This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Romano, P. R.
Right arrow Articles by Hinnebusch, A. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Romano, P. R.
Right arrow Articles by Hinnebusch, A. G.

 Previous Article  |  Next Article 

Molecular and Cellular Biology, December 1998, p. 7304-7316, Vol. 18, No. 12
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Inhibition of Double-Stranded RNA-Dependent Protein Kinase PKR by Vaccinia Virus E3: Role of Complex Formation and the E3 N-Terminal Domain

Patrick R. Romano,1,dagger Fan Zhang,1 Seng-Lai Tan,2 Minerva T. Garcia-Barrio,1 Michael G. Katze,2 Thomas E. Dever,1 and Alan G. Hinnebusch1,*

Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, Bethesda, Maryland 20892,1 and Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington 981952

Received 3 June 1998/Returned for modification 3 August 1998/Accepted 18 August 1998

The human double-stranded RNA (dsRNA)-dependent protein kinase PKR inhibits protein synthesis by phosphorylating translation initiation factor 2alpha (eIF2alpha ). Vaccinia virus E3L encodes a dsRNA binding protein that inhibits PKR in virus-infected cells, presumably by sequestering dsRNA activators. Expression of PKR in Saccharomyces cerevisiae inhibits protein synthesis by phosphorylation of eIF2alpha , dependent on its two dsRNA binding motifs (DRBMs). We found that expression of E3 in yeast overcomes the lethal effect of PKR in a manner requiring key residues (Lys-167 and Arg-168) needed for dsRNA binding by E3 in vitro. Unexpectedly, the N-terminal half of E3, and residue Trp-66 in particular, also is required for anti-PKR function. Because the E3 N-terminal region does not contribute to dsRNA binding in vitro, it appears that sequestering dsRNA is not the sole function of E3 needed for inhibition of PKR. This conclusion was supported by the fact that E3 activity was antagonized, not augmented, by overexpressing the catalytically defective PKR-K296R protein containing functional DRBMs. Coimmunoprecipitation experiments showed that a majority of PKR in yeast extracts was in a complex with E3, whose formation was completely dependent on the dsRNA binding activity of E3 and enhanced by the N-terminal half of E3. In yeast two-hybrid assays and in vitro protein binding experiments, segments of E3 and PKR containing their respective DRBMs interacted in a manner requiring E3 residues Lys-167 and Arg-168. We also detected interactions between PKR and the N-terminal half of E3 in the yeast two-hybrid and lambda  repressor dimerization assays. In the latter case, the N-terminal half of E3 interacted with the kinase domain of PKR, dependent on E3 residue Trp-66. We propose that effective inhibition of PKR in yeast requires formation of an E3-PKR-dsRNA complex, in which the N-terminal half of E3 physically interacts with the protein kinase domain of PKR.

* Corresponding author. Mailing address: Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health & Human Development, Bldg. 6A, Room B1A-13A, Bethesda, MD 20892. Phone: (301) 496-4480. Fax: (301) 496-6828. E-mail: ahinnebusch{at}nih.gov.

dagger Present address: Small Molecule Therapeutics, Inc., Monmouth Junction, NJ 08852.

Molecular and Cellular Biology, December 1998, p. 7304-7316, Vol. 18, No. 12
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Marq, J.-B., Hausmann, S., Luban, J., Kolakofsky, D., Garcin, D. (2009). The Double-stranded RNA Binding Domain of the Vaccinia Virus E3L Protein Inhibits Both RNA- and DNA-induced Activation of Interferon {beta}. J. Biol. Chem. 284: 25471-25478 [Abstract] [Full Text]  
  • Myskiw, C., Arsenio, J., van Bruggen, R., Deschambault, Y., Cao, J. (2009). Vaccinia Virus E3 Suppresses Expression of Diverse Cytokines through Inhibition of the PKR, NF-{kappa}B, and IRF3 Pathways. J. Virol. 83: 6757-6768 [Abstract] [Full Text]  
  • Short, J. A.L. (2009). Viral evasion of interferon stimulated genes. Bioscience Horizons 2: 212-224 [Abstract] [Full Text]  
  • Child, S. J., Geballe, A. P. (2009). Binding and Relocalization of Protein Kinase R by Murine Cytomegalovirus. J. Virol. 83: 1790-1799 [Abstract] [Full Text]  
  • Deng, L., Dai, P., Parikh, T., Cao, H., Bhoj, V., Sun, Q., Chen, Z., Merghoub, T., Houghton, A., Shuman, S. (2008). Vaccinia Virus Subverts a Mitochondrial Antiviral Signaling Protein-Dependent Innate Immune Response in Keratinocytes through Its Double-Stranded RNA Binding Protein, E3. J. Virol. 82: 10735-10746 [Abstract] [Full Text]  
  • Zhang, P., Jacobs, B. L., Samuel, C. E. (2008). Loss of Protein Kinase PKR Expression in Human HeLa Cells Complements the Vaccinia Virus E3L Deletion Mutant Phenotype by Restoration of Viral Protein Synthesis. J. Virol. 82: 840-848 [Abstract] [Full Text]  
  • Domingo-Gil, E., Perez-Jimenez, E., Ventoso, I., Najera, J. L., Esteban, M. (2008). Expression of the E3L Gene of Vaccinia Virus in Transgenic Mice Decreases Host Resistance to Vaccinia Virus and Leishmania major Infections. J. Virol. 82: 254-267 [Abstract] [Full Text]  
  • Van Quyen, D., Ha, S. C., Lowenhaupt, K., Rich, A., Kim, K. K., Kim, Y.-G. (2007). Characterization of DNA-binding activity of Z{alpha} domains from poxviruses and the importance of the -wing regions in converting B-DNA to Z-DNA. Nucleic Acids Res 35: 7714-7720 [Abstract] [Full Text]  
  • Guerra, S., Najera, J. L., Gonzalez, J. M., Lopez-Fernandez, L. A., Climent, N., Gatell, J. M., Gallart, T., Esteban, M. (2007). Distinct Gene Expression Profiling after Infection of Immature Human Monocyte-Derived Dendritic Cells by the Attenuated Poxvirus Vectors MVA and NYVAC. J. Virol. 81: 8707-8721 [Abstract] [Full Text]  
  • Fenner, B. J., Goh, W., Kwang, J. (2007). Dissection of Double-Stranded RNA Binding Protein B2 from Betanodavirus. J. Virol. 81: 5449-5459 [Abstract] [Full Text]  
  • Ostertag, D., Hoblitzell-Ostertag, T. M., Perrault, J. (2007). Overproduction of Double-Stranded RNA in Vesicular Stomatitis Virus-Infected Cells Activates a Constitutive Cell-Type-Specific Antiviral Response. J. Virol. 81: 503-513 [Abstract] [Full Text]  
  • Garcia, M. A., Gil, J., Ventoso, I., Guerra, S., Domingo, E., Rivas, C., Esteban, M. (2006). Impact of Protein Kinase PKR in Cell Biology: from Antiviral to Antiproliferative Action. Microbiol. Mol. Biol. Rev. 70: 1032-1060 [Abstract] [Full Text]  
  • Hakki, M., Marshall, E. E., De Niro, K. L., Geballe, A. P. (2006). Binding and Nuclear Relocalization of Protein Kinase R by Human Cytomegalovirus TRS1. J. Virol. 80: 11817-11826 [Abstract] [Full Text]  
  • Deng, L., Dai, P., Ding, W., Granstein, R. D., Shuman, S. (2006). Vaccinia virus infection attenuates innate immune responses and antigen presentation by epidermal dendritic cells.. J. Virol. 80: 9977-9987 [Abstract] [Full Text]  
  • Langland, J. O., Kash, J. C., Carter, V., Thomas, M. J., Katze, M. G., Jacobs, B. L. (2006). Suppression of Proinflammatory Signal Transduction and Gene Expression by the Dual Nucleic Acid Binding Domains of the Vaccinia Virus E3L Proteins.. J. Virol. 80: 10083-10095 [Abstract] [Full Text]  
  • Child, S. J., Hanson, L. K., Brown, C. E., Janzen, D. M., Geballe, A. P. (2006). Double-Stranded RNA Binding by a Heterodimeric Complex of Murine Cytomegalovirus m142 and m143 Proteins.. J. Virol. 80: 10173-10180 [Abstract] [Full Text]  
  • Mulvey, M., Arias, C., Mohr, I. (2006). Resistance of mRNA translation to acute endoplasmic reticulum stress-inducing agents in herpes simplex virus type 1-infected cells requires multiple virus-encoded functions.. J. Virol. 80: 7354-7363 [Abstract] [Full Text]  
  • Guerra, S., Lopez-Fernandez, L. A., Garcia, M. A., Zaballos, A., Esteban, M. (2006). Human Gene Profiling in Response to the Active Protein Kinase, Interferon-induced Serine/threonine Protein Kinase (PKR), in Infected Cells: INVOLVEMENT OF THE TRANSCRIPTION FACTOR ATF-3 IN PKR-INDUCED APOPTOSIS. J. Biol. Chem. 281: 18734-18745 [Abstract] [Full Text]  
  • Cardenas, W. B., Loo, Y.-M., Gale, M. Jr., Hartman, A. L., Kimberlin, C. R., Martinez-Sobrido, L., Saphire, E. O., Basler, C. F. (2006). Ebola Virus VP35 Protein Binds Double-Stranded RNA and Inhibits Alpha/Beta Interferon Production Induced by RIG-I Signaling.. J. Virol. 80: 5168-5178 [Abstract] [Full Text]  
  • Kwon, J.-A., Rich, A. (2005). Biological function of the vaccinia virus Z-DNA-binding protein E3L: Gene transactivation and antiapoptotic activity in HeLa cells. Proc. Natl. Acad. Sci. USA 102: 12759-12764 [Abstract] [Full Text]  
  • Nie, Y., Ding, L., Kao, P. N., Braun, R., Yang, J.-H. (2005). ADAR1 Interacts with NF90 through Double-Stranded RNA and Regulates NF90-Mediated Gene Expression Independently of RNA Editing. Mol. Cell. Biol. 25: 6956-6963 [Abstract] [Full Text]  
  • Hakki, M., Geballe, A. P. (2005). Double-Stranded RNA Binding by Human Cytomegalovirus pTRS1. J. Virol. 79: 7311-7318 [Abstract] [Full Text]  
  • Hung, M.-L., Chao, P., Chang, K.-Y. (2003). dsRBM1 and a proline-rich domain of RNA helicase A can form a composite binder to recognize a specific dsDNA. Nucleic Acids Res 31: 5741-5753 [Abstract] [Full Text]  
  • Hornemann, S., Harlin, O., Staib, C., Kisling, S., Erfle, V., Kaspers, B., Hacker, G., Sutter, G. (2003). Replication of Modified Vaccinia Virus Ankara in Primary Chicken Embryo Fibroblasts Requires Expression of the Interferon Resistance Gene E3L. J. Virol. 77: 8394-8407 [Abstract] [Full Text]  
  • Kim, Y.-G., Muralinath, M., Brandt, T., Pearcy, M., Hauns, K., Lowenhaupt, K., Jacobs, B. L., Rich, A. (2003). A role for Z-DNA binding in vaccinia virus pathogenesis. Proc. Natl. Acad. Sci. USA 100: 6974-6979 [Abstract] [Full Text]  
  • Gonzalez-Lopez, C., Martinez-Costas, J., Esteban, M., Benavente, J. (2003). Evidence that avian reovirus {sigma}A protein is an inhibitor of the double-stranded RNA-dependent protein kinase. J. Gen. Virol. 84: 1629-1639 [Abstract] [Full Text]  
  • Khoo, D., Perez, C., Mohr, I. (2002). Characterization of RNA Determinants Recognized by the Arginine- and Proline-Rich Region of Us11, a Herpes Simplex Virus Type 1-Encoded Double-Stranded RNA Binding Protein That Prevents PKR Activation. J. Virol. 76: 11971-11981 [Abstract] [Full Text]  
  • Xiang, Y., Condit, R. C., Vijaysri, S., Jacobs, B., Williams, B. R. G., Silverman, R. H. (2002). Blockade of Interferon Induction and Action by the E3L Double-Stranded RNA Binding Proteins of Vaccinia Virus. J. Virol. 76: 5251-5259 [Abstract] [Full Text]  
  • Smith, V. P., Alcami, A. (2002). Inhibition of Interferons by Ectromelia Virus. J. Virol. 76: 1124-1134 [Abstract] [Full Text]  
  • Gil, J., Rullas, J., Alcami, J., Esteban, M. (2001). MC159L protein from the poxvirus molluscum contagiosum virus inhibits NF-{kappa}B activation and apoptosis induced by PKR. J. Gen. Virol. 82: 3027-3034 [Abstract] [Full Text]  
  • Bergmann, M., Romirer, I., Sachet, M., Fleischhacker, R., Garcia-Sastre, A., Palese, P., Wolff, K., Pehamberger, H., Jakesz, R., Muster, T. (2001). A Genetically Engineered Influenza A Virus with ras-Dependent Oncolytic Properties. Cancer Res. 61: 8188-8193 [Abstract] [Full Text]  
  • He, Y., Tan, S.-L., Tareen, S. U., Vijaysri, S., Langland, J. O., Jacobs, B. L., Katze, M. G. (2001). Regulation of mRNA Translation and Cellular Signaling by Hepatitis C Virus Nonstructural Protein NS5A. J. Virol. 75: 5090-5098 [Abstract] [Full Text]  
  • Brandt, T. A., Jacobs, B. L. (2001). Both Carboxy- and Amino-Terminal Domains of the Vaccinia Virus Interferon Resistance Gene, E3L, Are Required for Pathogenesis in a Mouse Model. J. Virol. 75: 850-856 [Abstract] [Full Text]  
  • PARKER, L.M., FIERRO-MONTI, I., REICHMAN, T.W., GUNNERY, S., MATHEWS, M.B. (2001). Double-stranded RNA-binding Proteins and the Control of Protein Synthesis and Cell Growth. Cold Spring Harb Symp Quant Biol 66: 485-498 [Abstract]  
  • Poppers, J., Mulvey, M., Khoo, D., Mohr, I. (2000). Inhibition of PKR Activation by the Proline-Rich RNA Binding Domain of the Herpes Simplex Virus Type 1 Us11 Protein. J. Virol. 74: 11215-11221 [Abstract] [Full Text]  
  • Basler, C. F., Wang, X., Mühlberger, E., Volchkov, V., Paragas, J., Klenk, H.-D., García-Sastre, A., Palese, P. (2000). The Ebola virus VP35 protein functions as a type I IFN antagonist. Proc. Natl. Acad. Sci. USA 10.1073/pnas.220398297v1 [Abstract] [Full Text]  
  • Coolidge, C. J., Patton, J. G. (2000). A new double-stranded RNA-binding protein that interacts with PKR. Nucleic Acids Res 28: 1407-1417 [Abstract] [Full Text]  
  • Sood, R., Porter, A. C., Olsen, D., Cavener, D. R., Wek, R. C. (2000). A Mammalian Homologue of GCN2 Protein Kinase Important for Translational Control by Phosphorylation of Eukaryotic Initiation Factor-2{alpha}. Genetics 154: 787-801 [Abstract] [Full Text]  
  • Smith, E. J., Marie, I., Prakash, A., Garcia-Sastre, A., Levy, D. E. (2001). IRF3 and IRF7 Phosphorylation in Virus-infected Cells Does Not Require Double-stranded RNA-dependent Protein Kinase R or Ikappa B Kinase but Is Blocked by Vaccinia Virus E3L Protein. J. Biol. Chem. 276: 8951-8957 [Abstract] [Full Text]  
  • Zhang, F., Romano, P. R., Nagamura-Inoue, T., Tian, B., Dever, T. E., Mathews, M. B., Ozato, K., Hinnebusch, A. G. (2001). Binding of Double-stranded RNA to Protein Kinase PKR Is Required for Dimerization and Promotes Critical Autophosphorylation Events in the Activation Loop. J. Biol. Chem. 276: 24946-24958 [Abstract] [Full Text]  
  • Parker, L. M., Fierro-Monti, I., Mathews, M. B. (2001). Nuclear Factor 90 Is a Substrate and Regulator of the Eukaryotic Initiation Factor 2 Kinase Double-stranded RNA-activated Protein Kinase. J. Biol. Chem. 276: 32522-32530 [Abstract] [Full Text]  
  • Basler, C. F., Wang, X., Muhlberger, E., Volchkov, V., Paragas, J., Klenk, H.-D., Garcia-Sastre, A., Palese, P. (2000). The Ebola virus VP35 protein functions as a type I IFN antagonist. Proc. Natl. Acad. Sci. USA 97: 12289-12294 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»