We have been studying the regulation of protein synthesis in influenza virus-infected cells to decipher the molecular mechanisms by which the virus maintains both the selective and efficient translation of its mRNAs. Our studies have included an identification of nucleotide sequences which allow viral mRNAs to be translated at times when host cell protein synthesis is inhibited. In the current proposal we intend to extend these observations by precisely identifying the nucleotide sequences within the influenza viral 5' untranslated region (UTR) which confer this resistance to the host shut-off. A systematic mutagenesis study will be performed on the nucleocapsid protein (NP) 5'UTR. Protein expression will be measured in our in vivo assay in which the appropriate cDNAs are first transfected into COS-1 cells followed by infection with influenza virus. Further, we propose to analyze potential viral and/or cellular transacting factors interacting with 5'UTRs by mobility gel shift analysis and ultraviolet (UV) cross-linking. We also have been engaged in studies defining the mechanisms by which influenza maintains high overall levels of viral translation inside the infected cells. This is accomplished through the downregulation of the interferon-induced, double stranded (ds) RNA activated protein kinase, referred to as PKR. Once activated, the protein kinase phosphorylates its natural substrate, eukaryotic initiation factor 2 (eIF-2), which could then lead to dramatic declines in protein synthetic rates. Since viral RNAs have the capacity to activate PKR, influenza virus has evolved complex strategies to counteract this pathway and avoid its harmful effects on virus replication. A cellular 58,000 dalton protein (P58), which inhibits PKR activity, is activated during viral infection probably as a result of the dissociation of P58 from another cellular factor (termed I-P58 for inhibitor of P58). To further unravel the molecular details of these PKR regulatory pathways we propose the following. We will perform a structure-function analysis of the influenza virus activated PKR inhibitor, P58. This will be facilitated by our recent isolation of a full-length P58 cDNA clone which can direct the synthesis of a functional P58 protein in Escherichia coli. Utilizing both in vitro and in vivo approaches and monoclonal antibodies specific for P58 and PKR, we will determine whether P58 acts by directly interacting with PKR or other cellular and viral proteins. In addition we intend to examine the regulation of P58 itself, both in uninfected and influenza virus infected cells. We will test whether P58, which has homology to heat shock proteins and is a member of the TPR (tricotetrapeptide) family of proteins, is regulated by phosphorylation, stress, or changes in cell localization. Further, we will purify and characterize I-P58, the cellular inhibitor of P58 as well as attempt to determine how influenza virus activates P58. Recent evidence suggests that PKR regulates cellular gene expression even in the absence of virus infection. For example, PKR, possibly due to its growth restrictive properties, may function as a tumor suppressor gene. Thus our proposed studies on the cellular regulation of PKR by these complex pathways will contribute towards a more general understanding of cellular gene expression at the translational level.
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