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.
Highlights
Dengue virus (DENV) causes tens of millions of cases of dengue annually and is a major international health concern
The second hypothesis is that the rearrangement and expansion of the endoplasmic reticulum (ER) early during DENV-2 infection requires the induction of pathways involved in lipid and ER biogenesis
The results presented here for DENV-2 support our first hypothesis; namely, that viral protein synthesis is sufficient to drive the early expansion of the ER without inducing increased transcription and/or translation of resident ER proteins and genes involved in membrane biogenesis
Summary
Dengue virus (DENV) causes tens of millions of cases of dengue annually and is a major international health concern. Infection with DENV, as well as other members of the Flaviviridae family, has been shown to induce rearrangement of cellular membranes known as cubic membranes (often described as convoluted membranes and vesicle packets that contain the replication complex) [2,3,4]. The ER is an extensive membranous network that is contiguous with the nuclear membrane and is responsible for the synthesis, maturation and proper folding of a wide range of proteins. It plays a critical role in Ca2+ and lipid homeostasis as well as intracellular signal transduction. The unfolded protein response (UPR) functions through the PKR-like ER kinase (PERK), kinase/ endoribonuclease inositol requiring 1 (IRE1)/X-box binding protein 1 (XBP1), and/or activation transcription factor 6 (ATF6) pathways. Despite the UPR’s protective role, prolonged and persistent ER stress can result in caspase activation and cellular apoptosis [15]
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