Pattanakitsakul, Sa-ngaSa-ngaPattanakitsakulPoungsawai, JesdapornJesdapornPoungsawaiKanlaya, RattiyapornRattiyapornKanlayaSinchaikul, SupachokSupachokSinchaikulChen, Shui-TeinShui-TeinChenThongboonkerd, VisithVisithThongboonkerd2012-10-202018-07-062012-10-202018-07-062010http://ntur.lib.ntu.edu.tw//handle/246246/243472The most severe form of dengue virus (DENV) infection is dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), which is accompanied by increased vascular permeability indicating that endothelial cells are the targets of DENV infection. However, molecular mechanisms underlying DENV replication in endothelial cells remained poorly understood. We therefore examined changes in subcellular proteomes of different cellular compartments (including cytosolic, membrane/organelle, nucleus, and cytoskeleton) of human endothelial (EA.hy926) cells upon DENV2 infection using a 2-DE-based proteomics approach followed by Q-TOF MS and MS/MS. A total of 35 altered proteins were identified in these subcellular locales, including an increase in the level of Alix (apoptosis-linked gene-2-interacting protein X) in the cytosolic fraction of DENV2-infected cells compared to mock control cells. Double immunofluorescence staining revealed colocalization of Alix with late endosomal lysobisphosphatidic acid (LBPA). This complex has been proposed to be involved in the export of DENV proteins from late endosomes to the cytoplasm. Subsequent functional study revealed that pretreatment with an anti-LBPA antibody prior to DENV challenge significantly reduced the level of viral envelope protein synthesis and DENV replication. Our data indicate that Alix plays a pivotal role in the early phase of DENV replication, particularly when it arrives at the late endosome stage. Blocking this step may lead to a novel therapeutic approach to reducing the level of DENV replication in vivo. ? 2010 American Chemical Society.en-USAlix; dengue; endothelial cells; host responses; LBPA; proteome; proteomics[SDGs]SDG3lysobisphosphatidic acid; phosphatidic acid; protein alix; unclassified drug; virus envelope protein; article; cell nucleus; cytoplasm; cytoskeleton; cytosol; dengue; Dengue virus; endosome; endothelium cell; human; human cell; immunofluorescence; mass spectrometry; priority journal; protein localization; proteomics; virus replication; Apoptosis; Calcium-Binding Proteins; Cell Cycle Proteins; Cell Line; Dengue; Electrophoresis, Gel, Two-Dimensional; Endosomal Sorting Complexes Required for Transport; Endosomes; Endothelial Cells; Humans; Lysophospholipids; Microscopy, Fluorescence; Monoglycerides; Proteins; Proteome; Proteomics; Reproducibility of Results; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Subcellular Fractions; Tandem Mass Spectrometry; Virus Replication; Dengue virusjournal article10.1021/pr100357fhttp://ntur.lib.ntu.edu.tw/bitstream/246246/243472/-1/118.pdf