Abstract:
Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is a major cause of viral encephalitis in Asia. Even though the principle target cells for JEV in the central nervous system are neurons, the microglia is activated in response to JEV infection. Viral attachment to a host cell is the first step of the viral entry process and is a critical mediator of tissue tropism. This research aimed to identify molecules associated with JEV entry to microglial cells and examine their function as viral receptor. One dimensional and Two-dimensional gel electrophoresis were applied to separate the membrane proteins. Virus overlay protein binding assay (VOPBA) and liquid chromatography-mass spectrometry (LC/MS/MS) identified the laminin receptor protein and nucleolin as a potential JEV binding proteins. These newly identified JEV binding proteins were further characterized for their function as viral receptors by infection inhibition assay using anti-laminin receptor and anti-nucleolin. Other possible candidate receptor molecules including Hsp70, Hsp90, GRP78, CD14 and CD4 were also applied in the antibody mediated inhibition of infection experiments. Results showed that both anti-laminin receptor and anti-CD4 antibodies significantly reduced virus entry (25-40%). Significant inhibition of virus entry (up to 80%) was observed in the presence of lipopolysaccharide (LPS) which resulted in a complete down regulation of CD4. These results suggest that multiple receptor proteins may mediate the entry of JEV to microglial cells, with LPS receptor molecules playing a major role.
