The Study of M olecular M echanism of DEN2 Binding to Endothelial Cells

Abstract：A human endothelial cell line(ECV304)derived from umbilical vein was used to study the mechanism of Dengue virus type 2 fDEN2)infection on endothelial cells．VimS growth curve on the ECV304 cells showed that the cells were sensitive to DEN2 infection．M embrane preparations from ECV304 cells were collected either by mechanical scraping or by trypsin digestion．SDS-PAGE an alysis showed that there lacks of a protein with a molecular size 43 kDa in the membran e preparation treated by trypsin．Virus overlay protein binding assays(VOPBA)w． ere performed by binding∞S-M et labeled DEN2 with separated membrane proteins of ECV304 cells．Three proteins each of 29．34 an d 43 kDa located on the surface of ECV304 were found to bind with the labeled viru s．The virus．binding efects of the 34 and 43 kDa proteins could be inhibited when the ECV304 cells were treated with trypsin． Preincubation of ECV304 cells with rEgp could inhibit DEN2．binding an d block viru s infection． Preincubation of the protein electro—transferred nitrocellulose membran e with the rEgP could alSO block DEN2 binding with the three identified proteins on the surface of ECV304 cells in VOPBA．Th e result suggests that three proteins each of 29．34 and 43 kDa on the surface of ECV304 cell might associate with the receptor complex for DEN2 binding，and envelope E glycoprotein could mediate initial binding of DEN2 to human vascular endothelial cells．