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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 activates the IRE1 and ATF6 pathways of UPR.(A) Left panel: analysis scheme for the splicing of XBP1 mRNA: the approximate location of the 26-nt intron, the PstI digestion site, and PCR amplification primers are shown. Right panel: the reverse transcripts of total XBP1 mRNA were analyzed by PstI digestion. Resulting DNA products were separated on 2% agrose gel. Transcripts of β-actin were included as loading controls. (B) The total XBP1 mRNA level was determined by quantitative RT-PCR. (C) Reporter assay was performed using the XBP1u-fluc (XBP-1 splicing reporter) and were analyzed as described in Fig. 1A. (D) Reporter assay was similarly performed using the 5XATF6-fluc plasmid. (E) 293T cells were transfected with plasmids encoding M1 or the control vector for 24 hours, treated with 20 nM Thapsigargin (TG) (lane 1) or DMSO (lane 2, 3, 4) for 30 minutes, and harvested for western blot analysis using antibodies specific for phosphorylated-eIF2a, total eIF2a and β-actin (loading control) as indicated.
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f5: M1 activates the IRE1 and ATF6 pathways of UPR.(A) Left panel: analysis scheme for the splicing of XBP1 mRNA: the approximate location of the 26-nt intron, the PstI digestion site, and PCR amplification primers are shown. Right panel: the reverse transcripts of total XBP1 mRNA were analyzed by PstI digestion. Resulting DNA products were separated on 2% agrose gel. Transcripts of β-actin were included as loading controls. (B) The total XBP1 mRNA level was determined by quantitative RT-PCR. (C) Reporter assay was performed using the XBP1u-fluc (XBP-1 splicing reporter) and were analyzed as described in Fig. 1A. (D) Reporter assay was similarly performed using the 5XATF6-fluc plasmid. (E) 293T cells were transfected with plasmids encoding M1 or the control vector for 24 hours, treated with 20 nM Thapsigargin (TG) (lane 1) or DMSO (lane 2, 3, 4) for 30 minutes, and harvested for western blot analysis using antibodies specific for phosphorylated-eIF2a, total eIF2a and β-actin (loading control) as indicated.

Mentions: To test whether M1 affects the IRE1 pathway, we probed for the unconventional splicing of XBP1 mRNA using RT-PCR and reporter assay. XBP1 cDNA was amplified using primers flanking the splicing sites and the PCR products were made subject to PstI digestion. PstI cuts a site within the 26-nt intron in the unspliced XBP1 (XBP1u) but leaves the spliced XBP1 (XBP1s) intact. In cells expressing M1, an elevated level of XBP1s was observed, manifested by an increased amount of spliced products that are resistant to PstI digestion (Fig. 5A). To quantitatively measure the splicing of XBP1, we employed a splicing-specific reporter system (pXBP1u-fluc). pXBP1u-fluc consists the coding sequence of firefly luciferase conjugated to the second ORF of XBP1u. Therefore, the luciferase is expressed only after IRE1-induced splicing removes the 26-nt intron33. Consistent with the PstI digestion results, M1 expression markedly increased the reporter activity to about 9-fold that of vector control (Fig. 5C).


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 activates the IRE1 and ATF6 pathways of UPR.(A) Left panel: analysis scheme for the splicing of XBP1 mRNA: the approximate location of the 26-nt intron, the PstI digestion site, and PCR amplification primers are shown. Right panel: the reverse transcripts of total XBP1 mRNA were analyzed by PstI digestion. Resulting DNA products were separated on 2% agrose gel. Transcripts of β-actin were included as loading controls. (B) The total XBP1 mRNA level was determined by quantitative RT-PCR. (C) Reporter assay was performed using the XBP1u-fluc (XBP-1 splicing reporter) and were analyzed as described in Fig. 1A. (D) Reporter assay was similarly performed using the 5XATF6-fluc plasmid. (E) 293T cells were transfected with plasmids encoding M1 or the control vector for 24 hours, treated with 20 nM Thapsigargin (TG) (lane 1) or DMSO (lane 2, 3, 4) for 30 minutes, and harvested for western blot analysis using antibodies specific for phosphorylated-eIF2a, total eIF2a and β-actin (loading control) as indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663489&req=5

f5: M1 activates the IRE1 and ATF6 pathways of UPR.(A) Left panel: analysis scheme for the splicing of XBP1 mRNA: the approximate location of the 26-nt intron, the PstI digestion site, and PCR amplification primers are shown. Right panel: the reverse transcripts of total XBP1 mRNA were analyzed by PstI digestion. Resulting DNA products were separated on 2% agrose gel. Transcripts of β-actin were included as loading controls. (B) The total XBP1 mRNA level was determined by quantitative RT-PCR. (C) Reporter assay was performed using the XBP1u-fluc (XBP-1 splicing reporter) and were analyzed as described in Fig. 1A. (D) Reporter assay was similarly performed using the 5XATF6-fluc plasmid. (E) 293T cells were transfected with plasmids encoding M1 or the control vector for 24 hours, treated with 20 nM Thapsigargin (TG) (lane 1) or DMSO (lane 2, 3, 4) for 30 minutes, and harvested for western blot analysis using antibodies specific for phosphorylated-eIF2a, total eIF2a and β-actin (loading control) as indicated.
Mentions: To test whether M1 affects the IRE1 pathway, we probed for the unconventional splicing of XBP1 mRNA using RT-PCR and reporter assay. XBP1 cDNA was amplified using primers flanking the splicing sites and the PCR products were made subject to PstI digestion. PstI cuts a site within the 26-nt intron in the unspliced XBP1 (XBP1u) but leaves the spliced XBP1 (XBP1s) intact. In cells expressing M1, an elevated level of XBP1s was observed, manifested by an increased amount of spliced products that are resistant to PstI digestion (Fig. 5A). To quantitatively measure the splicing of XBP1, we employed a splicing-specific reporter system (pXBP1u-fluc). pXBP1u-fluc consists the coding sequence of firefly luciferase conjugated to the second ORF of XBP1u. Therefore, the luciferase is expressed only after IRE1-induced splicing removes the 26-nt intron33. Consistent with the PstI digestion results, M1 expression markedly increased the reporter activity to about 9-fold that of vector control (Fig. 5C).

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