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Many cellular proteins are altered through the addition of carbohydrate residues attached through either N- or O-linkages when they are translocated into the endoplasmic reticulum. Enveloped viruses use this cellular pathway to produce proteins that are glycosylated as they mature in the ER and Golgi compartment. In Japanese encephalitis virus (JEV), both structure protein E and non-structure protein NS1 are glycosylated. To determine the effect of glycosylation on the antigenicity and expression of envelope protein (E), the only glycosylation site on E protein open reading frame was point- mutated and resulted protein was shown not glycosylated. The ability of thirteen anti -E mouse monoclonal antibodies, however, showed no difference when determined by radio-immunoprecipitation in binding with glycosylated and non-glycosylated E protein. Using immunofluorescence assay, we found that the pattern of internal labeling was similar in both glycosylated and non-glycosylated E protein. These result infer that the carbohydrate moiety does not seem to play a major role in the antigenic structure and maturation of the JEV E protein. An E protein mutant clone, pE277, produced by linker insertion mutagenesis on E protein open reading frame expressed mutated E protein, E277, in cells and accumulated around the nucleus instead of the transport vehicle. The mutant clone can therefore be used as a tool to study the maturation process ofE protein.
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