Functional interactions of the E7 oncoprotein encoded by HPV-16 with cellular target proteins: Determination of structural requirements

Research project P 14288 Interactions of HPV-16 E7 with cellular target proteins Pidder JANSEN-DÜRR 06.03.2000 Human papillomaviruses (HPVs) of the "high-risk" type are etiological agents responsible for benign epithelial proliferative disorders including genital warts, and are a contributory factor in the pathogenesis of cervical cancer. The oncogenic potential of papillomaviruses is mediated through two viral gene products, referred to as E6 and E7. Both proteins show autonomous cell transformation capability, and it was shown that the ability of the E7 protein to override cellular growth control correlates with its physical interaction with a group of cellular growth regulatory proteins; it is conceivable that the binding of E7 to these cellular proteins modulates the activity of growth regulatory pathways in the host cell. The goal of the present application is to characterize at the molecular level the interaction of the E7 oncoprotein encoded by HPV-16 with two cellular target proteins, IGFBP-3 and M2- PK. Both interactions were discovered by this laboratory, and we found that E7 binding modulates the function of both proteins. To clarify the structural basis for these interactions, we will identify the protein domains in IGFBP-3 and M2-PK that are required for the interaction with HPV-16 E7, using site-directed mutagenesis, followed by in vitro and in vivo interaction experiments. It is also planned to establish an electrophoretic mobility shift assay (EMSA) for the biophysical analysis of E7- containing complexes. We will also investigate the role of a recently described Zn finger motif in IGFBP-3 for the interaction with E7. We will analyze if IGFBP-3 mutants which fail to interact with E7 can still induce apoptosis and, if so, whether E7 can override this activity. Artificial E7-binding peptide aptamers, which were developed in the laboratory, will be tested for their ability to competitively inhibit the binding of E7 to IGFBP-3 and M2-PK. Such peptide aptamers may serve as specific tools to block the interaction of E7 with individual cellular target proteins. In a separate project, the solution structure of HPV-16 E7 will be established by NMR spectroscopy, with particular emphasis on the Zn finger motifs in the E7 C-terminus. If the solution structure of E7 can be resolved; we plan to use NMR spectroscopy also in the present project for the analysis of complexes between E7 and selected E7-binding proteins. The experiments described here may allow the identification of high-affinity E7-binding compounds, with the goal to disrupt the interaction of E7 with relevant cellular target proteins within tumor cells.

The main goal of this project was to analyze by molecular methods the interaction of the E7 oncoprotein encoded by oncogenic human papillomavirus type 16 with cellular regulatory proteins. E7 is the major transforming protein of HPV-16 and plays a key role in virus-induced carcinogenesis. In the present project, we attempted to obtain new information on the interactions of E7 with cellular partner proteins that are known to control physiologic processes in the cell that are relevant in the context of cell transformation. For HPV-16 E7, we have obtained a series of synthetic artificial peptides which bind to various regions in the E7 protein. We have also been able to demonstrate that these peptides can be used to modulate the interaction of E7 with cellular regulatory proteins in vitro as well as in vivo. During the course of this project, we have also described a novel interaction between E7 and the glycolytic enzyme complex and have shown that E7, via binding to M2 pyruvate kinase, can modulate the composition of the glycolytic enzyme complex, resulting in a reprogramming of the cellular carbohydrate metabolism. These results have established a new paradigm in which E7 and an activated Ras oncogene cooperate to create the tumor metabolome that is essential for successful cell transformation. We have also established a protocol to recombinantly express and purify to homogeneity the B? proteins encoded by three distinct high risk human papillomaviruses, namely HPV-16, HPV-18 and HPV-45. We have performed a biophysical analysis of the HPV- 16 E7 oncoprotein. This resulted in the elucidation of structural characteristics of E7; in particular, these results have revealed that E7 is one of the few known proteins which natively exist in a partially unfolded conformation. This property is probably linked to the documented ability of E7 to interact with a large variety of cellular partner proteins. We have also used the purified recombinant E7 proteins to produce and affinity-purify polyclonal antibodies. These antibodies were shown in further experiments to be suitable for detection of E7 protein in biopsies obtained from cervical carcinoma. The data obtained clearly indicate that expression of E7 and the retinoblastoma protein is mutually exclusive in cervical carcinoma biopsies which is a first indication that the ability of E7 to degrade the retinoblastoma protein, as shown before in in vitro experiments, is also relevant in the context of cervical carcinogenesis. Together, the results obtained in this part of the project will lay the ground for improved diagnostic procedures for the early detection of cervical carcinoma.