The long-term goal of this proposal is to understand the regulation of protein synthesis in mammalian viruses. Our laboratory has focused on the role of mRNA structure on translation. The P/C mRNA of Sendai virus provides an excellent model to dissect translation machinery. It uses five translation start sites to express five proteins (P, C, C', Yl and Y2) in differential amounts. Expression of these proteins is not in accordance with the tenets of ribosome scanning, internal ribosome entry, or the ribosome shunting model. Our studies indicated that the C-AUG must occur in +1 orientation in relation to the P-AUG for the efficient synthesis of C protein. Importantly, all the known viral bicistronic mRNAs with overlapping reading frames have their second AUG in +1 orientation. Thus, one aim of the proposal is to test the hypothesis that the +1 orientation of the downstream reading frame in a bicistronic mRNA is crucial for its efficient initiation. This concept will be examined by constructing mRNAs with three overlapping open reading frames. Appropriate placement of AUG start sites will test the validity of this hypothesis. Second, non-AUG start sites are used efficiently in the P/C mRNA. The 5' UTR of the mRNA appears to be important for translation initiation at non-AUG codons. Third, the Y2 start site (fourth AUG) locus has a high propensity for translation initiation. Chimeric mRNAs will be constructed to define the loci that enhance translation of C' and Y2 proteins. Preliminary results have indicated that long-range interactions in the mRNA play a role in the initiation of all P/C mRNA encoded proteins. To test this, secondary structure of the entire P/C mRNA will be analyzed using both secondary and higher order structure-probing reagents to establish structure-function relationships. Creating deletions within the coding regions of P and C proteins will localize internal regulatory sequences. Fourth, synthesis of C' and certain cellular proteins is resistant to cycloheximide inhibition. Elements and mechanism that allow cycloheximide resistance will be defined. Finally, P/C mRNA binding proteins that are involved in translation regulation will be identified and characterized. Recent studies have shown that the internal initiation can occur on the A site of the ribosome without the initiator methionine tRNA, eIF2 or GTP hydrolysis. This initiation mechanism can explain some of our results. We will test this model for the P/C mRNA. In essence, the proposed studies will define the mechanisms that allow the regulated expression of the polycistronic P/C mRNA. Moreover, these studies will provide insights as to how mRNA structure and its interacting proteins regulate the protein synthesis machinery in mammalian cells.
