Identification and Characterization of Carbohydrate Molecules in Mammalian Cells Recognized by Dengue Virus Type 2

Abstract

The interaction between cell surface receptors and the envelope glycoprotein (EGP) on the viral membrane surface is the initial step of Dengue virus infection. To understand the host range, tissue tropism, and virulence of this pathogen, it is critical to elucidate the molecular mechanisms of the interaction of EGP with receptor molecules. Here, using a TLC/virus-binding assay, we isolated and characterized a carbohydrate molecule on mammalian cell surfaces that is recognized by dengue virus type 2 (DEN2). Structural determination by immunochemical methods showed that the carbohydrate structure of the purified glycosphingolipid was neolactotetraosylceramide (nLc4Cer). This glycosphingolipid was expressed on the cell surface of susceptible cells, such as human erythroleukemia K562 and baby hamster kidney BHK-21. All serotypes of DEN viruses, DEN1 to DEN4, reacted with nLc4Cer, and the non-reducing terminal disaccharide residue Galbeta1-4GlcNAcbeta1- was found to be a critical determinant for the binding of DEN2. Chemically synthesized derivatives carrying multiple carbohydrate residues of nLc4, but not nLc4 oligosaccharide, inhibited DEN2 infection of BHK-21 cells. These findings strongly suggested that multivalent nLc4 oligosaccharide could act as a competitive inhibitor against the binding of DEN2 to the host cells.

Anti-Hepatitis C Virus Activity of a Crude Extract from Longan (<i>Dimocarpus longan</i> Lour.) Leaves

“…Cultured cells were incubated at 379 C in a 5z CO 2 humidified chamber. A cell culture-adapted HCV variant (JFH1 strain of genotype 2a) was propagated as described previously (27,28). An Indonesian strain of dengue virus type 2 (DENV, DS-18/09 strain) was propagated in mosquito-derived C6/36 cells and African green monkey kidney-derived Vero cells as described previously (29,30).…”

“…Virus titration: Virus titrations were performed as described previously (19,27). Cells were seeded in 96-well plates and then inoculated with culture supernatants serially diluted in culture medium.…”

Role of Membrane Rafts in Viral Infection

“…DEN, which is the most important arthropod-borne human pathogen, represents clinical manifestations from a simple self-limited febrile illness known as dengue fever to a hemorrhagic fever and potentially fatal hemorrhagic shock syndrome. Receptor candidates of DEN have been reported to be dendritic cell-specific ICAM 3-grabbing non-integrin (DC-SIGN) [131], heparan sulfate [132], heat shock proteins (HSPs) [88], and neolactotetraosylceramide [133]. HSP90 and HSP70 are part of a receptor complex of DEN, which are associated with membrane rafts required for virus entry in neuroblastoma and human monocytes/macrophages [88,89].…”

Unique heparan sulfate from shrimp heads exhibits a strong inhibitory effect on infections by dengue virus and Japanese encephalitis virus

“…The anti-DEN-2 and anti-JEV activity was examined according to the method described previously [17]. Briefly, DEN-2 (1,800 focus-forming units/ml as a final concentration) or JEV (Nakayama strain) was premixed with known concentrations of the shrimp HS preparation or Hep (as a positive control).…”

“…After three washes with serum-free Dulbecco's modified Eagle's medium (DMEM), DMEM containing 1% fetal bovine serum and 0.5% methyl cellulose was overlayed and the plate was incubated at 37 ºC for 24 or 43 h. Infected foci of cells were visualized by the focus-forming assay and counted under a light microscope [17].…”