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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 ATF6 pathway.ATF6+/+ (A–C) and ATF6−/− (D–F) MEF cells were infected with DENV-2 for 12 h and intracellularly stained for cellular proteins (A and D) GRP78, (B and E) GRP94, and (C and F) XBP1 (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-g003: DENV-induced ER rearrangement and expansion is independent of the ATF6 pathway.ATF6+/+ (A–C) and ATF6−/− (D–F) MEF cells were infected with DENV-2 for 12 h and intracellularly stained for cellular proteins (A and D) GRP78, (B and E) GRP94, and (C and F) XBP1 (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: To further demonstrate that rearrangement and expansion of the ER is ATF6-independent early after DENV-2 infection, we infected ATF6+/+ and ATF6−/− MEF cells with DENV-2, fixed them, and processed them for LSCM 12 h p.i. Consistent with the data in Fig. 1, we once again found expression of GRP78 (Fig. 3A and 3D) and GRP94 (Fig. 3B and 3E) to localize within a rearranged and enlarged ER in DENV-2-infected ATF6+/+ and ATF6−/− MEF cells. Furthermore, expression of DENV E and NS3 proteins was co-localized with GRP78 and GRP94 in the expanded ER. In addition, the expression of XBP1 is induced but remains localized to the ER in both ATF6+/+ (Fig. 3C) and ATF6−/− (Fig. 3F) MEF cells. These data show that rearrangement and expansion of the ER early during DENV-2 infection is independent of the ATF6 and IRE1/XBP1 pathways of the UPR.


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 ATF6 pathway.ATF6+/+ (A–C) and ATF6−/− (D–F) MEF cells were infected with DENV-2 for 12 h and intracellularly stained for cellular proteins (A and D) GRP78, (B and E) GRP94, and (C and F) XBP1 (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-g003: DENV-induced ER rearrangement and expansion is independent of the ATF6 pathway.ATF6+/+ (A–C) and ATF6−/− (D–F) MEF cells were infected with DENV-2 for 12 h and intracellularly stained for cellular proteins (A and D) GRP78, (B and E) GRP94, and (C and F) XBP1 (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: To further demonstrate that rearrangement and expansion of the ER is ATF6-independent early after DENV-2 infection, we infected ATF6+/+ and ATF6−/− MEF cells with DENV-2, fixed them, and processed them for LSCM 12 h p.i. Consistent with the data in Fig. 1, we once again found expression of GRP78 (Fig. 3A and 3D) and GRP94 (Fig. 3B and 3E) to localize within a rearranged and enlarged ER in DENV-2-infected ATF6+/+ and ATF6−/− MEF cells. Furthermore, expression of DENV E and NS3 proteins was co-localized with GRP78 and GRP94 in the expanded ER. In addition, the expression of XBP1 is induced but remains localized to the ER in both ATF6+/+ (Fig. 3C) and ATF6−/− (Fig. 3F) MEF cells. These data show that rearrangement and expansion of the ER early during DENV-2 infection is independent of the ATF6 and IRE1/XBP1 pathways of the UPR.

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