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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 expansion induces lipid droplet reabsorption.DENV-2-infected 2fTGH cells were fixed and stained with mouse MAbs against DENV-2 E-protein (4G2; pseudo-colored green) and NS3 (red). Lipid droplets (LD; pseudo-colored magenta) were stained using Bodipy 493/503 dye. (B) Three-dimensional reconstruction of the rearranged and expanded ER early after DENV-2 infection. The deconvolved image was rotated 180° on the X-axis relative to Fig. 7A. Images were obtained using a Zeiss 710 LSM as previously described. Z-stack series (61 panels) were deconvolved using Huygens software (Scientific Volume Imaging), and an iso-surface model was generated using Imaris 7.2 software (Bitplane Scientific Software). LD (pseudo-color magenta), E (pseudo-color green) and NS3 (red). Total magnification 600×.
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pone-0038202-g007: DENV-induced ER expansion induces lipid droplet reabsorption.DENV-2-infected 2fTGH cells were fixed and stained with mouse MAbs against DENV-2 E-protein (4G2; pseudo-colored green) and NS3 (red). Lipid droplets (LD; pseudo-colored magenta) were stained using Bodipy 493/503 dye. (B) Three-dimensional reconstruction of the rearranged and expanded ER early after DENV-2 infection. The deconvolved image was rotated 180° on the X-axis relative to Fig. 7A. Images were obtained using a Zeiss 710 LSM as previously described. Z-stack series (61 panels) were deconvolved using Huygens software (Scientific Volume Imaging), and an iso-surface model was generated using Imaris 7.2 software (Bitplane Scientific Software). LD (pseudo-color magenta), E (pseudo-color green) and NS3 (red). Total magnification 600×.

Mentions: In this study, we have demonstrated that DENV-2-induced ER rearrangement and expansion proceeds independently of cellular pathways involved in ER biogenesis. Additionally, our data demonstrates that DENV viral protein synthesis is sufficient to drive the rearrangement of preexisting intracellular membranes. However, the data presented so far do not fully account for the increased volume of the ER during DENV-2 infection. Given the sudden cellular demand for lipids after DENV infection and the non-involvement of the UPR and SREBP-2 pathways, we decided to investigate whether lipid droplets (LDs) might provide the cell with a pool of ready-made lipids for the expansion of the ER. LDs are organelles specialized for the storage of neutral lipids and regulation of cellular lipid metabolism, and they function as lipid processing centers that are readily available to the cell [40]. Therefore, we infected 2fTGH cells with DENV-2 for 12 h, fixed them, and stained for viral proteins E and NS3 and for LDs with Bodipy 493/503, a dye that specifically targets neutral lipids in LDs, and processed the cells for LSCM. Uninfected cells showed a staining pattern characteristic of LDs (Fig. 7A). Interestingly, these LD spots were not seen in DENV-infected cells; instead, we found the ER to be stained with the Bodipy 493/503 dye, suggesting the LD neutral lipids had been reabsorbed into the ER (Fig. 7A). As with our previous results, staining of E and NS3 viral proteins colocalized to the grossly enlarged ER of DENV-2 infected cells and was absent in adjacent non-infected cells (Fig. 7A). In order to gain more accurate information regarding the spatial localization of LDs in uninfected cells and the relative rearrangement of the ER in DENV-2-infected cells, we performed a deconvolution of the image stacks from Figure 7A and generated three-dimensional (3-D) views of the deconvoluted data with an iso-surface rendering based on the LDs stained with Bodipy 493/503. This 3-D image rendition allows for the visualization of the LDs in the uninfected cells while also allowing for the visualization of the neutral lipid staining within the enlarged ER as well as the DENV-2 viral proteins in the infected cells. In agreement with Figure 7A, LDs are only found in uninfected cells (Fig. 7B). These results suggest an early involvement of LDs in the rearrangement and expansion of the ER.


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 expansion induces lipid droplet reabsorption.DENV-2-infected 2fTGH cells were fixed and stained with mouse MAbs against DENV-2 E-protein (4G2; pseudo-colored green) and NS3 (red). Lipid droplets (LD; pseudo-colored magenta) were stained using Bodipy 493/503 dye. (B) Three-dimensional reconstruction of the rearranged and expanded ER early after DENV-2 infection. The deconvolved image was rotated 180° on the X-axis relative to Fig. 7A. Images were obtained using a Zeiss 710 LSM as previously described. Z-stack series (61 panels) were deconvolved using Huygens software (Scientific Volume Imaging), and an iso-surface model was generated using Imaris 7.2 software (Bitplane Scientific Software). LD (pseudo-color magenta), E (pseudo-color green) and NS3 (red). Total magnification 600×.
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pone-0038202-g007: DENV-induced ER expansion induces lipid droplet reabsorption.DENV-2-infected 2fTGH cells were fixed and stained with mouse MAbs against DENV-2 E-protein (4G2; pseudo-colored green) and NS3 (red). Lipid droplets (LD; pseudo-colored magenta) were stained using Bodipy 493/503 dye. (B) Three-dimensional reconstruction of the rearranged and expanded ER early after DENV-2 infection. The deconvolved image was rotated 180° on the X-axis relative to Fig. 7A. Images were obtained using a Zeiss 710 LSM as previously described. Z-stack series (61 panels) were deconvolved using Huygens software (Scientific Volume Imaging), and an iso-surface model was generated using Imaris 7.2 software (Bitplane Scientific Software). LD (pseudo-color magenta), E (pseudo-color green) and NS3 (red). Total magnification 600×.
Mentions: In this study, we have demonstrated that DENV-2-induced ER rearrangement and expansion proceeds independently of cellular pathways involved in ER biogenesis. Additionally, our data demonstrates that DENV viral protein synthesis is sufficient to drive the rearrangement of preexisting intracellular membranes. However, the data presented so far do not fully account for the increased volume of the ER during DENV-2 infection. Given the sudden cellular demand for lipids after DENV infection and the non-involvement of the UPR and SREBP-2 pathways, we decided to investigate whether lipid droplets (LDs) might provide the cell with a pool of ready-made lipids for the expansion of the ER. LDs are organelles specialized for the storage of neutral lipids and regulation of cellular lipid metabolism, and they function as lipid processing centers that are readily available to the cell [40]. Therefore, we infected 2fTGH cells with DENV-2 for 12 h, fixed them, and stained for viral proteins E and NS3 and for LDs with Bodipy 493/503, a dye that specifically targets neutral lipids in LDs, and processed the cells for LSCM. Uninfected cells showed a staining pattern characteristic of LDs (Fig. 7A). Interestingly, these LD spots were not seen in DENV-infected cells; instead, we found the ER to be stained with the Bodipy 493/503 dye, suggesting the LD neutral lipids had been reabsorbed into the ER (Fig. 7A). As with our previous results, staining of E and NS3 viral proteins colocalized to the grossly enlarged ER of DENV-2 infected cells and was absent in adjacent non-infected cells (Fig. 7A). In order to gain more accurate information regarding the spatial localization of LDs in uninfected cells and the relative rearrangement of the ER in DENV-2-infected cells, we performed a deconvolution of the image stacks from Figure 7A and generated three-dimensional (3-D) views of the deconvoluted data with an iso-surface rendering based on the LDs stained with Bodipy 493/503. This 3-D image rendition allows for the visualization of the LDs in the uninfected cells while also allowing for the visualization of the neutral lipid staining within the enlarged ER as well as the DENV-2 viral proteins in the infected cells. In agreement with Figure 7A, LDs are only found in uninfected cells (Fig. 7B). These results suggest an early involvement of LDs in the rearrangement and expansion of the ER.

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