The correlation between papillomavirus infection and the development of human cancer is well established and the viral genes involved in this process have been identified. It is becoming increasingly evident that the ability of the papillomaviruses to induce cellular proliferation involves complex formation with certain cellular proteins that are important for the regulation of cell growth. A consequence of these interactions appears to be an alteration of the function of these proteins resulting in mitogenesis and transformation. The E5 protein is the major transforming protein expressed by the prototype bovine papillomavirus type 1 (BPV-1). It is unique in that it is highly hydrophobic, localizes predominantly to Golgi and endomembrane compartments, and consists of only 44 amino acids. Many of the human papillomaviruses (HPVs) also encode small, hydrophobic E5 proteins. However, our current knowledge of the activity of these structurally related E5 proteins is only rudimentary. Several recent studies on both the HPV and BPV E5 proteins suggest an involvement of tyrosine kinase growth factor receptors in the induction of cellular mitogenesis. This research grant application proposes to demonstrate that mitogenetic and transforming activity induced by the BPV-1 E5 protein is a direct consequence of the activation of growth factor receptors. In addition, we intend to define the molecular and biochemical mechanisms involved in the process of receptor activation. The molecular cloning of growth factor receptor genes has made it feasible to investigate the ability of specific receptors to couple with intracellular components needed to evoke a functional response in foreign host cells. To investigate the ability of the E5 proteins to induce mitogenic signalling through growth factor receptors, a nontumorigenic, murine myeloid cell line (32D) which is strictly dependent on interleukin 3 (IL-3) for sustained proliferation in culture will be employed. The strict requirement for IL-3 for the growth of 32D cells can be functionally substituted by the stimulation of signal transduction pathways initiated by the expression of specific growth factor receptors and the addition of the appropriate ligand to the culture medium. This phenomenon will be exploited to identify and characterize those components of signal transduction pathways that are capable of responding to E5 expression to induce mitogenesis and cellular transformation in the absence of ligand. The data generated from these studies, in addition to assisting in the elucidation of the complex mechanism of papillomavirus transformation, may also provide new insights into molecular aspects of growth factor receptor tyrosine kinases, receptor-mediated signal transduction, and the control of cell growth. As a long-term goal of this project, a similar approach will be applied to the structurally homologous HPV E5 proteins. This approach should assist in the identification of the function(s) and cellular targets of these less well-characterized proteins.
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