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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 requires ER localization for ER chaperone induction.(A) Schematic diagram of the M1 mutant constructs. (B) 293T cells were transfected for 24 hours with the plasmids encoding the indicated M1 mutants and harvested for western blot analysis using antibodies against tublin (top), HA (middle) or FLAG (bottom). (C) NIH3T3 cells transfected with the indicated M1 mutant constructs were fixed and analyzed by IFA using antibodies specific for FLAG (red), conc A (green) and Hoechst (Blue). (D) 293T cells were transfected with the GRP78-fluc, PGK-renilla-luciferase, and plasmids encoding each indicated M1 mutants. Cell lysates were collected 24 hours posttransfection and were analyzed by dual-luciferase assays as described in Fig. 1A.
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f4: M1 requires ER localization for ER chaperone induction.(A) Schematic diagram of the M1 mutant constructs. (B) 293T cells were transfected for 24 hours with the plasmids encoding the indicated M1 mutants and harvested for western blot analysis using antibodies against tublin (top), HA (middle) or FLAG (bottom). (C) NIH3T3 cells transfected with the indicated M1 mutant constructs were fixed and analyzed by IFA using antibodies specific for FLAG (red), conc A (green) and Hoechst (Blue). (D) 293T cells were transfected with the GRP78-fluc, PGK-renilla-luciferase, and plasmids encoding each indicated M1 mutants. Cell lysates were collected 24 hours posttransfection and were analyzed by dual-luciferase assays as described in Fig. 1A.

Mentions: We then asked whether the ER localization is required for M1 to induce chaperone genes. To test this hypothesis, we generated an M1 mutant (M1ΔSP) by removing the 18-aa signal peptide from the protein (Fig. 4A). M1 without the peptide lost its ER-specific distribution (Fig. 4C). Importantly, expression of M1ΔSP did not activate luciferase expression from the GRP78 promoter, indicating that the ER localization of M1 is required for its function (Fig. 4D). These results also rule out the possibility that M1 acts as a transcriptional activator of the chaperone genes, because even though M1ΔSP displayed an increased nuclear localization, it did not activate the chaperone gene.


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 requires ER localization for ER chaperone induction.(A) Schematic diagram of the M1 mutant constructs. (B) 293T cells were transfected for 24 hours with the plasmids encoding the indicated M1 mutants and harvested for western blot analysis using antibodies against tublin (top), HA (middle) or FLAG (bottom). (C) NIH3T3 cells transfected with the indicated M1 mutant constructs were fixed and analyzed by IFA using antibodies specific for FLAG (red), conc A (green) and Hoechst (Blue). (D) 293T cells were transfected with the GRP78-fluc, PGK-renilla-luciferase, and plasmids encoding each indicated M1 mutants. Cell lysates were collected 24 hours posttransfection and were analyzed by dual-luciferase assays as described in Fig. 1A.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: M1 requires ER localization for ER chaperone induction.(A) Schematic diagram of the M1 mutant constructs. (B) 293T cells were transfected for 24 hours with the plasmids encoding the indicated M1 mutants and harvested for western blot analysis using antibodies against tublin (top), HA (middle) or FLAG (bottom). (C) NIH3T3 cells transfected with the indicated M1 mutant constructs were fixed and analyzed by IFA using antibodies specific for FLAG (red), conc A (green) and Hoechst (Blue). (D) 293T cells were transfected with the GRP78-fluc, PGK-renilla-luciferase, and plasmids encoding each indicated M1 mutants. Cell lysates were collected 24 hours posttransfection and were analyzed by dual-luciferase assays as described in Fig. 1A.
Mentions: We then asked whether the ER localization is required for M1 to induce chaperone genes. To test this hypothesis, we generated an M1 mutant (M1ΔSP) by removing the 18-aa signal peptide from the protein (Fig. 4A). M1 without the peptide lost its ER-specific distribution (Fig. 4C). Importantly, expression of M1ΔSP did not activate luciferase expression from the GRP78 promoter, indicating that the ER localization of M1 is required for its function (Fig. 4D). These results also rule out the possibility that M1 acts as a transcriptional activator of the chaperone genes, because even though M1ΔSP displayed an increased nuclear localization, it did not activate the chaperone gene.

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