As a model for understanding the regulation of eukaryotic protein synthesis, we have been studying the mechanisms of translational control established by influenza virus type A, a negative stranded RNA virus with a segmented genome. In cells infected by influenza virus, viral mRNAs are selectively and efficiently translated over the host cell mRNAs. The preferential translation of viral mRNAs is due in part to the failure of newly made cellular mRNAs to reach the cytoplasm of infected cells, and in part to the protein synthesis initiation and elongation blocks exerted on the preexisting cytoplasmic cellular mRNAs. Recent evidence has suggested that the structure of influenza viral mRNAs may contribute directly t the selective synthesis of viral proteins. We intend to identify the specific regions and/or nucleotide sequences which confer this selective viral mRNA advantage. For this analysis, viral and cellular mRNAs, as well as chimeras containing viral and cellular sequences, will be produced from RNA expression vectors and subsequently translated in cell-free protein synthesizing systems. We have found that influenza virus utilizes specific regulatory mechanisms to prevent a decrease in overall levels of protein synthesis resulting from a depletion of functional eukaryotic initiation factor, eIF-2. Influenza ensures that mRNA translation is not compromised during infection by preventing the autophosphorylation and activation of the cellular interferon-induced protein kinase, P68. When activated, the P68 kinase phosphorylates its natural substrate, the alpha subunit of eIF-2, leading to inhibition of its catalytic recycling and consequent decreases in protein synthesis initiation. We are currently attempting to biochemically purify and identify the influenza virus gene product responsible for P68 kinase repression. As might be expected, other viruses find it essential to downregulate P68 kinase activity to avoid the negative effects of eIF-2 alpha phosphorylation. For example, adenovirus prevents a decline in the rate of protein synthesis through the action of the virus encoded VAI RNA, which we have shown complexes with and inactivates P68. In contrast to the influenza and adenovirus systems, poliovirus cannot block P68 activation, but rather prevents the premature cessation of protein synthesis by inducing the degradation of the activated protein kinase. We intend to determine whether the poliovirus 2A or 3C proteases and/or cellular proteases are responsible of the degradation of P68. Finally, as a long range goal, we will utilize a full length P68 cDNA clone to express a functional protein kinase and perform a mutagenic analysis on the regions of P68 which interact with virus-encoded inhibitors, such as the adenovirus VAI RNA. We are confident that results obtained in these studies on mRNA structure and the cellular protein kinase will help elucidate general mechanisms of translational control in other eukaryotic systems.
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