A single structural alteration in a protein synthesis initiation factor has been identified in poliovirus-infected cells, and the effect of that alteration on messenger RNA discrimination induced by poliovirus is the subject of this proposal. These studies will focus on the cellular Mr 220,000 polypeptide (p220) which is associated with an activity required to initiate translation on messenger RNAs with 5' end cap structures, and is proteolytically cleaved in poliovirus-infected cells. The function of p220, its interactions with other translational components, and alterations in function and interactions which result from proteolytic cleavage by poliovirus infection will be investigated. A monoclonal antibody directed against p220 is available, and will be utilized to purify p220 and cleavage product-containing complexes and to analyze polypeptide interactions by immunoblot identification and immunoaffinity chromatography. The structure of p220 and its cleavage products will probed with the aid of monoclonal antibodies which will be generated against specific domains of the polypeptide. The association of p220 and its cleavage products with CBP I, eIF4A, eIF4B and eIF3 will be analyzed by purification of complexes under different conditions of denaturation, complex building in vitro, antibody inhibition of polypeptide interactions, and analysis of activities associated with these complexes. Monoclonal antibodies directed against selected polypeptides which associate with p220 will be generated for further structural and functional analysis of these initiation factor interactions. In addition, the mediator of p220 cleavage will be purified and identified, and differences in p220, and its protease in different cell lines will be analyzed in an attempt to correlate p220 cleavage with host cell susceptibility to poliovirus infection. Information generated by these studies will contribute to the field of picornavirology with respect to the specific interactions of poliovirus with its host cell; as well as to our basic understanding of the biochemical mechanisms involved in messenger RNA discrimination during protein synthesis.
