Improved Display of Synthetic IgG-Binding Domains on the Baculovirus Surface

Abstract

Improved display of foreign protein moieties in combination with beneficial alteration of the viral surface properties should be of value for targeted and enhanced gene delivery. Here, we describe a vector based on Autographa californica multiple nucleopolyhedrovirus (AcMNPV) displaying synthetic IgG-binding domains (ZZ) of protein A fused to the transmembrane anchor of vesicular stomatitis virus (VSV) G protein. This display vector was equipped with a GFP/EGFP expression cassette enabling fluorescent detection in both insect and mammalian cells. The virus construct displayed the biologically active fusion protein efficiently and showed increased binding capacity to IgG. As the display is carried out using a membrane anchor of foreign origin, gp64 is left intact for virus entry, which may increase gene expression in the transduced mammalian cells. In addition, the viral vector can be targeted to any desired cell type via binding of ZZ domains when an appropriate IgG antibody is available.Key words: Baculovirus display, ZZ domains, Protein A, VSV G protein, Targeting, Gene delivery, Gene therapy. IntroductionViral vectors have become important tools in the field of gene therapy and recently baculovirus has become a target of increasing interest also in this context. During the past years, baculoviruses, especially Autographa californica multiple nucleopolyhedrovirus (AcMNPV), have widened their applications from insect pesticides (reviewed by (1)) to protein production (reviewed by (2-4)) and further, by display modifications, to foreign protein (5-7) or antigen presentation (8)(9)(10)(11) and library screening (12). In most cases, the display has been carried out by fusion of the foreign protein to a second copy of the envelope glycoprotein, gp64 (5-12), but in some studies straightly to the only copy of the same protein (13, 14).In addition, baculoviruses are currently evaluated for gene transfer to various cell types, one of the goals being gene therapy applications. A mammalian promoter element such as CMV (15)(16)(17), CAG (18, 19) or RSV LTR (17, 20), is needed for gene expression in the target cells, since baculoviral promoters appear to be silent in mammalian cells (20). The first in vitro studies indicated that baculoviruses preferentially enter hepatocytic cells (15, 20, 21). However, additional studies have shown that baculoviruses can enter multiple cell lines (16)(17)(18)(19) 22). Further, baculoviruses have been tested for gene delivery in vivo, the target organs including rabbit carotid artery (23), mouse eye (24) and rat or mouse brain (25, 26). For more specific targeting, baculoviral vectors can be modified by displaying or pseudotyping the viral surface. By this method, recombinant viruses were shown to specifically bind to certain cell lines (27) As the envelope protein gp64 is necessary for the viral infection (28), fusions to this protein may interfere with the internalization of the virus, although this does not seem to affect virus titers in insect cells. The gp64 protein has an essential...

Truncated vesicular stomatitis virus G protein improves baculovirus transduction efficiency in vitro and in vivo

“…16 The 21 amino-acid ectodomain of VSV-GED may retain these membrane-binding characteristics and thus mediate a stronger interaction with the target cell membrane. 18 This could also explain the difference observed in the transduction pattern in the rat brain of the VSV-GED-pseudotyped virus when compared with the control virus. However, when it is taken into account that baculoviruses are able to enter numerous cell lines, 22,28,29 the reason for the observed enhanced gene expression in vivo is less likely due to the increased virus attachment than augmented endosomal release.…”

“…Previously, it has been demonstrated that the GS-region of VSV-G can be used as a membrane anchor in displaying fusion proteins on baculovirus surface. 17, 18 Yet, a vector displaying a fusion protein of IgG-domain (ZZ) of protein A fused to the VSV-GED did not result in increased transduction efficiency. 18 In the current study, we constructed a baculovirus vector displaying, in addition to gp64, a 21-amino-acid ectodomain in conjunction with the TM and CTD domains of VSV-G (VSV-GED) and studied its effect on the baculovirus infection and transduction rates in insect and vertebrate cells, respectively.…”

“…17, 18 Yet, a vector displaying a fusion protein of IgG-domain (ZZ) of protein A fused to the VSV-GED did not result in increased transduction efficiency. 18 In the current study, we constructed a baculovirus vector displaying, in addition to gp64, a 21-amino-acid ectodomain in conjunction with the TM and CTD domains of VSV-G (VSV-GED) and studied its effect on the baculovirus infection and transduction rates in insect and vertebrate cells, respectively. The resulting virus was efficiently produced and resulted in higher transduction efficiency several folds in HeLa, SKOV-3, HepG2, 293T and BT4C cell lines as compared to the control virus.…”

Ligand-Directed Gene Targeting to Mammalian Cells by Pseudotype Baculoviruses

“…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).…”

Virus-Coated Layer-by-Layer Colloids as a Multiplex Suspension Array for the Detection and Quantification of Virus-Specific Antibodies

“…We propagated Autographa californica nuclear polyhedrosis virus (AcNPV; ATCC); its derivative AcCOPSNo10 (16 ), which displays a 17-amino acid epitope of HIV-1 gp120 N-terminally fused with a second copy of the major envelope protein gp64; and the construct AcZZVSVgTM-EGFP (17 ) in Sf9 (Spodoptera frugiperda; ATCC) with standard methods (18 ). We used modified IPL-41 (16 ) from Sigma-Aldrich as medium.…”