The regulation of protein synthesis is central to a cells ability to respond to extracellular stimuli and intracellular cues, including mitogenic signals. Influenza virus infection results in the selective translation of viral mRNAs, dependent on sequences in the viral 5' UTR. Our laboratory has identified a host cell mRNA binding protein, G-rich sequence factor-1 (GRSF-1), which specifically bound the influenza 5' UTR and stimulated viral 5' UTR driven luciferase protein synthesis in vitro. To further examine the role of GRSF-1 in selective, cap-dependent translation initiation, we propose to determine the regions of GRSF-1 responsible for mRNA binding and increased translation, and to determine the nature of the interaction of GRSF-1 with proteins involved in the control of protein synthesis. We propose to determine the domains required for these functions using deletion and site-directed mutagenesis coupled with RNA electrophoretic shift assays and in vitro translation experiments. Of particular interest will be the identification of mutants which are impaired in translational stimulation, yet retain mRNA binding activity. We further hypothesize that GRSF-1 stimulates translation by binding to viral 5' UTRs and catalyzing the formation of the initiation complex by interacting with specific components of the translational machinery. To test this hypothesis, we will identify GRSF-1 interacting proteins using far-western analysis of both protein blots and lambda expression libraries, as well as using GST-pulldown analyses. The proposed studies will provide a much more detailed understanding of Influenza virus replication, as well as the regulation of protein synthesis in mammals.
