MUTATIONAL ANALYSIS OF THE N-LINKED GLYCANS ON AUTOGRAPHA-CALIFORNICANUCLEOPOLYHEDROVIRUS GP64

gp64 is the major envelope glycoprotein in the budded form of Autograp ha californica multicapsid nucleopolyhedrovirus (AcMNPV). gp64 is esse ntial for AcMNPV infection, as it mediates penetration of budded virus into host cells via the endocytic pathway. In this study, we used sit e-directed mutagenesis to map the positions of the N-linked glycans on AcMNPV gp64, characterize their structures, and evaluate their influe nce on gp64 function. We found that four of the five consensus N-glyco sylation 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 loca ted at different positions are processed to various degrees. Lectin bl otting analyses showed that each N-linked glycan on gp64 contains alph a-linked mannose, all but one contains alpha-linked fucose, and none c ontains detectable beta-linked galactose or alpha 2,6-linked sialic ac id. The amounts of infectious progeny produced by AcMNPV mutants lacki ng one, two, or three N-linked glycans on gp64 were about 10- to 100-f old lower than wild-type levels. This reduction did not correlate with reductions in the expression, transport, or inherent fusogenic activi ty of the mutant gp64s or in the gp64 content of mutant budded virus p articles. However, all of the mutant viruses bound more slowly than th e wild type. Therefore, elimination of one or more N-glycosylation sit es in AcMNPV gp64 impairs binding of budded virus to the cell, which e xplains why viruses containing these mutant forms of gp64 produce less infectious progeny.