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M1 of Murine Gamma-Herpesvirus 68 Induces Endoplasmic Reticulum Chaperone Production.

Feng J, Gong D, Fu X, Wu TT, Wang J, Chang J, Zhou J, Lu G, Wang Y, Sun R - Sci Rep (2015)

Bottom Line: We found that M1 protein selectively induces the chaperon-producing pathways (IRE1, ATF6) while, interestingly, sparing the translation-blocking arm (PERK).We identified, for the first time, a viral factor capable of selectively intervening the initiation of ER stress signaling to induce chaperon production.This finding provides a unique opportunity of using viral protein as a tool to define the activation mechanisms of individual UPR pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Medical Pharmacology, University of California, Los Angeles, California 90095.

ABSTRACT
Viruses rely on host chaperone network to support their infection. In particular, the endoplasmic reticulum (ER) resident chaperones play key roles in synthesizing and processing viral proteins. Influx of a large amount of foreign proteins exhausts the folding capacity in ER and triggers the unfolded protein response (UPR). A fully-executed UPR comprises signaling pathways that induce ER folding chaperones, increase protein degradation, block new protein synthesis and may eventually activate apoptosis, presenting both opportunities and threats to the virus. Here, we define a role of the MHV-68M1 gene in differential modulation of UPR pathways to enhance ER chaperone production. Ectopic expression of M1 markedly induces ER chaperone genes and expansion of ER. The M1 protein accumulates in ER during infection and this localization is indispensable for its function, suggesting M1 acts from the ER. We found that M1 protein selectively induces the chaperon-producing pathways (IRE1, ATF6) while, interestingly, sparing the translation-blocking arm (PERK). We identified, for the first time, a viral factor capable of selectively intervening the initiation of ER stress signaling to induce chaperon production. This finding provides a unique opportunity of using viral protein as a tool to define the activation mechanisms of individual UPR pathways.

No MeSH data available.


Related in: MedlinePlus

M1 induces expression of ER chaperone genes.(A) 293T cells were transfected with the GRP78-fluc or GRP78mut-fluc reporter plamids, the PGK-renilla-luciferase as an internal control, and increasing amounts of plasmids encoding M1 or M3. The cell lysates were prepared 24 hours posttransfection for dual-luciferase assay. The ratio of firefly luciferase activity to renilla luciferase activity was calculated based on the value of the vector control (set as 1). This assay and all following assays were performed in triplicates. Error bars show standard deviation. (B) Cells were transiently transfected with the M1 or M3 expression plasmids for 24 hours and were harvested for RNA extraction. Quantitative RT-PCR was performed using a primer set specific for GRP78 (Table 1). The levels of GRP78 mRNA were normalized to that of GAPDH mRNA. The fold changes shown were calculated relative to the values obtained in vector-transfected cells (set as 1). (C) Cells were transfected as described in Fig. 1B and were harvested for western blot analysis using antibodies specific for GRP78 and β-actin (loading control) as indicated. M1 and M3 genes were tagged with Human influenza hemagglutinin (HA) and the expressions were shown using anti-HA antibodies. The lower panel shows the intensity of bands that was quantified from western blot analysis by measuring the peak height of the bands using ImageJ software. The intensity of bands in top and middle panel was compared to the intensity of vector control (which was defined as 100%). The relative intensity was calculated by normalizing the intensity of upper panel bands (GRP78) to the middle panel bands (β-actin). (D) The promoter activities of GRP94 and ERdj4 were determined by reporter assay as described in Fig. 1A using the GRP78-fluc and ERdj4-fluc reporter plasmids, and mRNA levels determined quantitative RT-PCR analysis as in Fig. 1B using specific primer sets for indicated genes (Table 1). *P < 0.05; **P < 0.01; ***P < 0.001 by Student’s t test.
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f1: M1 induces expression of ER chaperone genes.(A) 293T cells were transfected with the GRP78-fluc or GRP78mut-fluc reporter plamids, the PGK-renilla-luciferase as an internal control, and increasing amounts of plasmids encoding M1 or M3. The cell lysates were prepared 24 hours posttransfection for dual-luciferase assay. The ratio of firefly luciferase activity to renilla luciferase activity was calculated based on the value of the vector control (set as 1). This assay and all following assays were performed in triplicates. Error bars show standard deviation. (B) Cells were transiently transfected with the M1 or M3 expression plasmids for 24 hours and were harvested for RNA extraction. Quantitative RT-PCR was performed using a primer set specific for GRP78 (Table 1). The levels of GRP78 mRNA were normalized to that of GAPDH mRNA. The fold changes shown were calculated relative to the values obtained in vector-transfected cells (set as 1). (C) Cells were transfected as described in Fig. 1B and were harvested for western blot analysis using antibodies specific for GRP78 and β-actin (loading control) as indicated. M1 and M3 genes were tagged with Human influenza hemagglutinin (HA) and the expressions were shown using anti-HA antibodies. The lower panel shows the intensity of bands that was quantified from western blot analysis by measuring the peak height of the bands using ImageJ software. The intensity of bands in top and middle panel was compared to the intensity of vector control (which was defined as 100%). The relative intensity was calculated by normalizing the intensity of upper panel bands (GRP78) to the middle panel bands (β-actin). (D) The promoter activities of GRP94 and ERdj4 were determined by reporter assay as described in Fig. 1A using the GRP78-fluc and ERdj4-fluc reporter plasmids, and mRNA levels determined quantitative RT-PCR analysis as in Fig. 1B using specific primer sets for indicated genes (Table 1). *P < 0.05; **P < 0.01; ***P < 0.001 by Student’s t test.

Mentions: In order to systematically identify viral components of MHV-68 that modulate the expression of ER chaperone genes, we conducted a genomic viral ORF screen using a reporter system based on GRP78 expression. GRP78 is the most abundant ER resident chaperone and is highly stress-inducible29. The reporter construct (GRP78-fluc) used in the study contains the promoter region of GRP78 driving the coding sequence of firefly luciferase gene3031. GRP78-fluc was co-transfected into the 293T cells with PGK_renilla-luciferase (an internal control plasmid in which the renilla luciferase expression is driven by the constitutively active PGK promoter), and either individual ORFs of MHV-68 or a vector control. The reporter activity was measured by dual-luciferase assay 24 hours post transfection. From the screen, a strong induction (>6-fold) on the reporter was consistently noted with multiple clones of M1 (Fig. 1A and data not shown); in contrast, transfection with the sequence-related M3 showed basal-level activity comparable to that of vector control. The observed changes in reporter activity were specific for the stress response elements within the promoter region of GRP78, because M1 had no effect on the mutant reporter plasmid (GRP78mut-fluc) in which the response elements were eliminated30. In line with the reporter assays, the endogenous mRNA and protein levels of GRP78 were also upregulated in a dose-dependent manner with M1 expression as shown, respectively, by real time RT-PCR (Fig. 1B) and western blot analyses (Fig. 1C).


M1 of Murine Gamma-Herpesvirus 68 Induces Endoplasmic Reticulum Chaperone Production.

Feng J, Gong D, Fu X, Wu TT, Wang J, Chang J, Zhou J, Lu G, Wang Y, Sun R - Sci Rep (2015)

M1 induces expression of ER chaperone genes.(A) 293T cells were transfected with the GRP78-fluc or GRP78mut-fluc reporter plamids, the PGK-renilla-luciferase as an internal control, and increasing amounts of plasmids encoding M1 or M3. The cell lysates were prepared 24 hours posttransfection for dual-luciferase assay. The ratio of firefly luciferase activity to renilla luciferase activity was calculated based on the value of the vector control (set as 1). This assay and all following assays were performed in triplicates. Error bars show standard deviation. (B) Cells were transiently transfected with the M1 or M3 expression plasmids for 24 hours and were harvested for RNA extraction. Quantitative RT-PCR was performed using a primer set specific for GRP78 (Table 1). The levels of GRP78 mRNA were normalized to that of GAPDH mRNA. The fold changes shown were calculated relative to the values obtained in vector-transfected cells (set as 1). (C) Cells were transfected as described in Fig. 1B and were harvested for western blot analysis using antibodies specific for GRP78 and β-actin (loading control) as indicated. M1 and M3 genes were tagged with Human influenza hemagglutinin (HA) and the expressions were shown using anti-HA antibodies. The lower panel shows the intensity of bands that was quantified from western blot analysis by measuring the peak height of the bands using ImageJ software. The intensity of bands in top and middle panel was compared to the intensity of vector control (which was defined as 100%). The relative intensity was calculated by normalizing the intensity of upper panel bands (GRP78) to the middle panel bands (β-actin). (D) The promoter activities of GRP94 and ERdj4 were determined by reporter assay as described in Fig. 1A using the GRP78-fluc and ERdj4-fluc reporter plasmids, and mRNA levels determined quantitative RT-PCR analysis as in Fig. 1B using specific primer sets for indicated genes (Table 1). *P < 0.05; **P < 0.01; ***P < 0.001 by Student’s t test.
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f1: M1 induces expression of ER chaperone genes.(A) 293T cells were transfected with the GRP78-fluc or GRP78mut-fluc reporter plamids, the PGK-renilla-luciferase as an internal control, and increasing amounts of plasmids encoding M1 or M3. The cell lysates were prepared 24 hours posttransfection for dual-luciferase assay. The ratio of firefly luciferase activity to renilla luciferase activity was calculated based on the value of the vector control (set as 1). This assay and all following assays were performed in triplicates. Error bars show standard deviation. (B) Cells were transiently transfected with the M1 or M3 expression plasmids for 24 hours and were harvested for RNA extraction. Quantitative RT-PCR was performed using a primer set specific for GRP78 (Table 1). The levels of GRP78 mRNA were normalized to that of GAPDH mRNA. The fold changes shown were calculated relative to the values obtained in vector-transfected cells (set as 1). (C) Cells were transfected as described in Fig. 1B and were harvested for western blot analysis using antibodies specific for GRP78 and β-actin (loading control) as indicated. M1 and M3 genes were tagged with Human influenza hemagglutinin (HA) and the expressions were shown using anti-HA antibodies. The lower panel shows the intensity of bands that was quantified from western blot analysis by measuring the peak height of the bands using ImageJ software. The intensity of bands in top and middle panel was compared to the intensity of vector control (which was defined as 100%). The relative intensity was calculated by normalizing the intensity of upper panel bands (GRP78) to the middle panel bands (β-actin). (D) The promoter activities of GRP94 and ERdj4 were determined by reporter assay as described in Fig. 1A using the GRP78-fluc and ERdj4-fluc reporter plasmids, and mRNA levels determined quantitative RT-PCR analysis as in Fig. 1B using specific primer sets for indicated genes (Table 1). *P < 0.05; **P < 0.01; ***P < 0.001 by Student’s t test.
Mentions: In order to systematically identify viral components of MHV-68 that modulate the expression of ER chaperone genes, we conducted a genomic viral ORF screen using a reporter system based on GRP78 expression. GRP78 is the most abundant ER resident chaperone and is highly stress-inducible29. The reporter construct (GRP78-fluc) used in the study contains the promoter region of GRP78 driving the coding sequence of firefly luciferase gene3031. GRP78-fluc was co-transfected into the 293T cells with PGK_renilla-luciferase (an internal control plasmid in which the renilla luciferase expression is driven by the constitutively active PGK promoter), and either individual ORFs of MHV-68 or a vector control. The reporter activity was measured by dual-luciferase assay 24 hours post transfection. From the screen, a strong induction (>6-fold) on the reporter was consistently noted with multiple clones of M1 (Fig. 1A and data not shown); in contrast, transfection with the sequence-related M3 showed basal-level activity comparable to that of vector control. The observed changes in reporter activity were specific for the stress response elements within the promoter region of GRP78, because M1 had no effect on the mutant reporter plasmid (GRP78mut-fluc) in which the response elements were eliminated30. In line with the reporter assays, the endogenous mRNA and protein levels of GRP78 were also upregulated in a dose-dependent manner with M1 expression as shown, respectively, by real time RT-PCR (Fig. 1B) and western blot analyses (Fig. 1C).

Bottom Line: We found that M1 protein selectively induces the chaperon-producing pathways (IRE1, ATF6) while, interestingly, sparing the translation-blocking arm (PERK).We identified, for the first time, a viral factor capable of selectively intervening the initiation of ER stress signaling to induce chaperon production.This finding provides a unique opportunity of using viral protein as a tool to define the activation mechanisms of individual UPR pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Medical Pharmacology, University of California, Los Angeles, California 90095.

ABSTRACT
Viruses rely on host chaperone network to support their infection. In particular, the endoplasmic reticulum (ER) resident chaperones play key roles in synthesizing and processing viral proteins. Influx of a large amount of foreign proteins exhausts the folding capacity in ER and triggers the unfolded protein response (UPR). A fully-executed UPR comprises signaling pathways that induce ER folding chaperones, increase protein degradation, block new protein synthesis and may eventually activate apoptosis, presenting both opportunities and threats to the virus. Here, we define a role of the MHV-68M1 gene in differential modulation of UPR pathways to enhance ER chaperone production. Ectopic expression of M1 markedly induces ER chaperone genes and expansion of ER. The M1 protein accumulates in ER during infection and this localization is indispensable for its function, suggesting M1 acts from the ER. We found that M1 protein selectively induces the chaperon-producing pathways (IRE1, ATF6) while, interestingly, sparing the translation-blocking arm (PERK). We identified, for the first time, a viral factor capable of selectively intervening the initiation of ER stress signaling to induce chaperon production. This finding provides a unique opportunity of using viral protein as a tool to define the activation mechanisms of individual UPR pathways.

No MeSH data available.


Related in: MedlinePlus