The envelope glycoproteins of dengue 1 and dengue 2 viruses grown in mosquito cells differ in their utilization of potential glycosylation sites.
We have previously isolated and characterized two dengue (DEN) 2 viruses mutant in their fusion-from-within (FFWI) phenotype in the insect cell line C6/36. Both viruses lost a potential glycosylation site (Asn-153) in the envelope (E) glycoprotein. To determine whether the change in FFWI phenotype was due to a change in E-glycoprotein glycosylation, we characterized the patterns of glycosylation on the E-glycoprotein of wild-type DEN 1 and DEN 2 viruses. The E-glycoproteins were isolated from purified virus grown in Aedes albopictus C6/36 cells, by use of high-performance size-exclusion chromatography. The tryptic maps of wild-type glycosylated and enzymatically (PNGase F) deglycosylated E-glycoproteins were compared by reverse-phase high-performance liquid chromatography. The DEN 1 virus E-glycoprotein was found to have two peaks in the tryptic map that exhibited shifts after deglycosylation, whereas the DEN 2 virus E-glycoprotein had only one. Besides the potential glycosylation site at Asn-153, both DEN 1 and DEN 2 virus E-glycoproteins have another potential site located at Asn-67. Amino-terminal sequencing of the shifted peaks revealed that DEN 2 virus E-glycoprotein is glycosylated only at Asn-67; however, DEN 1 virus E-glycoprotein is glycosylated at both Asn-67 and Asn-153. These DEN virus serotypes are thus heterogeneous in their use of glycosylation sites. We also determined by a lectin-binding assay that the attached carbohydrates for both viruses were likely to be of the high-mannose type.
Johnson
A J
AJ
Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado 80522.
Guirakhoo
F
F
Roehrig
J T
JT
D000330:Aedes; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D003716:Dengue Virus / Q000737:chemistry*; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014759:Viral Envelope Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification
D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins