The purpose of this investigation is to understand how the E2 protein from papillomavirus recognizes DNA. Three-dimensional structures will be determined using nuclear magnetic resonance (NMR) spectroscopy for the DNA-binding domain of bovine papillomavirus (BPV1) E2 protein, the DNA to which E2 protein binds, and mutants of the protein. The approach will be to: l, Purify recombinant forms of the protein; 2, Optimize conditions for NMR spectroscopy; 3, Obtain NMR data for the DNA-binding domain; 4, Study site-specific mutants of E2; 5, Obtain NMR data for the binding-site DNA; 6, Determine structures from the NMR data; 7, Measure dynamics in the protein; and 8, Perform molecular modeling studies on the structures determined. The DNA-binding domain encompasses the most C-terminal 101 residues (310-410) of bovine papillomavirus E2 protein. This includes a 16 amino acid residue segment that has not been studied before structurally and which clearly appears to be important for E2 protein function. A comparison of structures for the previously published protein-DNA complex to that of the unbound protein and unbound DNA will reveal the conformational changes that the protein and DNA must undergo in order to recognize each other. Furthermore, dynamics within the protein will be studied by amide proton exchange and relaxation time measurements to understand the role of flexibility in the E2 protein-DNA recognition process. An important feature of this application is the study of site- specific mutants of E2 protein. Although many of these mutants have been characterized as to their DNA-binding and transactivating ability little is known about the structural consequences of introducing the amino acid change. Alteration of structure caused by replacement of the cysteine in the DNA recognition helix will be examined in particular since the DNA- binding affinity of E2 may be regulated by modification of cysteinyl sulfur through a novel reduction/oxidation mechanism. The papillomaviruses are a small family of DNA viruses that cause epithelial tumors in a variety of vertebrate hosts. The genetics and biochemistry of bovine papillomavirus (BPV1) have been most extensively characterized. The human (HPV) and bovine viruses have many properties in common, including homologous DNA-binding domains of E2 protein. E2 plays essential roles in both viral DNA replication and viral gene expression and therefore regulates viral growth and transformation of the host cell. In humans the vast majority of cervical carcinomas contain HPV viral DNA. A better understanding of the way in which E2 regulates viral activity is an important first step in the development of novel methods to correct aberrant processes in cellular control such as those associated with cancer caused by the human virus.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
First Independent Research Support & Transition (FIRST) Awards (R29)
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Molecular and Cellular Biophysics Study Section (BBCA)
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Tufts University
Schools of Medicine
United States
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Chen, J J; Hong, Y; Rustamzadeh, E et al. (1998) Identification of an alpha helical motif sufficient for association with papillomavirus E6. J Biol Chem 273:13537-44