The E3L gene of vaccinia virus encodes an inhibitor of the interferon-induced, double-stranded RNA-dependent protein kinase.

Chang, Hwai-Wen; Watson, Julia C.; Jacobs, Bertram L.

doi:10.1073/pnas.89.11.4825

Cited by 428 publications

(333 citation statements)

References 31 publications

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“…Under our assay conditions, these antibodies also detected nonspecific proteins, but the p-PKR-specific band was identifiable by its appearance in cells infected with VV⌬E3L compared to mock-infected cells in both cytoplasmic and nuclear fractions (Fig. 3) (16). The amount of total PKR in the cytoplasmic extracts of VV⌬E3L-infected cells was reduced compared to mock-infected cells, possibly because of the strong shutoff of overall protein synthesis observed during VV⌬E3L infection of HeLa cells (45).…”

Section: Resultsmentioning

confidence: 80%

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

Hakki

Marshall²,

Niro³

et al. 2006

J Virol

104

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: Resultsmentioning

confidence: 80%

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

Hakki

Marshall²,

Niro³

et al. 2006

J Virol

104

100

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“…9,10 One of these regulatory proteins, E3L, can bind double-stranded (ds) RNA and inhibit the dsRNA-stimulated enzymes, protein kinase R and RNA-specific adenosine deaminase. 11,12 A vaccinia virus deficient in E3L is highly sensitive to the activity of type I interferons and restricted in growth in some cell lines such as HeLa but not in others such as chicken embryonic fibroblasts (CEF). 13,14 We have previously generated the mutant virus MVA-DE3L deficient in E3L coding sequences (open reading frame 050, transcribed leftward through nucleotides 43 269 to 42 697 in the MVA genome).…”

Section: Introductionmentioning

confidence: 99%

Modified vaccinia virus Ankara protein F1L is a novel BH3-domain-binding protein and acts together with the early viral protein E3L to block virus-associated apoptosis

Fischer¹,

Ludwig

Holzapfel³

et al. 2005

Cell Death Differ

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Infection with viruses often protects the infected cell against external stimuli to apoptosis. Here we explore the balance of apoptosis induction and inhibition for infection with the modified vaccinia virus Ankara (MVA), using two MVA mutants with experimentally introduced deletions. Deletion of the E3L-gene from MVA transformed the virus from an inhibitor to an inducer of apoptosis. Noxa-deficient mouse embryonic fibroblasts (MEF) were resistant to MVA-DE3L-induced apoptosis. When the gene encoding F1L was deleted from MVA, apoptosis resulted that required Bak or Bax. MVA-DF1L-induced apoptosis was blocked by Bcl-2. When expressed in HeLa cells, F1L blocked apoptosis induced by forced expression of the BH3-only proteins, Bim, Puma and Noxa. Finally, biosensor analysis confirmed direct binding of F1L to BH3 domains. These data describe a molecular framework of how a cell responds to MVA infection by undergoing apoptosis, and how the virus blocks apoptosis by interfering with critical steps of its signal transduction.

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“…Examples include the adenovirus-encoded VAI RNA, which forms an inhibitory complex with PKR by functioning as a competitive inhibitor of dsRNA binding (39). Both reovirus and vaccinia virus encode virus-specific dsRNA-binding proteins which function to sequester dsRNA activators of PKR (14,34). In addition, the 88-amino-acid (aa) K3L gene product of vaccinia virus, K3L, and the tat gene product of human immunodeficiency virus type 1 form a physical complex with PKR resulting in inhibition of kinase activity during viral infection (13,59,69).…”

mentioning

confidence: 99%

Interaction of the Interferon-Induced PKR Protein Kinase with Inhibitory Proteins P58^IPKand Vaccinia Virus K3L Is Mediated by Unique Domains: Implications for Kinase Regulation

Gale

Tan

Wambach

et al. 1996

Molecular and Cellular Biology

104

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Expression of the double-stranded RNA-activated protein kinase (PKR) is induced by interferons, with PKR activity playing a pivotal role in establishing the interferon-induced antiviral and antiproliferative states. PKR is directly regulated by physical association with the specific inhibitor, P58 IPK , a cellular protein of the tetratricopeptide repeat (TPR) family, and K3L, the product of the corresponding vaccinia virus gene. P58 IPK and K3L repress PKR activation and activity. To investigate the mechanism of P58 IPK -and K3L-mediated PKR inhibition, we have used a combination of in vitro and in vivo binding assays to identify the interactive regions of these proteins. The P58 IPK -interacting site of PKR was mapped to a 52-amino-acid aa segment (aa 244 to 296) spanning the ATP-binding region of the protein kinase catalytic domain. The interaction with PKR did not require the C-terminal DNA-J homology region of P58 IPK but was dependent on the presence of the eukaryotic initiation factor 2-␣ homology region, mapping to the 34 aa within the sixth P58 IPK TPR motif. Consistent with other TPR proteins, P58 IPK formed multimers in vivo: the N-terminal 166 aa were both necessary and sufficient for complex formation. A parallel in vivo analysis to map the K3L-binding region of PKR revealed that like P58 IPK , K3L interacted exclusively with the PKR protein kinase catalytic domain. In contrast, however, the K3L-binding region of PKR was localized to within aa 367 to 551, demonstrating that each inhibitor bound PKR in unique, nonoverlapping domains. These data, taken together, suggest that P58 IPK and K3L may mediate PKR inhibition by distinct mechanisms. Finally, we will propose a model of PKR inhibition in which P58 IPK or a P58 IPK complex binds PKR and interferes with nucleotide binding and autoregulation, while formation of a PKR-K3L complex interferes with active-site function and/or substrate association.

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The E3L gene of vaccinia virus encodes an inhibitor of the interferon-induced, double-stranded RNA-dependent protein kinase.

Cited by 428 publications

References 31 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

Modified vaccinia virus Ankara protein F1L is a novel BH3-domain-binding protein and acts together with the early viral protein E3L to block virus-associated apoptosis

Interaction of the Interferon-Induced PKR Protein Kinase with Inhibitory Proteins P58^IPKand Vaccinia Virus K3L Is Mediated by Unique Domains: Implications for Kinase Regulation

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The E3L gene of vaccinia virus encodes an inhibitor of the interferon-induced, double-stranded RNA-dependent protein kinase.

Cited by 428 publications

References 31 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

Modified vaccinia virus Ankara protein F1L is a novel BH3-domain-binding protein and acts together with the early viral protein E3L to block virus-associated apoptosis

Interaction of the Interferon-Induced PKR Protein Kinase with Inhibitory Proteins P58IPKand Vaccinia Virus K3L Is Mediated by Unique Domains: Implications for Kinase Regulation

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Interaction of the Interferon-Induced PKR Protein Kinase with Inhibitory Proteins P58^IPKand Vaccinia Virus K3L Is Mediated by Unique Domains: Implications for Kinase Regulation