EARLY INTERACTIONS OF DEN-2 VIRUS AND CELLS

Abstract

Early virus-cell interaction events are critical to initiate infection in cells. In this study, the early events of DEN-2 virus infection in mammalian Vero and mosquito C6/36 cells were studied. These early events include the mteractions of DEN-2 virus with cellular receptors and the mechanisms of DEN-2 v1rus entry into these cells. The binding between DEN-2 virus and Vero and C3/36, cells was specific and saturable. Radiolabelled DEN-2 virus competed effectively for the binding Sites on Vern and C6/36 cell surfaces in vitro with unlabelled DEN-2 virus. Scatchard plot analysis revealed that DEN-2 virus recognised only one class of binding sites on Vero cells. This seemed also true for the binding of DEN-2 virus onto C6/36 cells at the initial part of the binding time course. OG (Octyl-ll-D-glucopyranosidc) extracts from Vero and C6/36 cells could exert about 75% and 60% inhibition to the binding of DEN-2 virus with Vern and CM36 cells, respectively A 150 KDa protein in OG extract from Vero cells and a 32 KDa protein in OG extract from C6/36 cells were identified to be putative receptor for DEN-2 virus. Biochemical experiments using lysosomotropic agents (NH4CI and monensin). 'H-ATPase inhibitor (bafilomycin Al-BFLAI) and phagocytic inhibitor (cytochalasin B) were performed to elucidate the entry mechanisms of DEN-2 virus into both Vero and C6/36 cells. NH4CI and monensin could raise interior pH of endosomes which would block the fusion between virus envelope and endosomal membrane as low pH is needed. Besides raising pH of endosome, BFLAI also disrupts the 'H-gradient existing in vesicle system. Cytochalasin B inhibits phagocytic activity of cells by binding to actin, preventing its proper polymenzatron into mrcrofilament and promoting microfilament disassembly. The infection of Vero cells by DEN-2 virus was blocked by NH4Cl, monensin and BFLAI when added very early during infection. This suggested that entry pathway of DEN-2 virus into Vero cells was low pH-dependent and by receptor-mediated endocytosis. On the uther hand, these agents exerted no inhibitory effects on DEN-2 virus entry into C6/36 cells. This was probably because the entry pathway of DEN-2 virus into C6/36 cells did not involve a low-pH-dependent process. Thus, entry of DEN-2 virus into C6/36 cells most likely was by direct fusion at plasma membrane. Although C6/36 cells were highly vacuolated, classical phagocytosis could be excluded as an entry mode because DEN-2 virus entry into C6/36 cells was not inhibited by cytochalasin B