title: Eukaryotic virus display: engineering the major surface glycoprotein of the Autographa californica nuclear polyhedrosis virus (AcNPV) for the presentation of foreign proteins on the virus surface.
abstract: We describe the development of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) as a vector for the display of distinct proteins on the viral surface in a manner that is analogous to the established bacterial "phage display" systems. As a model system, the marker gene encoding the 26kDa protein glutathione-S-transferase (GST) was used to construct several fusions with the major baculovirus glycoprotein gp64 gene. Following expression in Spodoptera frugiperda (Sf9) cells, the yield and cellular distribution of each GST-gp64 protein was assessed by Western blot of both cell and supernatant fractions. One fusion, in which GST was inserted between the leader peptide and the nature protein, was found to be efficiently secreted into the cell medium. In the context of expression of the full length gp64, the hybrid GST-gp64 was shown by immunogold labelling to be incorporated onto the virion surface. In addition, the affinity purification of the soluble transmembrane gp64-GST fusion protein resulted in the co-purification of wild type gp64 suggesting that co-oligomerization of the GST-tagged fusion and the wild type molecule was the basis for virion incorporation. The HIV major surface glycoprotein, gp120 was also efficiently displayed in functional form on the viral surface following fusion to the amino terminus of gp64. A general expression vector, pAcSurf-2, was constructed in which multiple cloning sites were positioned in-phase between the gp64 signal sequence and the sequence encoding the mature protein under the control of the polyhedrin promoter.
authors.affiliation: NERC Institute of Virology, Oxford, UK.
authors.forename: Y
authors.initials: Y
authors.lastname: Boublik
authors.forename: P
authors.initials: P
authors.lastname: Di Bonito
authors.forename: I M
authors.initials: IM
authors.lastname: Jones
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015704:CD4 Antigens / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D017353:Gene Deletion; D015870:Gene Expression; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics; D015699:HIV Envelope Protein gp120 / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics* / Q000648:ultrastructure; D019151:Peptide Library; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D018411:Spodoptera / Q000378:metabolism; D014760:Viral Fusion Proteins / Q000235:genetics*
chemical_list: D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D014760:Viral Fusion Proteins; D005982:Glutathione Transferase