Association of Alix with late endosomal lysobisphosphatidic acid is important for dengue virus infection in human endothelial cells.
The 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.
Pattanakitsakul
Sa-nga
SN
Medical Molecular Biology Unit, Office for Research and Development, Department of Immunology and Immunology Graduate Program, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Poungsawai
Jesdaporn
J
Kanlaya
Rattiyaporn
R
Sinchaikul
Supachok
S
Chen
Shui-Tein
ST
Thongboonkerd
Visith
V
D017209:Apoptosis; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D003715:Dengue / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry* / Q000378:metabolism; D011992:Endosomes / Q000737:chemistry / Q000378:metabolism*; D042783:Endothelial Cells / Q000378:metabolism; D006801:Humans; D008246:Lysophospholipids / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D050178:Monoglycerides / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D020543:Proteome / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014779:Virus Replication
D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D008246:Lysophospholipids; D050178:Monoglycerides; C117020:PDCD6IP protein, human; D011506:Proteins; D020543:Proteome; C012786:bis(monoacylglyceryl)phosphate