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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 does not induce the SREBP-2 pathway or upreglation of HMGCR early after infection.2fTGH cells were mock-treated (A) or treated with DTT (B) for 6 h. Cells were then fixed and stained intracellularly for cellular proteins using antibodies against SREBP-2 (green) and HMGCR (red) followed by secondary antibodies conjugated to Alexa Fluor® 488 and Alexa Fluor® 546, respectively. 2fTGH cells were infected with DENV-2 for 12 h and stained intracellularly for (C) SREBP-2 (magenta) and (D) HMGCR (magenta) followed by secondary antibodies conjugated to Alexa Fluor® 647; mouse MAbs against DENV E (green) and NS3 (red) directly conjugated to Alexa Fluor® 488 and Alexa Fluor® 594, respectively, were used to detect DENV proteins. Nuclear staining (blue) was performed using DAPI stain. The multiple-exposure images were captured using a 40×/1.4 Plan-Apochromat DIC oil objective on a Zeiss 710 LSM using bandpass filter sets appropriate for DAPI, Alexa Fluor® 488, Alexa Fluor® 594 and Alexa Fluor® 647 and were processed as previously described in Figure 1. (E) SREBP-2 proteolyic processing by immunoblot analysis. Actin was used as a loading control. In (F), 2fTGH cells were infected with DENV-2 over a 3–12 h time-course. Lysates were collected and analyzed for SREBP-2 and HMGCR expression by immunoblot analysis. Actin was used as a loading control, and expression of DENV NS1 was used to confirm viral infection. A time-matched mock-infected control was included at each time-point. Total magnification 600×.
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pone-0038202-g004: DENV does not induce the SREBP-2 pathway or upreglation of HMGCR early after infection.2fTGH cells were mock-treated (A) or treated with DTT (B) for 6 h. Cells were then fixed and stained intracellularly for cellular proteins using antibodies against SREBP-2 (green) and HMGCR (red) followed by secondary antibodies conjugated to Alexa Fluor® 488 and Alexa Fluor® 546, respectively. 2fTGH cells were infected with DENV-2 for 12 h and stained intracellularly for (C) SREBP-2 (magenta) and (D) HMGCR (magenta) followed by secondary antibodies conjugated to Alexa Fluor® 647; mouse MAbs against DENV E (green) and NS3 (red) directly conjugated to Alexa Fluor® 488 and Alexa Fluor® 594, respectively, were used to detect DENV proteins. Nuclear staining (blue) was performed using DAPI stain. The multiple-exposure images were captured using a 40×/1.4 Plan-Apochromat DIC oil objective on a Zeiss 710 LSM using bandpass filter sets appropriate for DAPI, Alexa Fluor® 488, Alexa Fluor® 594 and Alexa Fluor® 647 and were processed as previously described in Figure 1. (E) SREBP-2 proteolyic processing by immunoblot analysis. Actin was used as a loading control. In (F), 2fTGH cells were infected with DENV-2 over a 3–12 h time-course. Lysates were collected and analyzed for SREBP-2 and HMGCR expression by immunoblot analysis. Actin was used as a loading control, and expression of DENV NS1 was used to confirm viral infection. A time-matched mock-infected control was included at each time-point. Total magnification 600×.

Mentions: SREBP-2 has been shown to regulate both lipid and cholesterol synthesis in vivo and in vitro[30], [31], [32]. Therefore, we next investigated whether DENV-2 infection led to activation of the SREBP-2 pathway and induction of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGCR), the rate-limiting enzyme involved in cholesterol biosynthesis. We first mock-treated or DTT-treated 2fTGH cells for 6 h to induce ER stress and stained for cellular distribution of SREBP-2 and HMGCR. Mock-treated 2fTGH cells demonstrated diffused staining of SREBP-2 throughout the cell, while low levels of HMGCR were detected around the nucleus (Fig. 4A). This contrasts with DTT-treated 2fTGH cells (Fig. 4B), which demonstrated activation of SREBP-2 by increased nuclear localization and increased expression of HMGCR around the nucleus. We next determined whether rearrangement of the ER during DENV-2 infection was driven by the activation of SREBP-2 and upregulation of HMGCR in infected cells. We infected 2fTGH cells with DENV-2 at an MOI of 5 for 12 h, fixed and stained the cells, and determined whether the SREBP-2 pathway was activated and expression of HMGCR upregulated. SREBP-2 staining of DENV-2-infected 2fTGH cells (Fig. 4C) demonstrated that SREBP-2, like ATF6, remained inactive and localized to the ER with E and NS3 viral proteins. HMGCR was redistributed to the enlarged ER in DENV-2-infected cells compared to its nuclear distribution in uninfected cells (Fig. 4D).


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 does not induce the SREBP-2 pathway or upreglation of HMGCR early after infection.2fTGH cells were mock-treated (A) or treated with DTT (B) for 6 h. Cells were then fixed and stained intracellularly for cellular proteins using antibodies against SREBP-2 (green) and HMGCR (red) followed by secondary antibodies conjugated to Alexa Fluor® 488 and Alexa Fluor® 546, respectively. 2fTGH cells were infected with DENV-2 for 12 h and stained intracellularly for (C) SREBP-2 (magenta) and (D) HMGCR (magenta) followed by secondary antibodies conjugated to Alexa Fluor® 647; mouse MAbs against DENV E (green) and NS3 (red) directly conjugated to Alexa Fluor® 488 and Alexa Fluor® 594, respectively, were used to detect DENV proteins. Nuclear staining (blue) was performed using DAPI stain. The multiple-exposure images were captured using a 40×/1.4 Plan-Apochromat DIC oil objective on a Zeiss 710 LSM using bandpass filter sets appropriate for DAPI, Alexa Fluor® 488, Alexa Fluor® 594 and Alexa Fluor® 647 and were processed as previously described in Figure 1. (E) SREBP-2 proteolyic processing by immunoblot analysis. Actin was used as a loading control. In (F), 2fTGH cells were infected with DENV-2 over a 3–12 h time-course. Lysates were collected and analyzed for SREBP-2 and HMGCR expression by immunoblot analysis. Actin was used as a loading control, and expression of DENV NS1 was used to confirm viral infection. A time-matched mock-infected control was included at each time-point. Total magnification 600×.
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Related In: Results  -  Collection

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pone-0038202-g004: DENV does not induce the SREBP-2 pathway or upreglation of HMGCR early after infection.2fTGH cells were mock-treated (A) or treated with DTT (B) for 6 h. Cells were then fixed and stained intracellularly for cellular proteins using antibodies against SREBP-2 (green) and HMGCR (red) followed by secondary antibodies conjugated to Alexa Fluor® 488 and Alexa Fluor® 546, respectively. 2fTGH cells were infected with DENV-2 for 12 h and stained intracellularly for (C) SREBP-2 (magenta) and (D) HMGCR (magenta) followed by secondary antibodies conjugated to Alexa Fluor® 647; mouse MAbs against DENV E (green) and NS3 (red) directly conjugated to Alexa Fluor® 488 and Alexa Fluor® 594, respectively, were used to detect DENV proteins. Nuclear staining (blue) was performed using DAPI stain. The multiple-exposure images were captured using a 40×/1.4 Plan-Apochromat DIC oil objective on a Zeiss 710 LSM using bandpass filter sets appropriate for DAPI, Alexa Fluor® 488, Alexa Fluor® 594 and Alexa Fluor® 647 and were processed as previously described in Figure 1. (E) SREBP-2 proteolyic processing by immunoblot analysis. Actin was used as a loading control. In (F), 2fTGH cells were infected with DENV-2 over a 3–12 h time-course. Lysates were collected and analyzed for SREBP-2 and HMGCR expression by immunoblot analysis. Actin was used as a loading control, and expression of DENV NS1 was used to confirm viral infection. A time-matched mock-infected control was included at each time-point. Total magnification 600×.
Mentions: SREBP-2 has been shown to regulate both lipid and cholesterol synthesis in vivo and in vitro[30], [31], [32]. Therefore, we next investigated whether DENV-2 infection led to activation of the SREBP-2 pathway and induction of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGCR), the rate-limiting enzyme involved in cholesterol biosynthesis. We first mock-treated or DTT-treated 2fTGH cells for 6 h to induce ER stress and stained for cellular distribution of SREBP-2 and HMGCR. Mock-treated 2fTGH cells demonstrated diffused staining of SREBP-2 throughout the cell, while low levels of HMGCR were detected around the nucleus (Fig. 4A). This contrasts with DTT-treated 2fTGH cells (Fig. 4B), which demonstrated activation of SREBP-2 by increased nuclear localization and increased expression of HMGCR around the nucleus. We next determined whether rearrangement of the ER during DENV-2 infection was driven by the activation of SREBP-2 and upregulation of HMGCR in infected cells. We infected 2fTGH cells with DENV-2 at an MOI of 5 for 12 h, fixed and stained the cells, and determined whether the SREBP-2 pathway was activated and expression of HMGCR upregulated. SREBP-2 staining of DENV-2-infected 2fTGH cells (Fig. 4C) demonstrated that SREBP-2, like ATF6, remained inactive and localized to the ER with E and NS3 viral proteins. HMGCR was redistributed to the enlarged ER in DENV-2-infected cells compared to its nuclear distribution in uninfected cells (Fig. 4D).

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