Protein kinase PKR mutants resistant to the poxvirus pseudosubstrate K3L protein

Seo, Eun Joo; Liu, Furong; Kawagishi-Kobayashi, Makiko; Ung, Tekly L.; Cao, Chune; Dar, Arvin C.; Sicheri, Frank; Dever, Thomas E.

doi:10.1073/pnas.0805524105

Cited by 33 publications

(47 citation statements)

References 20 publications

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“…5C). Consistent with previous results, poly(I·C) induced the autophosphorylation of each PKR mutant in control cells expressing GFP, demonstrating that each PKR mutant was catalytically active (3,4,27). Though the PKR F495 mutant was activated to lower levels than wild-type PKR in control cells, pTRS1 prevented autophosphorylation of the PKR F495R mutant.…”

Section: Resultssupporting

confidence: 91%

“…In addition, the ICP34.5 protein recruits the cellular protein phosphatase 1 (PP1) to dephosphorylate eIF2␣ and ensure continued synthesis of viral proteins (25). Similarly, the murine cytomegalovirus (MCMV) m142 and m143 proteins and the vaccinia virus (VV) K3L and E3L proteins each function to limit PKR activation (26)(27)(28)(29)(30)(31)(32)(33). Other viruses, including orthomyxoviruses, flaviviruses, and retroviruses, each encode at least one PKR antagonist, highlighting the important role of PKR antagonism for successful viral replication (15,17,34).…”

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confidence: 99%

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Mechanism of Protein Kinase R Inhibition by Human Cytomegalovirus pTRS1

Vincent

Ziehr

Moorman

2017

J Virol

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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 eIF2␣. 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 eIF2␣ binding domain of PKR disrupted pTRS1 binding and rendered PKR resistant to inhibition by pTRS1. Consistent with a critical role for the conserved eIF2␣ contact site in PKR binding, pTRS1 bound an additional eIF2␣ kinase, heme-regulated inhibitor (HRI), and inhibited eIF2␣ phosphorylation in response to an HRI agonist. Together our data suggest that pTRS1 inhibits PKR by binding to conserved amino acids in the PKR eIF2␣ binding site and blocking PKR kinase activity. IMPORTANCEThe 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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Section: Resultssupporting

confidence: 91%

mentioning

confidence: 99%

Mechanism of Protein Kinase R Inhibition by Human Cytomegalovirus pTRS1

Vincent

Ziehr

Moorman

2017

J Virol

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“…Elucidating the mechanism by which PKR function is subverted by viral products has enhanced our understanding of how PKR and other eIF2α family kinases naturally function. In particular, viral mimicry appeared as a recurring theme: analysis of the pseudosubstrate inhibitor K3L from vaccinia virus revealed key molecular determinants required for PKR recognition of its natural substrate eIF2α (10)(11)(12); analysis of the RNA binding protein E3L from vaccinia virus revealed how PKR itself senses dsRNA through comparable infrastructure (13)(14)(15); and analysis of the RNA inhibitor VAI from adenovirus showed how a structured RNA decoy can competitively engage the dsRNA-binding domains of PKR in a manner that circumvents kinase domain activation (16)(17)(18).…”

Section: Significancementioning

confidence: 99%

Baculovirus protein PK2 subverts eIF2α kinase function by mimicry of its kinase domain C-lobe

Cao

Fixsen

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by eIF2α family kinases is a conserved mechanism to limit protein synthesis under specific stress conditions. The baculovirus-encoded protein PK2 inhibits eIF2α family kinases in vivo, thereby increasing viral fitness. However, the precise mechanism by which PK2 inhibits eIF2α kinase function remains an enigma. Here, we probed the mechanism by which PK2 inhibits the model eIF2α kinase human RNA-dependent protein kinase (PKR) as well as native insect eIF2α kinases. Although PK2 structurally mimics the C-lobe of a protein kinase domain and possesses the required docking infrastructure to bind eIF2α, we show that PK2 directly binds the kinase domain of PKR (PKR KD ) but not eIF2α. The PKR KD -PK2 interaction requires a 22-residue N-terminal extension preceding the globular PK2 body that we term the "eIF2α kinase C-lobe mimic" (EKCM) domain. The functional insufficiency of the N-terminal extension of PK2 implicates a role for the adjacent EKCM domain in binding and inhibiting PKR. Using a genetic screen in yeast, we isolated PK2-activating mutations that cluster to a surface of the EKCM domain that in bona fide protein kinases forms the catalytic cleft through sandwiching interactions with a kinase N-lobe. Interaction assays revealed that PK2 associates with the N-but not the C-lobe of PKR KD . We propose an inhibitory model whereby PK2 engages the N-lobe of an eIF2α kinase domain to create a nonfunctional pseudokinase domain complex, possibly through a lobe-swapping mechanism. Finally, we show that PK2 enhances baculovirus fitness in insect hosts by targeting the endogenous insect heme-regulated inhibitor (HRI)-like eIF2α kinase. viral mimicry | lobe-swap | HRI | eIF2α kinase inhibition | PKR E ukaryotic cells possess diverse mechanisms for molecular adaptation to stress conditions. Eukaryotic translation initiation factor 2α (eIF2α) kinases are evolutionarily conserved enzymes that detect specific stress signals and induce changes in translation to produce an adaptive response. The four eIF2α kinases in humans possess divergent regulatory domains that enable response to different stress signals: (i) the RNA-dependent protein kinase (PKR) senses viral infection to mediate antiviral immunity; (ii) the PKR-like endoplasmic reticulum kinase (PERK) detects unfolded proteins in the endoplasmic reticulum to regulate the unfolded protein response; (iii) the heme-regulated inhibitor kinase (HRI) senses free heme to coordinate globin synthesis in red blood cells; and (iv) the general control nonrepressible-2 kinase (GCN2) detects amino acid insufficiencies to regulate metabolite homeostasis (1). Although eIF2α kinases respond to different stress stimuli, their protein kinase domains are highly similar and allow transmission of an overlapping signal that potently inhibits cellular translation. Upon stress-induced activation, eIF2α kinases phosphorylate a common cellular substrate, eIF2α, at a conserved site corresponding to Ser51 in human eIF2α. eIF2 is a...

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“…Construction of yeast strains H2544 (MATa ura3-52 leu2-3 leu2-112 trp1-Δ63 gcn2Δ <GAL-CYC1-PKR, LEU2>) and J110 (MATa ura3-52 leu2-3 leu2-112 trp1-Δ63 gcn2Δ <LEU2>) has been described previously (Romano et al 1995;Seo et al 2008). Strain H2766 (MATa ura3-52 leu2-3 leu2-112 trp1-Δ63 gcn2Δ gcd2Δ:: hisG p[GCD2-K627T, TRP1]) was used for expression and purification of Flag-and His 6 -tagged PKR (Krishnamoorthy et al 2001).…”

Section: Yeast Strainsmentioning

confidence: 99%

“…In addition to the E3L protein, many poxviruses also encode a pseudosubstrate inhibitor of PKR (Bratke et al 2013). For example, the vaccinia virus K3L protein (Davies et al 1992;Carroll et al 1993;Kawagishi-Kobayashi et al 1997;Dar and Sicheri 2002;Seo et al 2008), variola virus C3L protein (Seo et al 2008), swinepox virus C8L protein (Kawagishi-Kobayashi et al 2000), and ranavirus vIF2α protein (Rothenburg et al 2011) resemble the N-terminal OB-fold domain of eIF2α (Dar and Sicheri 2002;Dar et al 2005). Binding of these proteins to activated PKR (Dar et al 2005) is thought to prevent access to eIF2α and thus block eIF2α phosphorylation.…”

Section: Introductionmentioning

confidence: 99%

Variola virus E3L Zα domain, but not its Z-DNA binding activity, is required for PKR inhibition

Thakur

Seo

Dever

2013

RNA

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Responding to viral infection, the interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase PKR phosphorylates translation initiation factor eIF2α to inhibit cellular and viral protein synthesis. To overcome this host defense mechanism, many poxviruses express the protein E3L, containing an N-terminal Z-DNA binding (Zα) domain and a C-terminal dsRNA-binding domain (dsRBD). While E3L is thought to inhibit PKR activation by sequestering dsRNA activators and by directly binding the kinase, the role of the Zα domain in PKR inhibition remains unclear. Here, we show that the E3L Zα domain is required to suppress the growth-inhibitory properties associated with expression of human PKR in yeast, to inhibit PKR kinase activity in vitro, and to reverse the inhibitory effects of PKR on reporter gene expression in mammalian cells treated with dsRNA. Whereas previous studies revealed that the Z-DNA binding activity of E3L is critical for viral pathogenesis, we identified point mutations in E3L that functionally uncouple Z-DNA binding and PKR inhibition. Thus, our studies reveal a molecular distinction between the nucleic acid binding and PKR inhibitory functions of the E3L Zα domain, and they support the notion that E3L contributes to viral pathogenesis by targeting PKR and other components of the cellular anti-viral defense pathway.

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Protein kinase PKR mutants resistant to the poxvirus pseudosubstrate K3L protein

Cited by 33 publications

References 20 publications

Mechanism of Protein Kinase R Inhibition by Human Cytomegalovirus pTRS1

Mechanism of Protein Kinase R Inhibition by Human Cytomegalovirus pTRS1

Baculovirus protein PK2 subverts eIF2α kinase function by mimicry of its kinase domain C-lobe

Variola virus E3L Zα domain, but not its Z-DNA binding activity, is required for PKR inhibition

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