Disruption of cellular translational control by a viral truncated eukaryotic translation initiation factor 2α kinase homolog

Dever, Thomas E.; Rajaraman, S; McLachlin, Jeanne R.; Lü, Jing-Fang; Fabian, John R.; Kimball, Scot R.; Miller, Lois K.

doi:10.1073/pnas.95.8.4164

Cited by 59 publications

(56 citation statements)

References 42 publications

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“…For example, poliovirus infection results in the degradation of PKR (6,7). Several viruses block activation of PKR by sequestering dsRNA or by interfering with PKR dimerization or enzymatic activity (26,27,32,64,65,72). The herpes simplex virus ␥34.5 gene product blocks the effects of PKR by cooperating with cellular protein phosphatase 1␣ to dephosphorylate eIF2␣-phosphate (37).…”

Section: Discussionmentioning

confidence: 99%

Binding and Nuclear Relocalization of Protein Kinase R by Human Cytomegalovirus TRS1

Hakki

Marshall²,

Niro³

et al. 2006

J Virol

103

100

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The human cytomegalovirus (HCMV) TRS1 and IRS1 genes block the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2␣) and the consequent shutoff of cellular protein synthesis that occur during infection with vaccinia virus (VV) deleted of the double-stranded RNA binding protein gene E3L (VV⌬E3L). To further define the underlying mechanism, we first evaluated the effect of pTRS1 on protein kinase R (PKR), the double-stranded RNA (dsRNA)-dependent eIF2␣ kinase. Immunoblot analyses revealed that pTRS1 expression in the context of a VV⌬E3L recombinant decreased levels of PKR in the cytoplasm and increased its levels in the nucleus of infected cells, an effect not seen with wild-type VV or a VV⌬E3L recombinant virus expressing E3L. This effect of pTRS1 was confirmed by visualizing the nuclear relocalization of PKR-EGFP expressed by transient transfection. PKR present in both the nuclear and cytoplasmic fractions was nonphosphorylated, indicating that it was unactivated when TRS1 was present. PKR also accumulated in the nucleus during HCMV infection as determined by indirect immunofluorescence and immunoblot analysis. Binding assays revealed that pTRS1 interacted with PKR in mammalian cells and in vitro. This interaction required the same carboxy-terminal region of pTRS1 that is necessary to rescue VV⌬E3L replication in HeLa cells. The carboxy terminus of pIRS1 was also required for rescue of VV⌬E3L and for mediating an interaction of pIRS1 with PKR. These results suggest that these HCMV genes directly interact with PKR and inhibit its activation by sequestering it in the nucleus, away from both its activator, cytoplasmic dsRNA, and its substrate, eIF2␣.Double-stranded RNA (dsRNA) activates the host cell response to viral infection in numerous ways, one of which involves the interferon-induced, dsRNA-dependent protein kinase R (PKR) (reviewed in reference 43). After binding to dsRNA, PKR dimerizes, autophosphorylates, and then phosphorylates the eukaryotic initiation factor 2 (eIF2) on its ␣ subunit. Phosphorylated eIF2␣ sequesters the guanine nucleotide exchange factor eIF2B, resulting in the inhibition of protein synthesis at the level of translation initiation. Since viruses depend on the cellular translational machinery, the shutoff of host cell protein synthesis inhibits viral replication and spread.Many viruses have mechanisms for inhibiting the PKR-mediated phosphorylation of eIF2␣ (56). The vaccinia virus (VV) E3L protein prevents the activation of PKR by binding to and sequestering dsRNA via a carboxy-terminal double-stranded RNA binding domain (dsRBD) (39). VV from which the E3L gene has been deleted (VV⌬E3L) exhibits a dsRNA-dependent restriction of host cell range, and this phenotype can be reversed by expression of the dsRBD from pE3L or dsRNAbinding proteins from other viruses (4, 47, 62). We previously found that the products of the human cytomegalovirus (HCMV) genes TRS1 and IRS1 restore the host cell range of VV⌬E3L, inhibit the phosphorylation of eIF2␣, and prevent the shutoff...

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Section: Discussionmentioning

confidence: 99%

Binding and Nuclear Relocalization of Protein Kinase R by Human Cytomegalovirus TRS1

Hakki

Marshall²,

Niro³

et al. 2006

J Virol

103

100

View full text Add to dashboard Cite

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“…Because subsequent structural studies (7,8,21) revealed that dimerization-dependent activation of eIF2α kinases is the exclusive function of the kinase N-lobe, whereas substrate recognition is mediated by helix αG in the kinase C-lobe, one would reasonably predict that PK2 might function by competitively sequestering eIF2α. Surprisingly, however, PK2 coimmunoprecipitated with PKR when coexpressed in yeast and also inhibited PKR KD autophosphorylation (20). Further suggesting that PKR, and not eIF2α, was the interaction target of PK2, a yeast slow-growth phenotype caused by PK2 overexpression was rescued by overexpression of a catalytically inactive PKR K296R but not by eIF2α (20).…”

Section: Significancementioning

confidence: 81%

“…When PKR was overexpressed in baculovirus-infected caterpillar moth cell lines, viral PK2 reduced eIF2α phosphorylation and stimulated virus replication (20). In addition, when PKR was expressed in yeast, PK2 alleviated PKR-mediated translational arrest and toxicity by preventing phosphorylation of yeast eIF2α.…”

Section: Significancementioning

confidence: 99%

“…In addition, when PKR was expressed in yeast, PK2 alleviated PKR-mediated translational arrest and toxicity by preventing phosphorylation of yeast eIF2α. Comparative analysis of available sequences showed that PK2 is similar to rabbit reticulocyte HRI and Saccharomyces cerevisiae GCN2 kinase C-lobes (19,20), suggesting that PK2 will mimic the function of the C-lobe of eIF2α kinases. Because subsequent structural studies (7,8,21) revealed that dimerization-dependent activation of eIF2α kinases is the exclusive function of the kinase N-lobe, whereas substrate recognition is mediated by helix αG in the kinase C-lobe, one would reasonably predict that PK2 might function by competitively sequestering eIF2α.…”

Section: Significancementioning

confidence: 99%

“…PK2, which is encoded by insect baculoviruses, is a less wellcharacterized modulator of eIF2α kinase function (19,20). When PKR was overexpressed in baculovirus-infected caterpillar moth cell lines, viral PK2 reduced eIF2α phosphorylation and stimulated virus replication (20).…”

Section: Significancementioning

confidence: 99%

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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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The Vaccinia Virus E3L Gene Product Interacts with both the Regulatory and the Substrate Binding Regions of PKR: Implications for PKR Autoregulation

et al. 1998

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The vaccinia virus E3L gene product, pE3, is a dsRNA binding protein that prevents activation of the interferon-induced, dsRNA-activated protein kinase, PKR. Activation of PKR, which results in phosphorylation of the translation initiation factor, eIF2alpha, leads to the inhibition of protein synthesis, a process involved in defense against virus infection. The E3L gene product has a conserved dsRNA binding domain (DRBD) in its carboxyl-terminal region and has been shown to function in vitro by sequestration of dsRNA. We have utilized in vitro binding assays and the yeast two-hybrid system to demonstrate direct interactions of pE3 with PKR. By these methods, we demonstrate that pE3 interacts with two distinct regions in PKR, the amino-terminal (amino acids 1-99) located in the regulatory domain and the carboxyl-terminal (amino acids 367-523) located in the catalytic domain. The amino-terminal region of PKR that interacts with pE3 contains a conserved DRBD, suggesting that PKR can form nonfunctional heterodimers with pE3, analogous to those seen with other dsRNA binding proteins. Interaction of pE3 with the amino-terminal region of PKR is enhanced by dsRNA. In contrast, dsRNA reduces the interaction of pE3 with the carboxyl-terminal region of PKR. Competition experiments demonstrate that the carboxyl-terminal region of PKR, to which pE3 binds, overlaps the region with which eIF2alpha and the pseudosubstrate pK3 interact, suggesting that pE3 may also prevent PKR activation by masking the substrate binding domain. Like pE3, the amino-terminal region of PKR also interacts with the carboxyl-terminal domain of PKR. These interactions increase our understanding of the mechanisms by which pE3 downregulates PKR. In addition, the PKR-PKR interactions observed leads us to suggest a novel autoregulatory mechanism for activation of PKR in which dsRNA binding to the DRBD(s) induces a conformational change that results in release of the amino terminal region from the substrate binding domain, allowing access to eIF2alpha and its subsequent phosphorylation.

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Disruption of cellular translational control by a viral truncated eukaryotic translation initiation factor 2α kinase homolog

Cited by 59 publications

References 42 publications

Binding and Nuclear Relocalization of Protein Kinase R by Human Cytomegalovirus TRS1

Binding and Nuclear Relocalization of Protein Kinase R by Human Cytomegalovirus TRS1

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

The Vaccinia Virus E3L Gene Product Interacts with both the Regulatory and the Substrate Binding Regions of PKR: Implications for PKR Autoregulation

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