Host cell receptor binding by baculovirus GP64 and kinetics of virion entry.
GP64 is the major envelope glycoprotein from budded virions of the baculoviruses Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). To examine the potential role of GP64 as a viral attachment protein in host cell receptor binding, we generated, overexpressed, and characterized a soluble form of the OpMNPV GP64 protein, GP64solOp. Assays for trimerization, sensitivity to proteinase K, and reduction by dithiothreitol suggested that GP64solOp was indistinguishable from the ectodomain of the wild-type OpMNPV GP64 protein. Virion binding to host cells was analyzed by incubating virions with cells at 4 degrees C in the presence or absence of competitors, using a single-cell infectivity assay to measure virion binding. Purified soluble GP64 (GP64solOp) competed with a recombinant AcMNPV marker virus for binding to host cells, similar to control competition with psoralen-inactivated wild-type AcMNPV and OpMNPV virions. A nonspecific competitor protein did not similarly inhibit virion binding. Thus specific competition by GP64solOp for virion binding suggests that the GP64 protein is a host cell receptor-binding protein. We also examined the kinetics of virion internalization into endosomes and virion release from endosomes by acid-triggered membrane fusion. Using a protease sensitivity assay to measure internalization of bound virions, we found that virions entered Spodoptera frugiperda Sf9 cells between 10 and 20 min after binding, with a half-time of approximately 12.5 min. We used the lysosomotropic reagent ammonium chloride to examine the kinetics of membrane fusion and nucleocapsid release from endosomes after membrane fusion. Ammonium chloride inhibition assays indicated that AcMNPV nucleocapsids were released from endosomes between 15 and 30 min after binding, with a half-time of approximately 25 min.
Hefferon
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KL
Boyce Thompson Institute, Cornell University, Tower Road, Ithaca, New York, 14853-1801, USA.
Oomens
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AG
Monsma
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Finnerty
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Blissard
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GW
D000818:Animals; D016367:Baculoviridae / Q000378:metabolism* / Q000502:physiology; D001667:Binding, Competitive; D002460:Cell Line; D004229:Dithiothreitol; D019286:Endopeptidase K / Q000378:metabolism; D007700:Kinetics; D011499:Protein Processing, Post-Translational; D011991:Receptors, Virus / Q000378:metabolism*; D012995:Solubility; D018411:Spodoptera / Q000166:cytology; D014760:Viral Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014771:Virion
D011991:Receptors, Virus; D014760:Viral Fusion Proteins; D019286:Endopeptidase K; D004229:Dithiothreitol