Mutational analysis of the N-linked glycans on Autographa californica nucleopolyhedrovirus gp64.
gp64 is the major envelope glycoprotein in the budded form of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). gp64 is essential for AcMNPV infection, as it mediates penetration of budded virus into host cells via the endocytic pathway. In this study, we used site-directed mutagenesis to map the positions of the N-linked glycans on AcMNPV gp64, characterize their structures, and evaluate their influence on gp64 function. We found that four of the five consensus N-glycosylation sites in gp64 are used, and we mapped the positions of those sites to amino acids 198, 355, 385, and 426 in the polypeptide chain. Endoglycosidase H sensitivity assays showed that N-linked glycans located at different positions are processed to various degrees. Lectin blotting analyses showed that each N-linked glycan on gp64 contains alpha-linked mannose, all but one contains alpha-linked fucose, and none contains detectable beta-linked galactose or alpha2,6-linked sialic acid. The amounts of infectious progeny produced by AcMNPV mutants lacking one, two, or three N-linked glycans on gp64 were about 10- to 100-fold lower than wild-type levels. This reduction did not correlate with reductions in the expression, transport, or inherent fusogenic activity of the mutant gp64s or in the gp64 content of mutant budded virus particles. However, all of the mutant viruses bound more slowly than the wild type. Therefore, elimination of one or more N-glycosylation sites in AcMNPV gp64 impairs binding of budded virus to the cell, which explains why viruses containing these mutant forms of gp64 produce less infectious progeny.
Jarvis
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Department of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071-3944, USA. djarvis@uwyo.edu
Wills
L
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Burow
G
G
Bohlmeyer
D A
DA
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002459:Cell Fusion; D002460:Cell Line; D005814:Genes, Viral; D006031:Glycosylation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017924:Nucleopolyhedroviruses / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D011134:Polysaccharides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018411:Spodoptera; D014760:Viral Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014774:Virulence / Q000235:genetics
D011134:Polysaccharides; D014760:Viral Fusion Proteins