The double-stranded (ds) RNA-activated protein kinase, PKR, is one of several proteins induced by interferon and plays a pivotal role in the cellular antiviral response. PKR has also been implicated in other cellular processes including transformation, differentiation and apoptosis. There are also structural and functional connections between PKR and the RNA interference (RNAi) pathway. PKR is synthesized in a latent state and is activated upon binding to dsRNA to undergo autophosphorylation reactions that activate the kinase. In turn, activated PKR phosphorylates eukaryotic initiation factor 2a, resulting in the inhibition of protein synthesis in virally-infected cells. The importance of this antiviral pathway is highlighted by the diverse mechanisms that viruses have evolved to combat PKR. The broad objective of our research program is to define the molecular mechanisms for activation and inhibition of PKR. We will define the stoichiometries, affinities and free-energy coupling that govern formation of macromolecular complexes that regulate PKR activity using quantitative biophysical and structural methods. We will determine how NS5A from hepatitis C virus and NS1 from influenza virus interact with PKR to evade the antiviral pathway. Mutations in these proteins that affect virulence or confer interferon resistance will be correlated with PKR binding and inhibition. We will determine how short, heparin oligosaccharides function as PKR activators. Microarrays of synthetic oligosaccharides will be screened and structure-activity relationships will be generated to define novel small-molecule activators of PKR. These studies will provide the foundation for the design of therapeutic agents that target PKR for the treatment of viral infections and cancer.

Public Health Relevance

PKR plays a central role in the biological activity of interferons, which are widely employed in the treatment of viral, neoplastic and immunological diseases. However, the therapeutic effectiveness of interferons is compromised by severe side effects as well as viral resistance mechanisms that inhibit the PKR pathway. A detailed understanding of the molecular mechanism of PKR activation will help us to design new drugs that will selectively activate PKR to avoid side effects and to evade viral resistance.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Macromolecular Structure and Function B Study Section (MSFB)
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Dempsey, Walla L
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University of Connecticut
Schools of Arts and Sciences
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Mayo, Christopher B; Cole, James L (2017) Interaction of PKR with single-stranded RNA. Sci Rep 7:3335
Husain, Bushra; Mayo, Christopher; Cole, James L (2016) Role of the Interdomain Linker in RNA-Activated Protein Kinase Activation. Biochemistry 55:253-61
Mayo, Christopher B; Wong, C Jason; Lopez, Prisma E et al. (2016) Activation of PKR by short stem-loop RNAs containing single-stranded arms. RNA 22:1065-75
Husain, Bushra; Hesler, Stephen; Cole, James L (2015) Regulation of PKR by RNA: formation of active and inactive dimers. Biochemistry 54:6663-72
Launer-Felty, Katherine; Wong, C Jason; Cole, James L (2015) Structural analysis of adenovirus VAI RNA defines the mechanism of inhibition of PKR. Biophys J 108:748-57
Launer-Felty, Katherine; Cole, James L (2014) Domain interactions in adenovirus VAI RNA mediate high-affinity PKR binding. J Mol Biol 426:1285-95
Lyons, Daniel F; Lary, Jeffrey W; Husain, Bushra et al. (2013) Are fluorescence-detected sedimentation velocity data reliable? Anal Biochem 437:133-7
Husain, Bushra; Mukerji, Ishita; Cole, James L (2012) Analysis of high-affinity binding of protein kinase R to double-stranded RNA. Biochemistry 51:8764-70
Anderson, Eric; Pierre-Louis, Willythssa S; Wong, C Jason et al. (2011) Heparin activates PKR by inducing dimerization. J Mol Biol 413:973-84
Wong, C Jason; Launer-Felty, Katherine; Cole, James L (2011) Analysis of PKR-RNA interactions by sedimentation velocity. Methods Enzymol 488:59-79

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