient to exert these cytotoxic effects and that other viral gene products are not involved. Finally, placing E1A expression under the control of a strong promoter did not alter the requirement for E1B in the transformation of primary cells. One possibility is that the E1B 19K protein is required to overcome the cytotoxic effects of E1A protein expression and thereby enable primary cells to become transformed.     

Oligomerization properties of the viral oncoproteins adenovirus E1A and human papillomavirus E7 and their complexes with the retinoblastoma protein

Human papillomavirus 16 E7 (HPV16 E7) and adenovirus 5 E1A (Ad5 E1A) are encoded by highly divergent viruses yet are functionally similar in their ability to bind the retinoblastoma (pRB) tumor suppressor protein, causing the aberrant displacement of E2F trancription factors. The amino acid residues of HPV16 E7 that are necessary for stability, for inhibition of pRB function, and for cell transformation are also necessary for E7 oligomerization. However, neither the specific oligomerization state of HPV16 E7 nor of Ad5 E1A as a function of pRB-binding has been characterized. To gain insight into HPV16 E7 and Ad5 E1A oligomerization properties, sedimentation equilibrium experiments were performed with recombinant HPV16 E7 and Ad5 E1A proteins. These studies reveal that, despite the overall functional similarities between these proteins, monomers, dimers, and tetramers of HPV16 E7 were detected while only reversible monomer-dimer association was identified for Ad5 E1A. The apparent K(d(monomer)-(dimer)) of HPV16 E7 is approximately 100-fold lower than that of a comparable region of Ad5 E1A, and it is concluded that under physiological protein concentrations HPV16 E7 exists primarily as a dimer. Sedimentation equilibrium experiments of pRB/Ad5 E1A and of pRB/HPV16 E7 complexes demonstrate that the tight association of pRB with the viral oncoproteins does not disturb their inherent oligomerization properties. Taken together, this study demonstrates significant differences between the Ad5 E1A and HPV16 E7 oligomerization states that are potentially related to their distinct structures and specific mechanisms of pRB-inactivation.     

Cellular targets for transformation by the adenovirus E1A proteins

Three cellular proteins, including species of 300,000 daltons and 107,000 daltons as well as p105-RB, the product of the retinoblastoma susceptibility gene, stably interact with the adenovirus E1A proteins. To help determine the functional basis of these interactions, the regions of E1A that participate in these interactions were mapped using a series of deletion mutants. The 300,000 dalton and the 107,000 dalton proteins interacted with sequences within amino acids 1 to 76 and 121 to 127, respectively. Interaction with the third cellular protein, p105-RB, required the presence of sequences from two noncontiguous regions of the E1A polypeptide chain, amino acids 30 to 60 and 121 to 127. The regions of E1A that are required for these interactions coincided precisely with the regions of E1A that are required for its transforming function. These results suggest that the interactions with these cellular proteins are fundamental to the transforming activity of E1A.     

E1A induces phosphorylation of the retinoblastoma protein independently of direct physical association between the E1A and retinoblastoma products.

We have studied the initial effects of adenovirus E1A expression on the retinoblastoma (RB) gene product in normal quiescent cells. Although binding of the E1A products to pRB could, in theory, make pRB phosphorylation unnecessary for cell cycle progression, we have found that the 12S wild-type E1A product is capable of inducing phosphorylation of pRB in normal quiescent cells. The induction of pRB phosphorylation correlates with E1A-mediated induction of p34cdc2 expression and kinase activity, consistent with the possibility that p34cdc2 is a pRB kinase. Expression of simian virus 40 T antigen induces similar effects. Induction of pRB phosphorylation is independent of the pRB binding activity of the E1A products; E1A domain 2 mutants do not bind detectable levels of pRB but remain competent to induce pRB phosphorylation and to activate cdc2 protein kinase expression and activity. Although the kinetics of induction are slower, domain 2 mutants induce wild-type levels of pRB phosphorylation and host cell DNA synthesis and yet fail to induce cell proliferation. These results imply that direct physical interaction between the RB and E1A products does not play a required role in the early stages of E1A-mediated cell cycle induction and that pRB phosphorylation is not, of itself, sufficient to allow quiescent cells to divide. These results suggest that the E1A products do not need to bind pRB in order to stimulate resting cells to enter the cell cycle. Indeed, a more important role of the RB binding activity of the E1A products may be to prevent dividing cells from returning to G0.