Biosynthesis and processing of the Autographa californica nuclear polyhedrosis virus gp64 protein.
gp64 is a major virion envelope glycoprotein of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). gp64 plays an important role in AcMNPV infection, probably mediating penetration of one form of the virus into host cells through the endocytic pathway. gp64 also represents an excellent probe for studying the membrane glycoprotein processing capabilities of baculovirus-infected insect cells, which are used widely as a eucaryotic expression system. The goals of this study were to characterize gp64 biosynthesis and processing and determine how N-glycosylation and N-linked oligosaccharide processing influence the fate and function of gp64 in AcMNPV-infected insect 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 detectable budded virus (BV) production, suggesting that gp64 is shed from infected cells early in infection. Transcriptional regulation accounted for the biphasic mode of gp64 protein synthesis, as transcription initiated at a consensus early motif during early times of infection, at a late motif during late times of infection, and there was a lag between the peak of early and the onset of late transcription. In vitro transcription-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 cells. These experiments also revealed that at least two differentially processed 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 expression, its assembly into infectious BV, or its fusogenic activity. This suggested that any gp64 glycoform produced during infection, regardless of its N-linked carbohydrate structure, can have essentially normal biological properties. By contrast, transport of gp64 to the cell surface, production of infectious BV, and fusogenic activity were reduced in the absence of N-glycosylation, indicating that this modification is necessary for optimal gp64 function.
Department of Entomology, Texas A&M University, College Station 77843.
D L
DL
Jarvis
A
A
Garcia
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D006031:Glycosylation; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D018411:Spodoptera; D014760:Viral Fusion Proteins; D014763:Viral Matrix Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D009844:Oligosaccharides; D014760:Viral Fusion Proteins; D014763:Viral Matrix Proteins