Specific Binding of Baculoviruses Displaying gp64 Fusion Proteins to Mammalian Cells

Ojala, Kirsi; Mottershead, David G.; Suokko, Aki; Oker‐Blom, Christian

doi:10.1006/bbrc.2001.5048

Cited by 64 publications

(46 citation statements)

References 23 publications

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“…Although modification of the virion surface enhances the efficiency of gene transduction into a variety of cell lines, the utility of recombinant baculoviruses in cell-type-specific gene transduction is still unsatisfactory. Ojala et al demonstrated that, while baculoviruses bearing either a single chain antibody fragment specific for carcinoembryonic antigen or a synthetic immunoglobulin G (IgG) binding domain derived from protein A could specifically bind target cells, cell type-specific gene transduction was unsuccessful (44,45). Although gp64-null pseudotype baculoviruses expressing a foreign viral envelope protein, such as VSVG or fusion envelope glycoproteins from other baculoviruses, exhibited high infectivity to insect cells, their capacity for gene transduction into mammalian cells has yet to be explored (33,34).…”

mentioning

confidence: 99%

Ligand-Directed Gene Targeting to Mammalian Cells by Pseudotype Baculoviruses

Kitagawa

Tani

Lim

et al. 2005

J Virol

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confidence: 99%

Ligand-Directed Gene Targeting to Mammalian Cells by Pseudotype Baculoviruses

Kitagawa

Tani

Lim

et al. 2005

J Virol

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“…These findings have generated interest in the use of AcMNPV as a safe viral vector for gene delivery in humans (24). Recently, specific targeting of AcMNPV to mammalian cell types was demonstrated by fusing ligands to the N terminus or transmembrane domain of GP64, and it was shown that GP64 could mediate entry of a lentivirus into mammalian cell types (30,41). GP64 is substantially less cytotoxic than the vesicular stomatitis Indiana virus (VSIV) G protein that has been used in many pseudotyping approaches (22,30).…”

mentioning

confidence: 99%

“…GP64 is substantially less cytotoxic than the vesicular stomatitis Indiana virus (VSIV) G protein that has been used in many pseudotyping approaches (22,30). The GP64 protein has also become a model for fusion pore formation and is being exploited for eukaryotic surface display applications (5,11,14,15,18,28,37,41,46,47). However, in spite of the attractive features of GP64 and a range of proposed practical applications, GP64 expression and function in mammalian cells have been only minimally explored.…”

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confidence: 99%

The Baculovirus GP64 Protein Mediates Highly Stable Infectivity of a Human Respiratory Syncytial Virus Lacking Its Homologous Transmembrane Glycoproteins

Oomens

Wertz

2004

J Virol

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Baculovirus GP64 is a low-pH-dependent membrane fusion protein required for virus entry and cell-to-cell transmission. Recently, GP64 has generated interest for practical applications in mammalian systems. Here we examined the membrane fusion function of GP64 from Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressed in mammalian cells, as well as its capacity to functionally complement a mammalian virus, human respiratory syncytial virus (HRSV). Both authentic GP64 and GP 64/F , a chimeric protein in which the GP64 cytoplasmic tail domain was replaced with the 12 C-terminal amino acids of the HRSV fusion (F) protein, induced low-pH-dependent cell-cell fusion when expressed transiently in HEp-2 (human) cells. Levels of surface expression and syncytium formation were substantially higher at 33°C than at 37°C. The open reading frames (ORFs) encoding GP64 or GP 64/F , along with two marker ORFs encoding green fluorescent protein (GFP) and ␤-glucuronidase (GUS), were used to replace all three homologous transmembrane glycoprotein ORFs (small hydrophobic SH, attachment G, and F) in a cDNA of HRSV. Infectious viruses were recovered that lacked the HRSV SH, G, and F proteins and expressed instead the GP64 or GP 64/F protein and the two marker proteins GFP and GUS. The properties of these viruses, designated RS⌬SH,G,F/GP64 or RS⌬SH,G,F/GP 64/F , respectively, were compared to a previously described HRSV expressing GFP in place of SH but still containing the wild-type HRSV G and F proteins (RS⌬SH [A. G. Oomens, A. G. Megaw, and G. W. Wertz, J. Virol., 77:3785-3798, 2003]). By immunoelectron microscopy, the GP64 and GP 64/F proteins were shown to incorporate into HRSV-induced filaments at the cell surface. Antibody neutralization, ammonium chloride inhibition, and replication levels in cell culture showed that both GP64 proteins efficiently mediated infectivity of the respective viruses in a temperature-sensitive, low-pH-dependent manner. Furthermore, RS⌬SH,G,F/GP64 and RS⌬SH,G,F/GP 64/F replicated to higher levels and had significantly higher stability of infectivity than HRSVs containing the homologous HRSV G and F proteins. Thus, GP64 and a GP64/HRSV F chimeric protein were functional and efficiently complemented an unrelated human virus in mammalian cells, producing stable, infectious virus stocks. These results demonstrate the potential of GP64 for both practical applications requiring stable pseudotypes in mammalian systems and for studies of viral glycoprotein requirements in assembly and pathogenesis.

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“…Baculovirus display allows the presentation of complex proteins following the eukaryotic posttranslational processing and modification of insect cells. Reportedly, scFv fragments have been successfully displayed in a functional form on the AcNPV surface by fusion to gp64 (Mottershead et al 2000;Ojala et al 2001). However, there would be little advantage to the use of baculoviruses displaying scFv fragments for the selection of specific antibodies, because scFv phage displays have been successfully used.…”

Section: Introductionmentioning

confidence: 99%

Baculovirus display of functional antibody Fab fragments

et al. 2015

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The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an antigp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

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Specific Binding of Baculoviruses Displaying gp64 Fusion Proteins to Mammalian Cells

Cited by 64 publications

References 23 publications

Ligand-Directed Gene Targeting to Mammalian Cells by Pseudotype Baculoviruses

Ligand-Directed Gene Targeting to Mammalian Cells by Pseudotype Baculoviruses

The Baculovirus GP64 Protein Mediates Highly Stable Infectivity of a Human Respiratory Syncytial Virus Lacking Its Homologous Transmembrane Glycoproteins

Baculovirus display of functional antibody Fab fragments

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