Mechanism of Interaction of the Double-Stranded RNA (dsRNA) Binding Domain of Protein Kinase R with Short dsRNA Sequences†

Biochemistry ◽  
2007 ◽  
Vol 46 (1) ◽  
pp. 55-65 ◽  
Author(s):  
Jason W. Ucci ◽  
Yumiko Kobayashi ◽  
Gregory Choi ◽  
Andrei T. Alexandrescu ◽  
James L. Cole
Keyword(s):  
Protein Kinase ◽  
Binding Domain ◽  
Protein Kinase R ◽  
Double Stranded Rna ◽  
Dsrna Binding ◽  
Mechanism Of Interaction

scholarly journals Mutational Analysis of the Double-stranded RNA (dsRNA) Binding Domain of the dsRNA-activated Protein Kinase, PKR

1995 ◽  
Vol 270 (6) ◽  
pp. 2601-2606 ◽  
Author(s):  
Nigel A. J. McMillan ◽  
Bruce W. Carpick ◽  
Britton Hollis ◽  
W. Mark Toone ◽  
Maryam Zamanian-Daryoush ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mutational Analysis ◽  
Binding Domain ◽  
Double Stranded Rna ◽  
Dsrna Binding

scholarly journals Binding and Relocalization of Protein Kinase R by Murine Cytomegalovirus

2008 ◽  
Vol 83 (4) ◽  
pp. 1790-1799 ◽  
Author(s):  
Stephanie J. Child ◽  
Adam P. Geballe
Keyword(s):  
Protein Kinase ◽  
Initiation Factor ◽  
Murine Cytomegalovirus ◽  
Protein Kinase R ◽  
Mcmv Infection ◽  
Viral Dsrna ◽  
Double Stranded Rna ◽  
Dsrna Binding ◽  
Single Protein ◽  
Cellular Compartments

ABSTRACT Many viruses have evolved mechanisms to evade the repression of translation mediated by protein kinase R (PKR). In the case of murine cytomegalovirus (MCMV), the protein products of two essential genes, m142 and m143, bind to double-stranded RNA (dsRNA) and block phosphorylation of PKR and eukaryotic initiation factor 2α. A distinctive feature of MCMV is that two proteins are required to block PKR activation whereas other viral dsRNA-binding proteins that prevent PKR activation contain all the necessary functions in a single protein. In order to better understand the mechanism by which MCMV evades the PKR response, we investigated the associations of pm142 and pm143 with each other and with PKR. Both pm142 and pm143 interact with PKR in infected and transfected cells. However, the ∼200-kDa pm142-pm143 complex that forms in these cells does not contain substantial amounts of PKR, suggesting that the interactions between pm142-pm143 and PKR are unstable or transient. The stable, soluble pm142-pm143 complex appears to be a heterotetramer consisting of two molecules of pm142 associated with each other, and each one binds to and stabilizes a monomer of pm143. MCMV infection also causes relocalization of PKR into the nucleus and to an insoluble cytoplasmic compartment. These results suggest a model in which the pm142-pm143 multimer interacts with PKR and causes its sequestration in cellular compartments where it is unable to shut off translation and repress viral replication.

scholarly journals Double-Stranded RNA Binding of Influenza B Virus Nonstructural NS1 Protein Inhibits Protein Kinase R but Is Not Essential To Antagonize Production of Alpha/Beta Interferon

2006 ◽  
Vol 80 (23) ◽  
pp. 11667-11677 ◽  
Author(s):  
Bianca Dauber ◽  
Jana Schneider ◽  
Thorsten Wolff
Keyword(s):  
Protein Kinase ◽  
Influenza B Virus ◽  
Influenza B ◽  
Ns1 Protein ◽  
Protein Kinase R ◽  
Double Stranded Rna ◽  
Antiviral Responses ◽  
Dsrna Binding ◽  
B Virus ◽  
Alpha Beta

ABSTRACT Expression of alpha/beta interferon (IFN-α/β) in virus-infected vertebrate cells is a key event in the establishment of a sustained antiviral response, which is triggered by double-stranded RNA (dsRNA) produced during viral replication. These antiviral cytokines initiate the expression of cellular proteins with activities that limit the replication and spread of the invading viruses. Within this response, the dsRNA-dependent protein kinase R (PKR) that is expressed at constitutive levels and upregulated by IFN-α/β acts as an important antiviral effector that can block the cellular translational machinery. We previously demonstrated that efficient replication of influenza B virus depends on the viral dsRNA-binding NS1 protein that inhibits the transcriptional activation of IFN-α/β genes. Here we tested the postulate that the viral NS1 protein counteracts antiviral responses through sequestering intracellular dsRNA by analyzing a collection of recombinant influenza B viruses. As expected, viruses expressing dsRNA-binding-defective NS1 proteins were strongly attenuated for replication in IFN-competent hosts. Interestingly, these virus mutants failed to prevent activation of PKR but could effectively limit IFN induction. Conversely, a mutant virus expressing the N-terminal dsRNA-binding domain of NS1 prevented PKR activation, but not IFN induction, suggesting an important role for the NS1 C-terminal part in silencing the activation route of IFN-α/β genes. Thus, our findings indicate an unexpected mechanistic dichotomy of the influenza B virus NS1 protein in the suppression of antiviral responses, which involves at least one activity that is largely separable from dsRNA binding.

scholarly journals Ebola Virus VP35 Antagonizes PKR Activity through Its C-Terminal Interferon Inhibitory Domain

2009 ◽  
Vol 83 (17) ◽  
pp. 8993-8997 ◽  
Author(s):  
Michael Schümann ◽  
Thorsten Gantke ◽  
Elke Mühlberger
Keyword(s):  
Amino Acids ◽  
Ebola Virus ◽  
Regulatory Factor ◽  
Protein Kinase R ◽  
Double Stranded Rna ◽  
Basic Amino Acids ◽  
Dsrna Binding ◽  
Inhibitory Domain ◽  
Ebola Virus Infection ◽  
Terminal Cluster

ABSTRACT Ebola virus VP35 contains a C-terminal cluster of basic amino acids required for double-stranded RNA (dsRNA) binding and inhibition of interferon regulatory factor 3 (IRF3). VP35 also blocks protein kinase R (PKR) activation; however, the responsible domain has remained undefined. Here we show that the IRF inhibitory domain of VP35 mediates the inhibition of PKR and enhances the synthesis of coexpressed proteins. In contrast to dsRNA binding and IRF inhibition, alanine substitutions of at least two basic amino acids are required to abrogate PKR inhibition and enhanced protein expression. Moreover, we show that PKR activation is not only blocked but reversed by Ebola virus infection.

scholarly journals Two functionally distinct RNA-binding motifs in the regulatory domain of the protein kinase DAI.

1995 ◽  
Vol 15 (1) ◽  
pp. 358-364 ◽  
Author(s):  
S R Green ◽  
L Manche ◽  
M B Mathews
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Alpha Helix ◽  
Binding Domain ◽  
Single Amino Acid ◽  
Double Stranded Rna ◽  
C Terminus ◽  
Rna Binding Domain

The RNA-binding domain of the protein kinase DAI, the double-stranded RNA inhibitor of translation, contains two repeats of a motif that is also found in a number of other RNA-binding proteins. This motif consists of 67 amino acid residues and is predicted to contain a positively charged alpha helix at its C terminus. We have analyzed the effects of equivalent single amino acid changes in three conserved residues distributed over each copy of the motif. Mutants in the C-terminal portion of either repeat were severely defective, indicating that both copies of the motif are essential for RNA binding. Changes in the N-terminal and central parts of the motif were more debilitating if they were made in the first motif than in the second, suggesting that the first motif is the more important for RNA binding and that the second motif is structurally more flexible. When the second motif was replaced by a duplicate of the first motif, the ectopic copy retained its greater sensitivity to mutation, implying that the two motifs have distinct functions with respect to the process of RNA binding. Furthermore, the mutations have the same effect on the binding of double-stranded RNA and VA RNA, consistent with the existence of a single RNA-binding domain for both activating and inhibitory RNAs.

scholarly journals ADAR1 Interacts with NF90 through Double-Stranded RNA and Regulates NF90-Mediated Gene Expression Independently of RNA Editing

2005 ◽  
Vol 25 (16) ◽  
pp. 6956-6963 ◽  
Author(s):  
Yongzhan Nie ◽  
Li Ding ◽  
Peter N. Kao ◽  
Robert Braun ◽  
Jing-Hua Yang
Keyword(s):  
Gene Expression ◽  
Rna Editing ◽  
Nuclear Export ◽  
Fetal Liver ◽  
Regulation Of Gene Expression ◽  
Nuclear Export Signal ◽  
Binding Domain ◽  
Double Stranded Rna ◽  
Recognition Element ◽  
Dsrna Binding

ABSTRACT The RNA-editing enzyme ADAR1 modifies adenosines by deamination and produces A-to-I mutations in mRNA. ADAR1 was recently demonstrated to function in host defense and in embryonic erythropoiesis during fetal liver development. The mechanisms for these phenotypic effects are not yet known. Here we report a novel function of ADAR1 in the regulation of gene expression by interacting with the nuclear factor 90 (NF90) proteins, known regulators that bind the antigen response recognition element (ARRE-2) and have been demonstrated to stimulate transcription and translation. ADAR1 upregulates NF90-mediated gene expression by interacting with the NF90 proteins, including NF110, NF90, and NF45. A knockdown of NF90 with small interfering RNA suppresses this function of ADAR1. Coimmunoprecipitation and double-stranded RNA (dsRNA) digestion demonstrate that ADAR1 is associated with NF110, NF90, and NF45 through the bridge of cellular dsRNA. Studies with ADAR1 deletions demonstrate that the dsRNA binding domain and a region covering the Z-DNA binding domain and the nuclear export signal comprise the complete function of ADAR1 in upregulating NF90-mediated gene expression. These data suggest that ADAR1 has the potential both to change information content through editing of mRNA and to regulate gene expression through interacting with the NF90 family proteins.

scholarly journals Mechanism of Protein Kinase R Inhibition by Human Cytomegalovirus pTRS1

2016 ◽  
Vol 91 (5) ◽  
Author(s):  
Heather A. Vincent ◽  
Benjamin Ziehr ◽  
Nathaniel J. Moorman
Keyword(s):  
Protein Kinase ◽  
Binding Site ◽  
Human Cytomegalovirus ◽  
Kinase Activity ◽  
Critical Role ◽  
Binding Domain ◽  
Single Amino Acid ◽  
Protein Kinase R ◽  
Hcmv Disease ◽  
Pkr Kinase

ABSTRACT Double-stranded RNAs (dsRNA) produced during human cytomegalovirus (HCMV) infection activate the antiviral kinase protein kinase R (PKR), which potently inhibits virus replication. The HCMV pTRS1 and pIRS1 proteins antagonize PKR to promote HCMV protein synthesis and replication; however, the mechanism by which pTRS1 inhibits PKR is unclear. PKR activation occurs in a three-step cascade. First, binding to dsRNA triggers PKR homodimerizaton. PKR dimers then autophosphorylate, leading to a conformational shift that exposes the binding site for the PKR substrate eIF2α. Consistent with previous in vitro studies, we found that pTRS1 bound and inhibited PKR. pTRS1 binding to PKR was not mediated by an RNA intermediate, and mutations in the pTRS1 RNA binding domain did not affect PKR binding or inhibition. Rather, mutations that disrupted the pTRS1 interaction with PKR ablated the ability of pTRS1 to antagonize PKR activation by dsRNA. pTRS1 did not block PKR dimerization and could bind and inhibit a constitutively dimerized PKR kinase domain. In addition, pTRS1 binding to PKR inhibited PKR kinase activity. Single amino acid point mutations in the conserved eIF2α binding domain of PKR disrupted pTRS1 binding and rendered PKR resistant to inhibition by pTRS1. Consistent with a critical role for the conserved eIF2α contact site in PKR binding, pTRS1 bound an additional eIF2α kinase, heme-regulated inhibitor (HRI), and inhibited eIF2α phosphorylation in response to an HRI agonist. Together our data suggest that pTRS1 inhibits PKR by binding to conserved amino acids in the PKR eIF2α binding site and blocking PKR kinase activity. IMPORTANCE The antiviral kinase PKR plays a critical role in controlling HCMV replication. This study furthered our understanding of how HCMV evades inhibition by PKR and identified new strategies for how PKR activity might be restored during infection to limit HCMV disease.

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