Functional and structural analysis of GP64, the major envelope glycoprotein of the Budded Virus phenotype of Autographa californica and Orgyia pseudotsugata Multicapsid Nucleopolyhedroviruses

The Baculoviridae are a family of large, enveloped, double-stranded DNA viruses, that cause severe disease in the larvae of mostly lepidopteran insects. Baculoviruses have been studied with the aim of developing alternatives to chemical pest control, and later for their potential as systems for foreign gene expression and to address fundamental virological questions. The Baculoviridae are divided into two genera: the Granuloviruses (GVs) and the Nucleopolyhedroviruses (NPVs). The viruses studied in this thesis, Autographa californica , Orgyia pseudotsugata and Anticarsia gemmatalis Multicapsid Nucleopolyhedroviruses (AcMNPV, OpMNPV and AgMNPV), fall within the NPV genus. A striking characteristic of the NPVs is the presence of two different phenotypes with distinct roles in the infection cycle.Important differences between these two phenotypes are the lipid and protein compositions of their envelopes and the type of host cells they infect. Budded virus (BV) is the phenotype responsible for systemic infection of the insect host, and GP64 is the major envelope glycoprotein of this phenotype. GP64 is absent from the envelope of the other phenotype, the occlusion-derived virus (ODV), which serves to initiate baculovirus infection in midgut epithelial cells. GP64 was shown to be a glycosylated, phosphorylated, and acylated protein, present at early and late stages of the infection cycle. Based on estimations of mass on non-reducing SDS-PAGE, GP64 was speculated to exist as both trimeric and tetrameric forms. Several observations contributed to the belief that GP64 was important for BV entry into the host cell by endocytosis: 1) antibodies to GP64, as well as lipophilic amines, inhibited BV infectivity at a step beyond virus binding to host cells. 2) GP64 induced low-pH mediated membrane fusion when expressed alone at the surface of cells. 3) a monoclonal antibody to GP64 inhibited this low pH-induced membrane fusion capacity. Because of GP64's presence at sites where viral budding occurred, roles for GP64 in virus exit were suspected, but never demonstrated. In this thesis, structural and functional aspects of GP64 were examined with the aim to expand our knowledge about the role of GP64 in BV exit/transmission and entry, within the baculovirus infection cycle.In the first experimental chapter (chapter 2), an anti-GP64 antiserum was generated, and used to examine the kinetics of OpMNPV GP64 synthesis, oligomerization, and carbohydrate processing. Immunoprecipitation, pulse label, and pulse-chase analyses showed that OpMNPV GP64 is produced during a large part of the infection cycle, and undergoes a rapid but inefficient oligomerization step. While carbohydrate addition was rapid, carbohydrate processing occurred with half-times of 45 to 75 min and appeared to be the rate-limiting step in maturation of GP64. Mass spectrometry of a highly purified, soluble form of OpMNPV GP64 revealed that two high molecular weight forms of GP64, typically observed on non-reducing gels, are both homotrimers. The different forms may be the result of differential disulfide bonding.Next, the role of GP64 in the infection cycle was addressed by examining AcMNPV infectivity in the complete absence of GP64 (chapter 3). Because GP64 was suspected to be an essential BV protein, OpMNPV GP64-expressing Sf9 cells were used to complement propagation of an AcMNPV recombinant gp64null virus (vAc 64Z ). This gp64null virus was able to propagate in insect cells when GP64 was complemented. However, by detection of an included lacZ marker gene and comparison to wild-type AcMNPV, vAc 64Z failed to transmit from cell-to-cell in the absence of GP64 complementation, both in cell culture as in Trichoplusia ni larvae. Thus, this result demonstrated a critical role for GP64 in virus transmission.Whether the block in virus transmission was due a defect in BV production, or to production of non-infectious BV, was the next question we sought to address (chapter 4). A budding assay was designed to compare BV budding in the presence (wild-type AcMNPV) or absence of GP64 (gp64null virus). A second generation gp64null virus (vAc 64- ) was generated and used for these studies. In the absence of GP64, budding was reduced by approximately 98%. Hence, the earlier observed block in viral transmission was likely and predominantly a result of decreased virus budding. Because the highly charged GP64 cytoplasmic tail domain (CTD) is predicted to be in a position to interact with cytoplasmic viral and/or cellular factors, it became the next target in the pursuit of the role of GP64 in BV budding. Recombinant viruses carrying deletions or modifications in the GP64 CTD and/or portions of the transmembrane (TM) domain were generated and examined by the earlier developed budding assay. The complete, seven amino acid, predicted CTD was found to be dispensable for propagation of AcMNPV in cell culture, for GP64 surface localization, and low pH-induced membrane fusion. Virus budding however was reduced by approximately 50%, and incorporation of GP64 into virions by 63%, thus indicating that the presence of the CTD domain may confer an advantage. Removal of 11 and 14 amino acids resulted in more dramatic effects. Membrane-anchoring of these GP64s was significantly affected, and viral budding was 4-22% and 2% respectively compared to a virus with a wild-type GP64. The virus carrying a truncation of 14 amino acids was unable to propagate efficiently in Sf9 cells, indicating a possible role for amino acids -12, -13, and -14 from the C-terminus. A recombinant virus, vAc-CΔ3Ra, in which all arginines were replaced with alanines, was in all measurable aspects indistinguishable from virus carrying a wild-type GP64, demonstrating that the CTD charge is not essential.In chapter 5, the role of GP64 in BV binding and entry into host cells was examined by a single cell infectivity assay. In competition studies, inactivated virus as well as a highly purified soluble form of OpMNPV GP64, competed with AcMNPV during a 1 hour viral adsorption period at 4°C, resulting in a reduced number of infected Sf9 cells. This suggests that AcMNPV GP64 is a host cell receptor binding protein. In addition, entry kinetics of the BV phenotype were examined. After binding, BV entered Sf9 cells with a half-time of approximately 12.5 min, and virions were released from endosomes with a half-time of approximately 25 min.The last experimental chapter (chapter 6) describes the mapping and analysis of the gp 64 genomic region in baculovirus Anticarsia gemmatalis Multicapsid Nucleopolyhedrovirus (AgMNPV). This virus is extensively utilized in the protection of soybean crop in Brazil, and demonstrates well the baculovirus potential for insect pest control. To examine the relatedness of AgMNPV to other NPVs, the gp64 genomic region of AgMNPV (containing 19 ORFs of ≥50 amino acids) was analyzed and compared to the corresponding regions in four other baculoviruses. In addition, a multiple alignment of GP64s from seven baculoviruses and envelope proteins of two orthomyxoviruses was performed, demonstrating that 1) baculovirus GP64s are highly conserved (74.5 to 78.6% amino acid identity, 2) among baculoviruses, AgMNPV GP64 is the least conserved in the TM domain and its CTD is truncated, 3) Between baculovirus GP64 and Thogoto/Dhori virus envelope proteins, clusters of amino acid conservation can be identified, which may be useful in designing approaches to map key functional domains of GP64.Finally, because virus budding is a poorly understood area of baculovirology, the general discussion includes a review of selected literature regarding the role of viral spike proteins in exit of enveloped viruses from host cells. In addition, a small selection of GP64 research topics that are not addressed experimentally in this thesis, are included to complement our understanding of GP64. Next, evolutionary implications of the various experimental results are discussed with regard to the significance of the GP64 protein, and the relationship between GP64 and Thogoto and Dhori virus (Orthomyxoviridae) envelope proteins. The discussion is concluded with a note on what the current avenues of GP64 research are and what these may lead to in the future.<br/

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

AbstractGP64 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°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