The Role of the PKR-Inhibitory Genes, E3L and K3L, in Determining Vaccinia Virus Host Range

Langland, Jeffrey; Jacobs, Bertram L.

doi:10.1006/viro.2002.1479

Cited by 169 publications

(193 citation statements)

References 26 publications

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“…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). Regardless, in clear contrast to VV⌬E3L-infected cells extracts, VVeq904 extracts contained no detectable p-PKR in the cytoplasmic or nuclear fractions.…”

Section: Resultsmentioning

confidence: 95%

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: 95%

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

Hakki

Marshall²,

Niro³

et al. 2006

J Virol

104

100

View full text Add to dashboard Cite

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“…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). 15,16 Intriguingly, unlike MVA, MVA-DE3L caused apoptosis in infected CEF cells.…”

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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“…Vaccinia virus encodes two gene products, E3L and K3L, both of which confer viral resistance to interferon. [73][74][75] The E3L protein, synthesized early during viral infection, contains an amino-terminal Z-DNA-binding domain and a carboxyl-terminal domain with a typical dsRNA-binding motif. 74 The carboxyl-terminus of E3L sequesters dsRNA and prevents the activation of PKR and phosphorylation of eIF-2a.…”

Section: Virus Inhibition Of the Interferon Response Mediated By Pkrmentioning

confidence: 99%

Viruses, endoplasmic reticulum stress, and interferon responses

2006

Cell Death Differ

373

316

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Viral infection induces endoplasmic reticulum (ER) stress and interferon responses. While viral double-stranded RNA intermediates trigger interferon responses, viral polypeptides synthesized during infection stimulate ER stress. Among the interferon-regulated gene products, the double-stranded RNA-dependent protein kinase (PKR) plays a key role in limiting viral replication. Thus, to establish productive infection, viruses have evolved mechanisms to overcome the deleterious effects of PKR. It has become clear that ER stress causes translational attenuation and transcriptional upregulation of genes encoding proteins that facilitate folding or degradation of proteins. Notably, prolonged ER stress triggers apoptosis. Therefore, viruses are confronted with the consequences of ER stress. Emerging evidence suggests that viruses not only interfere with the interferon system involving PKR but also manipulate the programs emanating from the ER in a complex way, which may facilitate viral replication or pathogenesis. This review highlights recent progress in these areas.

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The Role of the PKR-Inhibitory Genes, E3L and K3L, in Determining Vaccinia Virus Host Range

Cited by 169 publications

References 26 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

Viruses, endoplasmic reticulum stress, and interferon responses

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