BIOSYNTHESIS AND PROCESSING OF THE AUTOGRAPHA-CALIFORNICA NUCLEAR POLYHEDROSIS-VIRUS GP64 PROTEIN

gp64 is a major virion envelope glycoprotein of the baculovirus Autogr apha californica multicapsid nuclear polyhedrosis virus (AcMNPV). gp64 plays an important role in AcMNPV infection, probably mediating penet ration of one form of the virus into host cells through the endocytic pathway. gp64 also represents an excellent probe for studying the memb rane glycoprotein processing capabilities of baculovirus-infected inse ct cells, which are used widely as a eucaryotic expression system. The goals of this study were to characterize gp64 biosynthesis and proces sing and determine how N-glycosylation and N-linked oligosaccharide pr ocessing influence the fate and function of gp64 in AcMNPV-infected in sect cells. We found that gp64 was synthesized in a biphasic fashion, with peaks at 8 and 24 hr postinfection in both the intracellular and extracellular fractions. Interestingly, the first peak preceded detect able budded virus (BV) production, suggesting that gp64 is shed from i nfected cells early in infection. Transcriptional regulation accounted for the biphasic mode of gp64 protein synthesis, as transcription ini tiated at a consensus early motif during early times of infection, at a late motif during late times of infection, and there was a lag betwe en the peak of early and the onset of late transcription. In vitro tra nscription-translation assays showed that the second ATG in the AcMNPV gp64 long open reading frame is used as the translational initiation codon and that downstream sequences encode a functional signal peptide . Pulse-chase analyses, endoglycosidases, and various inhibitors were used to show that some N-linked oligosaccharides on gp64 are processed by glucosidases and alpha-mannosidases in AcMNPV-infected insect cell s. These experiments also revealed that at least two differentially pr ocessed gp64 glycoforms are produced in these cells and that both can reach the cell surface and assemble into progeny BV. However, N-linked oligosaccharide processing was not required for gp64 cell surface exp ression, its assembly into infectious BV, or its fusogenic activity. T his suggested that any gp64 glycoform produced during infection, regar dless of its N-linked carbohydrate structure, can have essentially nor mal biological properties. By contrast, transport of gp64 to the cell surface, production of infectious BV, and fusogenic activity were redu ced in the absence of N-glycosylation, indicating that this modificati on is necessary for optimal gp64 function. (C) 1994 Academic Press, In c.