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Virus-Cell Interactions

Human Cytomegalovirus Infection Activates and Regulates the Unfolded Protein Response

Jennifer A. Isler, Alison H. Skalet, James C. Alwine
Jennifer A. Isler
1Department of Cancer Biology, Abramson Family Cancer Research Institute
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Alison H. Skalet
2Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
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James C. Alwine
1Department of Cancer Biology, Abramson Family Cancer Research Institute
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  • For correspondence: alwine@mail.med.upenn.edu
DOI: 10.1128/JVI.79.11.6890-6899.2005
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    FIG. 1.

    Schematic illustration of the UPR. Three ER-resident transmembrane proteins are activated in response to ER stress: PERK, ATF6 and IRE-1. PERK, an eIF2α kinase, dimerizes, becomes phosphorylated and then phosphorylates eIF2α. Phospho-eIF2α: 1) inhibits global translation; and 2) stimulates translation of ATF4. ATF4 transcriptionally activates the expression of genes encoding metabolism and redox proteins. ATF6 (90kDa) is cleaved in the Golgi forming an active 50-kDa transcription factor which transcriptionally activates genes encoding chaperones. Lastly, IRE-1 dimerizes, becomes phosphorylated and then mediates the removal of an intron from Xbp-1 mRNA. This spliced form of Xbp-1 mRNA encodes a transcription factor which transcriptionally activates genes encoding proteins involved in ER-stress associated protein degradation (e.g., EDEM).

  • FIG. 2.
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    FIG. 2.

    HCMV induces the PERK pathway but limits phosphorylation of eIF2α. HCMV-infected HFFs were harvested at the indicated days postinfection (Dpi). Cell lysates were evaluated by Western analysis using antibodies specific for the phosphorylated forms of PERK and eIF2α, total eIF2α and actin (loading control). Panels to the right are UPR induction controls which demonstrate the effect of 1 h treatment with thapsigargin (+) on the indicated proteins. Arrowheads indicate the band corresponding to each protein. Note that the phosphorylated form of PERK runs slightly faster than a nonspecific protein (present prior to 3 days postinfection) and the phosphorylated form of eIF2α is represented by the upper band of a doublet.

  • FIG. 3.
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    FIG. 3.

    Translation is not attenuated by HCMV infection, despite phosphorylation of PERK and eIF2α. HCMV-infected HFFs were pulse-labeled with a 35S-labeled methionine and cysteine mix for 30 min. at the indicated times postinfection. Total cell lysates were analyzed by SDS-PAGE, and the intensity of protein profiles was compared using autoradiography. Cells treated with thapsigargin (Thap) for 1 h prior to labeling were used as a control to demonstrate the effect of definitive UPR induction on translation. 35S-labeled Met/Cys incorporation was also quantitated using trichloroacetic acid precipitation and is indicated as a percentage of mock-infected cells below each lane.

  • FIG. 4.
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    FIG. 4.

    Translation of ATF4 is upregulated in HCMV-infected cells. (A) HCMV-infected HFFs were harvested at the times indicated and evaluated by Western analysis using antibodies specific for ATF4 and actin (loading control). Arrowheads indicate the band corresponding to each protein. The UPR induction control shown is the level of ATF4 in HFFs after 12 h in the absence (-) or presence (+) of thapsigargin (Thap). (B) RNA was extracted from HCMV-infected HFFs harvested at the times indicated and analyzed by quantitative RT-PCR using primers specific for ATF4. The fold change in ATF4 mRNA levels is relative to mock-infected controls (set at 1) taken at each time point or to untreated cells in the case of thapsigargin treatment.

  • FIG. 5.
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    FIG. 5.

    HCMV does not activate ATF6, but regulates the expression of ATF6 target genes in a gene-specific manner. (A) HCMV-infected HFFs were harvested at the times indicated and evaluated by Western analysis using an antibody specific for ATF6. Thapsigargin (Thap)-treated HFFs were used as a positive control for ATF6 cleavage under conditions of definitive UPR induction. (B) HCMV-infected HFFs were harvested at the times indicated and evaluated by Western analysis using antibodies specific for BiP, GRP94 and actin (loading control). In the second panel, 5 days postinfection samples and thapsigargin-treated samples were used to compare the levels of induction under infection conditions and conditions of definitive UPR induction. Arrowheads indicate the band corresponding to each protein. (C) RNA was extracted from HCMV-infected HFFs harvested at the times indicated and analyzed by quantitative real-time RT-PCR using primers specific for Xbp-1. Thapsigargin (Thap)-treated HFFs were used to demonstrate the effect of ATF6 on Xbp-1 transcription under conditions of definitive UPR activation. The fold change in Xbp-1 mRNA levels is relative to mock-infected controls (set at 1) taken at each time point or to untreated cells in the case of thapsigargin.

  • FIG. 6.
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    FIG. 6.

    HCMV induces splicing of Xbp-1 but inhibits transcriptional activation of the Xbp-1 target gene EDEM. (A) RNA extracted from HCMV-infected HFFs harvested at the times indicated was analyzed by RT-PCR using primers capable of amplifying both the unspliced [Xbp-1(u)] and spliced form of Xbp-1 [Xbp-1(s)]. Mock-infected HFFs and thapsigargin (Thap)-treated HFFs were used as negative and positive controls for Xbp-1 splicing, respectively. (B) RNA was extracted from HCMV-infected HFFs harvested at the times indicated and was analyzed by quantitative real-time RT-PCR using primers specific for EDEM mRNA. Thapsigargin (Thap)-treated HFFs were used as a control to show transcriptional activation of EDEM under conditions of definitive UPR induction. The fold change in EDEM mRNA levels is relative to mock-infected controls (set at 1) taken at each time point, or to untreated cells in the case of thapsigargin.

  • FIG. 7.
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    FIG. 7.

    Schematic illustrating the effects of HCMV on the UPR. Our data demonstrate that HCMV activates a modified UPR in which outcomes that may be detrimental to HCMV replication are inhibited while outcomes that may be beneficial to HCMV infection are maintained.

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Human Cytomegalovirus Infection Activates and Regulates the Unfolded Protein Response
Jennifer A. Isler, Alison H. Skalet, James C. Alwine
Journal of Virology May 2005, 79 (11) 6890-6899; DOI: 10.1128/JVI.79.11.6890-6899.2005

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Human Cytomegalovirus Infection Activates and Regulates the Unfolded Protein Response
Jennifer A. Isler, Alison H. Skalet, James C. Alwine
Journal of Virology May 2005, 79 (11) 6890-6899; DOI: 10.1128/JVI.79.11.6890-6899.2005
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KEYWORDS

cytomegalovirus
Cytomegalovirus Infections
Protein Denaturation

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