Host Cell Receptor Binding by Baculovirus GP64 and Kinetics of Virion Entry

Abstract

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.

A Discrete Stage of Baculovirus GP64-mediated Membrane Fusion

“…This protein is both necessary and sufficient for mediating pH-dependent membrane fusion (Blissard and Wenz, 1992), is essential for cell-to-cell transmission of the enveloped virion (Monsma et al, 1996), and has host cell receptor-binding activity (Hefferon et al, 1999). Mutagenesis and anti-peptide antibody studies identified a putative hydrophobic membrane fusion peptide between amino acids 220 and 230 of the highly homologous Orgyia pseudotsugata MNPV (OpMNPV) GP64 protein .…”

“…GP64 is temporally expressed during both the early and late phases of viral infection (Blissard and Rohrmann, 1989;Jarvis and Garcia, 1994;Kogan et al, 1994;Oomens et al, 1995;Garrity et al, 1997) and oligomerizes to form two electrophoretically distinct trimers (Oomens et al, 1995;Markovic et al, 1998). This protein is both necessary and sufficient for mediating pH-dependent membrane fusion (Blissard and Wenz, 1992), is essential for cell-to-cell transmission of the enveloped virion (Monsma et al, 1996), and has host cell receptor-binding activity (Hefferon et al, 1999). Mutagenesis and anti-peptide antibody studies identified a putative hydrophobic membrane fusion peptide between amino acids 220 and 230 of the highly homologous Orgyia pseudotsugata MNPV (OpMNPV) GP64 protein .…”

Recent Advances in Our Knowledge of Baculovirus Molecular Biology and Its Relevance for the Registration of Baculovirus-Based Products for Insect Pest Population Control

“…Insect cells have different receptors for GP64 and Fprotein and these two proteins appear to act separately (Hefferon et al, 1999;Westenberg et al, 2007;Wickham et al, 1999). GP64 tropism is so broad in fact, that BVs from AcMNPV and other baculovirus species such as BmNPV have been shown to be taken up by numerous non-lepidopteran cell lines including mammalian and dipteran cell lines (Carbonell et al, 1985;Shoji et al, 1997).…”

Open reading frame Bm21 of Bombyx mori nucleopolyhedrovirus is not essential for virus replication in vitro, but its deletion extends the median survival time of infected larvae

“…Many of the unique group I genes have been characterized to some extent. For example, three genes encode structural proteins: PTP1 is present in both the budded virus (BV) and occlusion-derived virus (ODV) (Li & Miller, 1995), and can induce enhanced locomotory activity in infected larvae (Kamita et al, 2005); BV/ODV-E26 is a structural protein of the envelope of BV and ODV (Beniya et al, 1998), and is a palmitoylated multifunctional structural protein associated with DNA binding (Burks et al, 2007) and co-localizes with IE1 (Imai et al, 2004); and gp64 encodes a viral envelope fusion protein that is responsible for viral attachment, fusion and egress (Blissard & Wenz, 1992;Hefferon et al, 1999;. Two of the unique group I genes are involved in viral DNA replication and gene expression: ie2 encodes a protein that functions as an activator of viral gene expression (Carson et al, 1988;Yoo & Guarino, 1994a, b;Shippam-Brett et al, 2001;Mainz et al, 2002) and is involved in viral DNA replication (Kool et al, 1994;Lu & Miller, 1995) and arrest of the cell cycle (Prikhod'ko & Miller, 1998), whilst gta has been suggested to function in opening up viral chromatin and to aid in the binding of virus-specific transcription factors to viral DNA based on their sequence homology to a family of DNA-stimulated Open reading frame 21 of BmNPV (Bm21) is a homologue of ac30, one of the six uncharacterized unique genes of group I NPVs.…”