The overall objective of the proposed investigation is to elucidate the role of the interferon-inducible double-stranded RNA-dependent protein kinase (PKR) in the actions which natural and recombinant interferons mediate on viral and host functions.
The SPECIFIC AIMS of our proposed continuation investigation of protein phosphorylation and interferon (IFN) action are as follows: (1) To continue our characterization of the structure and function of the 5'-flanking region of the human and mouse Pkr genes. To delineate the structure of the 5'-flanking region of the Pkr gene required for IFN-inducible as well as basal transcriptional activity. To attempt to map the major transcription sites by S1 nuclease protection and primer extension analyses, and to characterize protein binding by DNase I footprint analysis and gel mobility shift assays. To elucidate the importance of potential protein binding sites in promoter activity through mutagenesis. To examine Pkr regulation by cytokines other than interferons alpha and gamma. (2) To further characterize the biochemical and biophysical properties of the PKR kinase. To identify the sites of PKR phosphorylation associated with activation of kinase catalytic activity through the use of molecular genetic and chemical approaches. To study the RNA-structure(s) capable of modulating kinase activity (autophosphorylation; eIF-2alpha phosphorylation). To elucidate the mechanism of heparin-mediated activation of PKR, and to characterize the structural basis and functional significance of PKR protein-protein associations that occur in uninfected and virus- infected cells (3) to further characterize the expression of wild-type and mutant forms of PKR cDNA in cells in culture. To examine the PKR- expressing cell lines for phenotype and growth properties, and for their ability to support virus replication and protein synthesis relative to cell lines devoid of PKR. The health relatedness of the proposed research stems from the likelihood that the work may contribute to a better understanding of regulatory mechanisms involving phosphorylation possibly operative in normal cells as well as virus-infected cells. Furthermore, the elucidation of the actions of IFN at the molecular level is of immediate importance in view of the potential applications of IFN in the clinic.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI020611-14
Application #
2003288
Study Section
Virology Study Section (VR)
Project Start
1983-12-01
Project End
2002-04-30
Budget Start
1997-05-01
Budget End
1998-04-30
Support Year
14
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of California Santa Barbara
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
City
Santa Barbara
State
CA
Country
United States
Zip Code
93106
Ma, Dzwokai; George, Cyril X; Nomburg, Jason et al. (2017) Upon Infection the Cellular WD Repeat-containing Protein 5 (WDR5) Localizes to Cytoplasmic Inclusion Bodies and Enhances Measles Virus Replication. J Virol :
Kainulainen, Markus; Lau, Simone; Samuel, Charles E et al. (2016) NSs Virulence Factor of Rift Valley Fever Virus Engages the F-Box Proteins FBXW11 and ?-TRCP1 To Degrade the Antiviral Protein Kinase PKR. J Virol 90:6140-7
George, Cyril X; Ramaswami, Gokul; Li, Jin Billy et al. (2016) Editing of Cellular Self-RNAs by Adenosine Deaminase ADAR1 Suppresses Innate Immune Stress Responses. J Biol Chem 291:6158-68
George, Cyril X; Samuel, Charles E (2015) STAT2-dependent induction of RNA adenosine deaminase ADAR1 by type I interferon differs between mouse and human cells in the requirement for STAT1. Virology 485:363-70
Wu, Chengjun; Bai, Lufeng; Li, Zhiqun et al. (2015) Poor growth of human adenovirus-12 compared to adenovirus-2 correlates with a failure to impair PKR activation during the late phase of infection. Virology 475:120-8
Pfaller, Christian K; Mastorakos, George M; Matchett, William E et al. (2015) Measles Virus Defective Interfering RNAs Are Generated Frequently and Early in the Absence of C Protein and Can Be Destabilized by Adenosine Deaminase Acting on RNA-1-Like Hypermutations. J Virol 89:7735-47
Pfaller, Christian K; Radeke, Monte J; Cattaneo, Roberto et al. (2014) Measles virus C protein impairs production of defective copyback double-stranded viral RNA and activation of protein kinase R. J Virol 88:456-68
John, Lijo; Samuel, Charles E (2014) Induction of stress granules by interferon and down-regulation by the cellular RNA adenosine deaminase ADAR1. Virology 454-455:299-310
Okonski, Kristina M; Samuel, Charles E (2013) Stress granule formation induced by measles virus is protein kinase PKR dependent and impaired by RNA adenosine deaminase ADAR1. J Virol 87:756-66
Taghavi, Nora; Samuel, Charles E (2013) RNA-dependent protein kinase PKR and the Z-DNA binding orthologue PKZ differ in their capacity to mediate initiation factor eIF2?-dependent inhibition of protein synthesis and virus-induced stress granule formation. Virology 443:48-58

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