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Modulations of PACT-PKR Pathway by Cellular Stresses and the NS1 Protein of Influenza A Virus

Li, Shoudong
http://rave.ohiolink.edu/etdc/view?acc_num=case1114788508

Abstract Details

2005, Doctor of Philosophy, Case Western Reserve University, Molecular Virology.
PACT contains three modular domains. While PACT domains 1 and 2 can mediate strong interactions with PKR and dsRNA, the C-terminal domain 3 binds to PKR weakly but is necessary and sufficient for activating PKR in vitro. In vivo, PACT-mediated PKR activation requires either domain 1 or domain 2 for anchoring domain 3 to PKR following cellular stress. The mechanism of PACT-mediated PKR activation was not well understood. The studies on PACT function in this dissertation led to the following findings: 1. We showed that PACT suppressed eIF2α phosphorylation without stress through domains 1 and 2, but enhanced eIF2α phosphorylation with stress through domain 3. We also showed that PACT was phosphorylated by stress, and there might be two phosphorylation sites in PACT, as shown by two dimensional gel analysis. 2. Latent PKR is thought to adopt an inactive confirmation by virtue of an intramolecular inhibitory interaction between the second of the two N-terminal dsRNA-binding motifs (dsRBM2) and the C-terminal kinase domain. Using combined structural and functional approaches, we characterized that this autoinhibitory interaction is mediated by residues 328-336 within the eIF2α kinase insertion region of PKR and a surface on dsRBM2 overlapping with putative dsRNA-binding sites. Residues 328-336 were also found to comprise the region to which PACT domain 3 bound. These results suggested that dsRNA and PACT domain 3 may competitively bind to either of the intramolecular interaction components of PKR to cause PKR activation. This model was supported by our mutational analysis, which showed that mutations within residues 328-335 could activate PKR. 3. We showed that PKR activation by PACT or dsRNA could be blocked by the RNA-binding NS1 protein of influenza A virus both in vitro and in vivo. PKR-binding, but neither dsRNA-binding nor PACT-binding, was essential for this inhibitory effect of NS1. Furthermore, we mapped the PKR-binding region in NS1 and showed that recombinant influenza A viruses encoding PKR-binding deficient NS1 point mutants could not block PKR activation in virus-infected cells. The results presented here provide models to study dsRNA- and PACT-mediated PKR activation pathways.
Ganes Sen (Advisor)
Biology, Molecular
PKR; PACT; NS1; Activation of PKR; dsRNA; PACT domain; PROTEIN

Recommended Citations

Citations

  • Li, S. (2005). Modulations of PACT-PKR Pathway by Cellular Stresses and the NS1 Protein of Influenza A Virus [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1114788508

    APA Style (7th edition)

  • Li, Shoudong. Modulations of PACT-PKR Pathway by Cellular Stresses and the NS1 Protein of Influenza A Virus. 2005. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1114788508.

    MLA Style (8th edition)

  • Li, Shoudong. "Modulations of PACT-PKR Pathway by Cellular Stresses and the NS1 Protein of Influenza A Virus." Doctoral dissertation, Case Western Reserve University, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=case1114788508

    Chicago Manual of Style (17th edition)

case1114788508
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© 2005, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.