The OVERALL OBJECTIVE of the proposed investigation is to elucidate the roles of the interferon-inducible RNA-regulated protein kinase (PKR) in the actions that interferons (IFN) mediate on viral and host functions. The three SPECIFIC AIMS of our proposed continuation studies are as follows: (1) To further delineate the roles of the PKR protein in the host responses to virus infection. To characterize the function of the PKR protein in human cells in culture, through the use of established HeLa, U, and Huh cell clones that are stably deficient in PKR because of targeted gene silencing compared to appropriate knockdown-control cell clones. These human cell clones will be examined for the mechanisms by which PKR affects virus replication, with emphasis on vaccinia virus (wild-type and E3L mutant), and measles virus (wild- type, V and C mutants) and adenovirus (VAI mutant), viruses with human cell tropism, and for the cellular apoptotic and IFN inducing phenotypes in response to different stimuli of the knockdown compared to control cell clones. (2) To elucidate the biochemical mechanism by which the PKR protein confers the PKR-dependent biological phenotypes characterized under aim 1. To attempt to determine the biochemical functions of the PKR protein (RNA binding activity;catalytic activity;PKR domain) necessary to complement the biological phenotypes (including apoptosis, virus growth and IFN production) and biochemical changes characteristic of the human PKR knockdown cells, using wild-type and mutant forms of cDNA expression constructs for the human PKR protein engineered to circumvent knockdown, and the mouse PKR and fish PKZ proteins. (3) To further delineate the structure of the 5'-flanking region of the Pkr gene and identity of trans-acting factors required for interferon-inducible as well as basal transcriptional activity. To map the major Pkr transcription sites, using RNA from uninfected and infected and untreated and IFN-treated cells and tissues, and to elucidate the basis of the tissue-specific differences in Pkr mRNA size multiplicity. To delineate the importance of Sp1 and Sp3 protein binding to the novel 15-bp DNA element and upstream sites in IFN inducible as well as basal transcriptional activity.

Public Health Relevance

The health relatedness and relevance of the proposed research stems from the likelihood that the studies may contribute to a better understanding of regulatory mechanisms operative in normal cells as well as virus- infected cells, including cells infected with oncolytic or vaccine strain viruses for therapeutic or preventative strategies. Elucidation of the actions of interferon at the molecular level is of immediate importance in view of the use of interferon in the clinic.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI020611-27
Application #
8414820
Study Section
Special Emphasis Panel (ZRG1-IDM-Q (02))
Program Officer
Challberg, Mark D
Project Start
1983-12-01
Project End
2014-12-31
Budget Start
2013-01-01
Budget End
2013-12-31
Support Year
27
Fiscal Year
2013
Total Cost
$302,248
Indirect Cost
$92,863
Name
University of California Santa Barbara
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
094878394
City
Santa Barbara
State
CA
Country
United States
Zip Code
93106
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
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
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
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
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 eIF2α-dependent inhibition of protein synthesis and virus-induced stress granule formation. Virology 443:48-58
Ruggieri, Alessia; Dazert, Eva; Metz, Philippe et al. (2012) Dynamic oscillation of translation and stress granule formation mark the cellular response to virus infection. Cell Host Microbe 12:71-85
Taghavi, Nora; Samuel, Charles E (2012) Protein kinase PKR catalytic activity is required for the PKR-dependent activation of mitogen-activated protein kinases and amplification of interferon beta induction following virus infection. Virology 427:208-16

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