Genetic engineering of the major envelope glycoprotein of a baculovirus
Creators: name: Walton, Kelly Louise
Publisher: name: Swinburne
Year: 2024

description: Baculoviruses are large double-stranded DNA viruses that infect insects. Gp64 is the major envelope glycoprotein of the Budded Virus (BV) form of the AcMNPV baculovirus. Gp64 is responsible for cell-to-cell transmission of BV in cell culture and in vivo and mediates binding and entry of BV by pH dependant membrane fusion. Although an oligomerisation and a fusion domain have been identified in gp64, the exact mechanism by which gp64 mediates viral entry has not been determined and no crystal structure for gp64 exists. To assist further studies on the structural and functional relationships of gp64 in the infection cycle, a Gp64 replacement system was developed that allowed mutation of native gp64 sequences. A recombinant AcMNPV virus (BUD1) was constructed that contained two unique Bsu36I sites flanking the gp64 ORF and a polyhedron driven GFP reporter gene. The unique restriction sites allowed gp64 to be removed from this virus and replaced with mutated gp64 sequences, donated from a plasmid transfer vector. The gp64 replacement system enabled the identification of a novel permissive site in AcMNPV gp64 between Asp-295 and Thr-296. A transposon based system was employed to introduce single copies of 19 amino acids randomly into gp64 sequence targets. The baculovirus with a transposon insertion at Asp-295 in gp64 (Bac295-T) was isolated after a co-transfection with plasmid transfer vectors carrying the mutated gp64 ORF’s and BUD1 viral DNA in Sf-21 cells. The BUD1 gp64-replacement system was also used to construct recombinant baculoviruses with motif insertions in native gp64. Bac283R9 was engineered with a polypeptide of 9 arginine residues into the native gp64 ORF after Trp-283, a site previously demonstrated to tolerate the insertion of up to 23 amino acids. The effect of the gp64-R9 fusion protein on baculoviral entry into mammalian cells was examined in COS-7 kidney cells. The Bac283R9 and Bac295-T viruses both demonstrated lower titers and smaller plaques relative to a virus with native gp64. The inserted peptides appeared to have disrupted the structure and/or functions of gp64. Western blot analysis revealed that the amount of gp64 on the surface of Bac295-T BV particles and in infected host cells was comparable to baculovirus with native gp64, suggesting the transposon insertion did not significantly disrupt the oligomerisation and transportation of gp64 to the cell surface. Bac283R9 however, contained less gp64 on the surface of BV particles, relative to BV with native gp64. The polyarginine motif in Bac283R9 gp64 had introduced a protease cleavage site, as confirmed by protease inhibitors. Protease inhibitors were able to reduce the cleavage of Bac283R9 gp64, but no increase in the amount of full length gp64 was observed. At the concentration of inhibitor required for complete inhibition the viral yield was significantly reduced. The introduced R9 motif promoted proteolysis of gp64, and this was likely to be interfering with the formation of gp64 structures and subsequent transportation of gp64 to the cell surface.