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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

Infection by M1-deficient MHV-68 leads to reduced ER chaperon production.(A) Schematic diagram showing the construction of the M1stop MHV68 by introducing two stop codons (TAG) and two digestion sites (NheI, SpeI) into the M1 gene. (B) 293T cells were transfected for 24 hours with GRP78_fluc or GRP78mut_fluc and PGK_RL plasmids, and were mock infected or infected with wild-type (WT), M1stop (M1S) or M1 revertent (M1R) MHV-68 at MOI 5 for 18 hours. Cells were lysed and analyzed by dual-luciferase assay as in Fig. 1A. The ratio was calculated based on the unifected control. (C) Reporter assays were performed using the GRP94-fluc, ERdj4-fluc and XBP1u-fluc constructs. (D) NIH3T3 cells were mock infected or infected with WT, M1S or M1R MHV-68 at MOI 10. Cells were harvested at indicated time points for RNA extraction and analyzed by Quantitative RT-PCR using primer sets specific for indicated genes. The fold change is calculated based on the uninfected cells.
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f6: Infection by M1-deficient MHV-68 leads to reduced ER chaperon production.(A) Schematic diagram showing the construction of the M1stop MHV68 by introducing two stop codons (TAG) and two digestion sites (NheI, SpeI) into the M1 gene. (B) 293T cells were transfected for 24 hours with GRP78_fluc or GRP78mut_fluc and PGK_RL plasmids, and were mock infected or infected with wild-type (WT), M1stop (M1S) or M1 revertent (M1R) MHV-68 at MOI 5 for 18 hours. Cells were lysed and analyzed by dual-luciferase assay as in Fig. 1A. The ratio was calculated based on the unifected control. (C) Reporter assays were performed using the GRP94-fluc, ERdj4-fluc and XBP1u-fluc constructs. (D) NIH3T3 cells were mock infected or infected with WT, M1S or M1R MHV-68 at MOI 10. Cells were harvested at indicated time points for RNA extraction and analyzed by Quantitative RT-PCR using primer sets specific for indicated genes. The fold change is calculated based on the uninfected cells.

Mentions: To extend the study on the importance of M1 in virus-mediate ER chaperone production, we constructed two recombinant MHV-68: An M1-stop virus (M1S) that contains two stop codons close to the N-terminal of the coding sequence (Fig. 6A) and a revertant virus of the M1S (M1R) in which the two stop codons were reverted back to wild type sequence to ensure what we observed with M1S viruses can be attributed to M1 deficiency rather than other unintentional mutations in the viral genome. Removal of the M1 expression from the MHV-68 genome had no effect on viral growth kinetics in vitro (Fig. S3) consistent with previous observations2428.


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)

Infection by M1-deficient MHV-68 leads to reduced ER chaperon production.(A) Schematic diagram showing the construction of the M1stop MHV68 by introducing two stop codons (TAG) and two digestion sites (NheI, SpeI) into the M1 gene. (B) 293T cells were transfected for 24 hours with GRP78_fluc or GRP78mut_fluc and PGK_RL plasmids, and were mock infected or infected with wild-type (WT), M1stop (M1S) or M1 revertent (M1R) MHV-68 at MOI 5 for 18 hours. Cells were lysed and analyzed by dual-luciferase assay as in Fig. 1A. The ratio was calculated based on the unifected control. (C) Reporter assays were performed using the GRP94-fluc, ERdj4-fluc and XBP1u-fluc constructs. (D) NIH3T3 cells were mock infected or infected with WT, M1S or M1R MHV-68 at MOI 10. Cells were harvested at indicated time points for RNA extraction and analyzed by Quantitative RT-PCR using primer sets specific for indicated genes. The fold change is calculated based on the uninfected cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Infection by M1-deficient MHV-68 leads to reduced ER chaperon production.(A) Schematic diagram showing the construction of the M1stop MHV68 by introducing two stop codons (TAG) and two digestion sites (NheI, SpeI) into the M1 gene. (B) 293T cells were transfected for 24 hours with GRP78_fluc or GRP78mut_fluc and PGK_RL plasmids, and were mock infected or infected with wild-type (WT), M1stop (M1S) or M1 revertent (M1R) MHV-68 at MOI 5 for 18 hours. Cells were lysed and analyzed by dual-luciferase assay as in Fig. 1A. The ratio was calculated based on the unifected control. (C) Reporter assays were performed using the GRP94-fluc, ERdj4-fluc and XBP1u-fluc constructs. (D) NIH3T3 cells were mock infected or infected with WT, M1S or M1R MHV-68 at MOI 10. Cells were harvested at indicated time points for RNA extraction and analyzed by Quantitative RT-PCR using primer sets specific for indicated genes. The fold change is calculated based on the uninfected cells.
Mentions: To extend the study on the importance of M1 in virus-mediate ER chaperone production, we constructed two recombinant MHV-68: An M1-stop virus (M1S) that contains two stop codons close to the N-terminal of the coding sequence (Fig. 6A) and a revertant virus of the M1S (M1R) in which the two stop codons were reverted back to wild type sequence to ensure what we observed with M1S viruses can be attributed to M1 deficiency rather than other unintentional mutations in the viral genome. Removal of the M1 expression from the MHV-68 genome had no effect on viral growth kinetics in vitro (Fig. S3) consistent with previous observations2428.

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