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Early dengue virus protein synthesis induces extensive rearrangement of the endoplasmic reticulum independent of the UPR and SREBP-2 pathway.

Peña J, Harris E - PLoS ONE (2012)

Bottom Line: We then demonstrate that enlargement of the ER is independent of the SREBP-2 activation and upregulation of 3-hydroxy-3-methylglutaryl-Coenzyme-A reductase, the rate-limiting enzyme in the cholesterol biosynthesis pathway.Lastly, we demonstrate that viral infection induces the reabsorption of lipid droplets into the ER.This work paves the way for further study of virally-induced membrane rearrangements and formation of cubic membranes.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, California, United States of America. jpena360@yahoo.com

ABSTRACT
The rearrangement of intracellular membranes has been long reported to be a common feature in diseased cells. In this study, we used dengue virus (DENV) to study the role of the unfolded protein response (UPR) and sterol-regulatory-element-binding-protein-2 (SREBP-2) pathway in the rearrangement and expansion of the endoplasmic reticulum (ER) early after infection. Using laser scanning confocal and differential interference contrast microscopy, we demonstrate that rearrangement and expansion of the ER occurs early after DENV-2 infection. Through the use of mouse embryonic fibroblast cells deficient in XBP1 and ATF6, we show that ER rearrangement early after DENV infection is independent of the UPR. We then demonstrate that enlargement of the ER is independent of the SREBP-2 activation and upregulation of 3-hydroxy-3-methylglutaryl-Coenzyme-A reductase, the rate-limiting enzyme in the cholesterol biosynthesis pathway. We further show that this ER rearrangement is not inhibited by the treatment of DENV-infected cells with the cholesterol-inhibiting drug lovastatin. Using the transcription inhibitor actinomycin D and the translation elongation inhibitor cycloheximide, we show that de novo viral protein synthesis but not host transcription is necessary for expansion and rearrangement of the ER. Lastly, we demonstrate that viral infection induces the reabsorption of lipid droplets into the ER. Together, these results demonstrate that modulation of intracellular membrane architecture of the cell early after DENV-2 infection is driven by viral protein expression and does not require the induction of the UPR and SREBP-2 pathways. This work paves the way for further study of virally-induced membrane rearrangements and formation of cubic membranes.

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DENV-induced ER rearrangement and expansion is independent of the XBP-1 pathway.XBP1+/+ (A–D) and XBP1−/− (E–H) MEF cells were infected with DENV-2 for 12 h and stained intracellularly for cellular proteins using antibodies against (A and E) GRP78, (B and F) GRP94, (C) XBP1, and (G) ATF6 (magenta), followed by secondary antibodies conjugated to Alexa Fluor® 647. DENV proteins were detected with mouse MAbs against DENV E (green) and NS3 (red) directly conjugated to Alexa Fluor® 488 and Alexa Fluor® 594, respectively. Nuclear staining (blue) was performed using DAPI stain. Images were acquired and processed as described in Fig. 1. Total magnification 630×.
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pone-0038202-g002: DENV-induced ER rearrangement and expansion is independent of the XBP-1 pathway.XBP1+/+ (A–D) and XBP1−/− (E–H) MEF cells were infected with DENV-2 for 12 h and stained intracellularly for cellular proteins using antibodies against (A and E) GRP78, (B and F) GRP94, (C) XBP1, and (G) ATF6 (magenta), followed by secondary antibodies conjugated to Alexa Fluor® 647. DENV proteins were detected with mouse MAbs against DENV E (green) and NS3 (red) directly conjugated to Alexa Fluor® 488 and Alexa Fluor® 594, respectively. Nuclear staining (blue) was performed using DAPI stain. Images were acquired and processed as described in Fig. 1. Total magnification 630×.

Mentions: Previous studies have shown XBP1 to play a critical role in cell size expansion, organelle biogenesis, increased protein synthesis, and induction of secretory genes [11], [13], [14], [29]. In Fig. 1, we presented data that suggests that DENV-induced rearrangement and expansion of the ER is independent of the UPR. To further demonstrate that this ER rearrangement is independent of the XBP1 pathway, we infected XBP1+/+ and XBP1−/− mouse embryo fibroblast (MEF) cells with DENV-2 for 12 h and fixed and processed them for LSCM analysis. As demonstrated with 2fTGH cells (Fig. 1), we found that ER-resident proteins GRP78 and GRP94 co-localize with E and NS3 proteins within the enlarged ER of DENV-2-infected XBP1+/+ (Fig. 2A and 2B) and XBP1−/− (Fig. 2E and 2F) MEF cells. As expected, XBP1 is visible in DENV-2-infected XBP1+/+ MEF cells (Fig. 2C) where we again find it colocalized with E and NS3 in the ER, but it is not detected in XBP1−/− MEF cells (Fig. 2G). A recent study demonstrated that the ATF6 pathway can induce expansion and upregulation of pathways involved in lipid biosynthesis and ER biogenesis in XBP1−/− MEFs [12]. When we examined the activation of the ATF6 pathway in both XBP1+/+ (Fig. 2D) and XBP1−/− (Fig. 2H) MEF cells, we found that ATF6 remains uninduced and localized to the ER of DENV-2-infected cells, also consistent we our previous results [26].


Early dengue virus protein synthesis induces extensive rearrangement of the endoplasmic reticulum independent of the UPR and SREBP-2 pathway.

Peña J, Harris E - PLoS ONE (2012)

DENV-induced ER rearrangement and expansion is independent of the XBP-1 pathway.XBP1+/+ (A–D) and XBP1−/− (E–H) MEF cells were infected with DENV-2 for 12 h and stained intracellularly for cellular proteins using antibodies against (A and E) GRP78, (B and F) GRP94, (C) XBP1, and (G) ATF6 (magenta), followed by secondary antibodies conjugated to Alexa Fluor® 647. DENV proteins were detected with mouse MAbs against DENV E (green) and NS3 (red) directly conjugated to Alexa Fluor® 488 and Alexa Fluor® 594, respectively. Nuclear staining (blue) was performed using DAPI stain. Images were acquired and processed as described in Fig. 1. Total magnification 630×.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366941&req=5

pone-0038202-g002: DENV-induced ER rearrangement and expansion is independent of the XBP-1 pathway.XBP1+/+ (A–D) and XBP1−/− (E–H) MEF cells were infected with DENV-2 for 12 h and stained intracellularly for cellular proteins using antibodies against (A and E) GRP78, (B and F) GRP94, (C) XBP1, and (G) ATF6 (magenta), followed by secondary antibodies conjugated to Alexa Fluor® 647. DENV proteins were detected with mouse MAbs against DENV E (green) and NS3 (red) directly conjugated to Alexa Fluor® 488 and Alexa Fluor® 594, respectively. Nuclear staining (blue) was performed using DAPI stain. Images were acquired and processed as described in Fig. 1. Total magnification 630×.
Mentions: Previous studies have shown XBP1 to play a critical role in cell size expansion, organelle biogenesis, increased protein synthesis, and induction of secretory genes [11], [13], [14], [29]. In Fig. 1, we presented data that suggests that DENV-induced rearrangement and expansion of the ER is independent of the UPR. To further demonstrate that this ER rearrangement is independent of the XBP1 pathway, we infected XBP1+/+ and XBP1−/− mouse embryo fibroblast (MEF) cells with DENV-2 for 12 h and fixed and processed them for LSCM analysis. As demonstrated with 2fTGH cells (Fig. 1), we found that ER-resident proteins GRP78 and GRP94 co-localize with E and NS3 proteins within the enlarged ER of DENV-2-infected XBP1+/+ (Fig. 2A and 2B) and XBP1−/− (Fig. 2E and 2F) MEF cells. As expected, XBP1 is visible in DENV-2-infected XBP1+/+ MEF cells (Fig. 2C) where we again find it colocalized with E and NS3 in the ER, but it is not detected in XBP1−/− MEF cells (Fig. 2G). A recent study demonstrated that the ATF6 pathway can induce expansion and upregulation of pathways involved in lipid biosynthesis and ER biogenesis in XBP1−/− MEFs [12]. When we examined the activation of the ATF6 pathway in both XBP1+/+ (Fig. 2D) and XBP1−/− (Fig. 2H) MEF cells, we found that ATF6 remains uninduced and localized to the ER of DENV-2-infected cells, also consistent we our previous results [26].

Bottom Line: We then demonstrate that enlargement of the ER is independent of the SREBP-2 activation and upregulation of 3-hydroxy-3-methylglutaryl-Coenzyme-A reductase, the rate-limiting enzyme in the cholesterol biosynthesis pathway.Lastly, we demonstrate that viral infection induces the reabsorption of lipid droplets into the ER.This work paves the way for further study of virally-induced membrane rearrangements and formation of cubic membranes.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, California, United States of America. jpena360@yahoo.com

ABSTRACT
The rearrangement of intracellular membranes has been long reported to be a common feature in diseased cells. In this study, we used dengue virus (DENV) to study the role of the unfolded protein response (UPR) and sterol-regulatory-element-binding-protein-2 (SREBP-2) pathway in the rearrangement and expansion of the endoplasmic reticulum (ER) early after infection. Using laser scanning confocal and differential interference contrast microscopy, we demonstrate that rearrangement and expansion of the ER occurs early after DENV-2 infection. Through the use of mouse embryonic fibroblast cells deficient in XBP1 and ATF6, we show that ER rearrangement early after DENV infection is independent of the UPR. We then demonstrate that enlargement of the ER is independent of the SREBP-2 activation and upregulation of 3-hydroxy-3-methylglutaryl-Coenzyme-A reductase, the rate-limiting enzyme in the cholesterol biosynthesis pathway. We further show that this ER rearrangement is not inhibited by the treatment of DENV-infected cells with the cholesterol-inhibiting drug lovastatin. Using the transcription inhibitor actinomycin D and the translation elongation inhibitor cycloheximide, we show that de novo viral protein synthesis but not host transcription is necessary for expansion and rearrangement of the ER. Lastly, we demonstrate that viral infection induces the reabsorption of lipid droplets into the ER. Together, these results demonstrate that modulation of intracellular membrane architecture of the cell early after DENV-2 infection is driven by viral protein expression and does not require the induction of the UPR and SREBP-2 pathways. This work paves the way for further study of virally-induced membrane rearrangements and formation of cubic membranes.

Show MeSH
Related in: MedlinePlus