The double stranded RNA-activated protein kinase, PKR, is an interferon (IFN)-regulated host defense enzyme essential for higher vertebrates to suppress the spread of different viruses. PKR is a key regulator of protein synthesis and gene transcription. PKR phosphorylates the protein synthesis initiation factor, eIF2alpha regulating its involvement in mRNA translation. In response to specific signals, PKR also mediates phosphorylation of important transcriptional regulators,including the inhibitor of NF-kappaB, IkappaB, and the transcription factor IRF-1. The long term objectives of this proposal are to understand how PKR functions to control viral infections and cellular homeostasis.
The specific aims are as follows: 1. To probe the mechanisms of viral or cellular dsRNA activation and inhibition of PKR, the three dimensional structures of PKR double stranded RNA binding domain (dsRBD) will be determined in a complex with the PKR activator, HIV-1 TAR RNA using multidimensional heteronuclear NMR spectroscopy. The global organization and detailed structure of the PKR domains and their interaction with dsRNA, will be determined by co- crystallization of full-length PKR with TAR RNA and the structure solved using multiple wavelength anomalous diffraction (MAD). 2. To investigate the hypothesis that PKR functions upstream of IRF-1 to regulate gene transcription and apoptosis, molecular and biochemical approaches will be used to study the interactions between PKR and IRF-1. Novel PKR-regulated genes will be identified which may play a role in mediating PKR activities in antiviral activity, and apoptosis. 3. To test the hypothesis that PKR plays a role both in the innate resistance to virus infection and in immunoregulation, the differences in the response of wild type and PKR knockout mice to different virus infections will be studied. The ability of PKR to be regulated by dsRNA provides a unique mechanism of activation of an antiviral enzyme to control viral infections. An understanding of the mechanisms of activation and of downstream targets of PKR may lead to the discovery and development of novel therapeutics for viral infections, cancer and immune dysfunction.
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