RGD motifs on the surface of baculovirus enhance transduction of human lung carcinoma cells.

Baculovirus vectors have been shown to enter a variety of mammalian cell lines and gene transfer with wild-type baculovirus (WT) has been demonstrated both in vitro and in vivo. Different protein motifs have been displayed on the viral surface to serve as ligands for cell-specific receptor molecules. We have generated recombinant baculovirus vectors displaying an RGD-motif, recognized by alphaV integrin, on the viral surface. The RGD motifs within the C-terminus of coxsackie virus A9 and human parechovirus 1 VP1 proteins were fused to the N-terminus of the major envelope glycoprotein, gp64, of Autographa californica multiple nucleopolyhedrovirus. The recombinant RGD-presenting viruses bound…

Parvovirus capsid disorders cholesterol-rich membranes.

In this study canine parvovirus, CPV, was found to induce disorder in DPPC:cholesterol membranes in acidic conditions. This acidicity-induced fluidizing effect is suggested to originate from the N-terminus of the viral capsid protein VP1. In accordance with the model membrane studies, a fluidizing effect was seen also in the endosomal membranes during CPV infection implying an important functional role of the fluidization in the endocytic entry of the virus.

Desipramine induces disorder in cholesterol-rich membranes:implications for viral trafficking

In this study, the effect of desipramine (DMI) on phospholipid bilayers and parvoviral entry was elucidated. In atomistic molecular dynamics simulations, DMI was found to introduce disorder in cholesterol-rich phospholipid bilayers. This was manifested by a decrease in the deuterium order parameter S(CD) as well as an increase in the membrane area. Disordering of the membrane suggested DMI to destabilize cholesterol-rich membrane domains (rafts) in cellular conditions. To relate the raft disrupting ability of DMI with novel biological relevance, we studied the intracellular effect of DMI using canine parvovirus (CPV), a virus known to interact with endosomal membranes and sphingomyelin, as …

Expression and trafficking of fluorescent viral membrane proteins in baculovirus-transduced BHK cells

Baculovirus vectors show promise as a novel tool for gene delivery into mammalian cells and gene transfer with wild-type baculovirus has been demonstrated both in vitro and in vivo. To study expression and intracellular trafficking of foreign viral membrane proteins in baculovirus-transduced mammalian cells, the envelope proteins, E1 and E2, of rubella virus (RV) were chosen as a model. The enhanced green fluorescent protein (EGFP) and a red fluorescent protein (RFP) were fused to the C-terminus of E1 and E2, respectively. The proteins were cloned under a cytomegalovirus (CMV) promoter and expressed as fluorescent fusion proteins in baculovirus-transduced baby hamster kidney (BHK) cells. Ex…

Tumor targeting of baculovirus displaying a lymphatic homing peptide.

Background Tumor-associated cells and vasculature express attractive molecular markers for site-specific vector targeting. To attain tumor-selective tropism, we recently developed a baculovirus vector displaying the lymphatic homing peptide LyP-1, originally identified by ex vivo/in vivo screening of phage display libraries, on the viral envelope by fusion to the transmembrane anchor of vesicular stomatitis virus G-protein. Methods In the present study, we explored the specificity and kinetics of viral binding and internalization as well as in vivo tumor homing of the LyP-1 displaying virus to elucidate the applicability of baculovirus for targeted therapies. Results We demonstrated that th…

Occlusion-derived baculovirus: interaction with human cells and evaluation of the envelope protein P74 as a surface display platform.

To develop complementary baculovirus-based tools for gene delivery and display technologies, the interaction of occlusion-derived baculovirus (ODV) with human cells, and the functionality of the P74 ODV envelope protein for display of the IgG-binding Z domains (ZZP74) were evaluated. The cellular binding of ODV was concentration-dependent and saturable. Only minority of the bound virions were internalized at both 37 and 4 degrees C, suggesting usage of direct membrane fusion as the entry mode. The intracellular transport of ODV was confined in vesicular structures peripheral to the plasma membrane, impeding subsequent nuclear entry and transgene expression. Transduction of ODV was not rescu…

Molecular and structural characterization of fluorescent human parvovirus B19 virus-like particles

Although sharing a T = 1 icosahedral symmetry with other members of the Parvoviridae family, it has been suggested that the fivefold channel of the human parvovirus B19 VP2 capsids is closed at its outside end. To investigate the possibility of placing a relatively large protein moiety at this site of B19, fluorescent virus-like particles (fVLPs) of B19 were developed. The enhanced green fluorescent protein (EGFP) was inserted at the N-terminus of the structural protein VP2 and assembly of fVLPs from this fusion protein was obtained. Electron microscopy revealed that these fluorescent protein complexes were very similar in size when compared to wild-type B19 virus. Further, fluorescence cor…

The baculovirus display technology--an evolving instrument for molecular screening and drug delivery.

High throughput screening is a core technology in drug discovery. During the past decade, several strategies have been developed to screen (poly)peptide libraries for diverse applications including disease diagnosis and profiling, imaging, as well as therapy. The recently established baculovirus display vector system (BDVS) represents a eukaryotic screening platform that combines the positive attributes of both cell and virus-based display approaches, allowing presentation of complex polypeptides on cellular and viral surfaces. Compared to microbial display systems, the BDVS has the advantage of correct protein folding and post-translational modifications similar to those in mammals, facili…

Baculovirus capsid display: a novel tool for transduction imaging

Baculoviruses are enveloped insect viruses that can carry large quantities of foreign DNA in their genome. Baculoviruses have proved to be very promising gene therapy vectors but little is known about their transduction mechanisms in mammalian cells. We show in this study that Autographa californica multiple nuclear polyhedrosis virus capsid is compatible with the incorporation of desired proteins in large quantities. Fusions can be made to the N-terminus or C-terminus of the major capsid protein vp39 without compromising the viral titer or functionality. As an example of the baculovirus capsid display we show a tracking of the baculovirus transduction in mammalian cells by an enhanced gree…

Baculoviral display of functional scFv and synthetic IgG-binding domains.

Viral vectors displaying specific ligand binding moities such as scFv fragments or intact antibodies hold promise for the development of targeted gene therapy vectors. In this report we describe baculoviral vectors displaying either functional scFv fragments or the synthetic Z/ZZ IgG binding domain derived from protein A. Display on the baculovirus surface was achieved via fusion of the scFv fragment or Z/ZZ domain to the N-terminus of gp64, the major envelope protein of the Autographa californica nuclear polyhedrosis virus, AcNPV. As examples of scFv fragments we have used a murine scFv specific for the hapten 2-phenyloxazolone and a human scFv specific for carcinoembryonic antigen. In pri…

Assembly of fluorescent chimeric virus-like particles of canine parvovirus in insect cells

Canine parvovirus (CPV) is a small non-enveloped ssDNA virus composed of the viral proteins VP1, VP2, and VP3 with a T=1 icosahedral symmetry. VP2 is nested in VP1 and the two proteins are produced by differential splicing of a primary transcript of the right ORF of the viral genome. The VP2 protein can be further proteolytically cleaved to form VP3. Previous studies have shown that VP1 and VP3 are unnecessary for capsid formation and consequently, that VP2 alone is sufficient for assembly. We have hypothesized that insertion of the enhanced green fluorescent protein (EGFP) at the N-terminus of VP2 could be carried out without altering assembly. To investigate the possibility to develop flu…

Baculovirus Display: A Multifunctional Technology for Gene Delivery and Eukaryotic Library Development

For over a decade, phage display has proven to be of immense value, allowing selection of a large variety of genes with novel functions from diverse libraries. However, the folding and modification requirements of complex proteins place a severe constraint on the type of protein that can be successfully displayed using this strategy, a restriction that could be resolved by similarly engineering a eukaryotic virus for display purposes. The quite recently established eukaryotic molecular biology tool, the baculovirus display vector system (BDVS), allows combination of genotype with phenotype and thereby enables presentation of eukaryotic proteins on the viral envelope or capsid. Data have sho…

Clathrin-independent entry of baculovirus triggers uptake of E. coli in non-phagocytic human cells

The prototype baculovirus, Autographa californica multiple nucleopolyhedrovirus, an insect pathogen, holds great potential as a gene therapy vector. To develop transductional targeting and gene delivery by baculovirus, we focused on characterizing the nature and regulation of its uptake in human cancer cells. Baculovirus entered the cells along fluid-phase markers from the raft areas into smooth-surfaced vesicles devoid of clathrin. Notably, regulators associated with macropinocytosis, namely EIPA, Pak1, Rab34, and Rac1, had no significant effect on viral transduction, and the virus did not induce fluid-phase uptake. The internalization and nuclear uptake was, however, affected by mutants o…

Baculovirus entry into human hepatoma cells.

ABSTRACT Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a prototype member of the Baculoviridae family, has gained increasing interest as a potential vector candidate for mammalian gene delivery applications. AcMNPV is known to enter both dividing and nondividing mammalian cell lines in vitro, but the mode and kinetics of entry as well as the intracellular transport of the virus in mammalian cells is poorly understood. The general objective of this study was to characterize the entry steps of AcMNPV- and green fluorescent protein-displaying recombinant baculoviruses in human hepatoma cells. The viruses were found to bind and transduce the cell line efficiently, and electron …

Improved display of synthetic IgG-binding domains on the baculovirus surface.

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 …

Expression and subcellular targeting of canine parvovirus capsid proteins in baculovirus-transduced NLFK cells

AbstractA mammalian baculovirus delivery system was developed to study targeting in Norden Laboratories feline kidney (NLFK) cells of the capsid proteins of canine parvovirus (CPV), VP1 and VP2, or corresponding counterparts fused to EGFP. VP1 and VP2, when expressed alone, both had equal nuclear and cytoplasmic distribution. However, assembled form of VP2 had a predominantly cytoplasmic localization. When VP1 and VP2 were simultaneously present in cells, their nuclear localization increased. Thus, confocal immunofluorescence analysis of cells transduced with the different baculovirus constructs or combinations thereof in the absence or presence of infecting CPV revealed that the VP1 protei…

Monitoring human parvovirus B19 virus-like particles and antibody complexes in solution by fluorescence correlation spectroscopy

AbstractFluorescence correlation spectroscopy (FCS) was used in monitoring human parvovirus B19 virus-like particle (VLP) antibody complexes from acute phase and pastimmunity serum samples. The Oregon Green 488-labeled VLPs gave an average diffusion coefficient of 1.7x10exp-7 cm(2)s(-1) with an apparent hydrodynamic radius of 14 nm. After incubation of the fluorescent VLPs with an acute phase serum sample, the mobility information obtained from the fluorescence intensity fluctuation by autocorrelation analysis showed an average diffusion coefficient of 1.5x10exp-8 cm(2)s(-1), corresponding to an average radius of 157 nm. In contrast, incubation of the fluorescent VLPs with a pastimmunity se…

Properties of baculovirus particles displaying GFP analyzed by fluorescence correlation spectroscopy.

Abstract Recombinant baculovirus particles displaying green fluorescent protein (GFP) fused to the major envelope glycoprotein gp64 of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were characterized by fluorescence correlation spectroscopy (FCS). FCS detected Brownian motion of single, intact recombinant baculovirus display particles with a diffusion coefficient (D) of (2.89±0.74)10 8 cm2s 1 and an apparent hydrodynamic radius of 83.35±21.22 nm. In the presence of sodium dodecyl sulfate (SDS), Triton X-100, and octylglucoside, the diffusion time was reduced to the 0.2 ms range (D = 7.5710 7 cm2s 1), showing that the fusion proteins were anchored in the viral envelope…

Agonist potency differentiates G protein activation and Ca2+ signalling by the orexin receptor type 1.

The G protein coupling characteristics of a flag epitope-tagged orexin receptor type 1 (OX1R) was investigated in HEK293 cells. Immunoprecipitation of the OX1R and immunoblotting revealed interactions with Gq/G11 proteins as well as with Gs and Gi proteins. Stimulation with orexin-A did not affect the ability of the OX1R to coprecipitate Gq/G11 proteins, but it robustly elevated the intracellular concentration of Ca2+, [Ca2+]i. No changes in cAMP levels could be detected upon receptor stimulation. To get further insight into the functional correlation of G protein activation and Ca2+ signalling, we used baculovirus transduction to express chimeric G proteins, containing the Galphas protein …

Functional display of an alpha2 integrin-specific motif (RKK) on the surface of baculovirus particles.

The use of baculovirus vectors shows promise as a tool for gene delivery into mammalian cells. These insect viruses have been shown to transduce a variety of mammalian cell lines, and gene transfer has also been demonstrated in vivo. In this study, we generated two recombinant baculovirus vectors displaying an integrin-specific motif, RKK, as a part of two different loops of the green fluorescent protein (GFP) fused with the major envelope protein gp64 of Autographa californica M nucleopolyhedrovirus. By enzyme linked immunosorbent assays, these viruses were shown to bind a peptide representing the receptor binding site of an α2 integrin, the α2I-domain. However, the interaction was not st…

Improving baculovirus transduction of mammalian cells by surface display of a RGD-motif

An RGD-containing peptide, comprising 23 amino acids from the foot-and-mouth disease virus (FMDV) VP1 protein was engineered into the envelope of Autographa californica nuclear polyhedrosis virus surface (AcNPV) using two different display strategies. The RGD-motif is a well-described tripeptide, that by binding to cell surface integrins facilitates virus entry into cells. This epitope was displayed, either by directly modifying the native major envelope protein gp64 of AcNPV, or by incorporating a second, modified version of gp64 onto the virus surface. Transduction efficiencies of four mammalian cell lines were compared by detecting the expression of the reporter gene green fluorescent pr…

Expression and glycosylation studies of human FGF receptor 4

Fibroblast growth factor receptor subtype 4 (FGFR4) has been shown to have special activation properties and just one splicing form, unlike the other FGFRs. FGFR4 overexpression is correlated with breast cancer and therefore FGFR4 is a target for drug design. Our aim is to overexpress high amounts of homogeneous FGFR4 extracellular domain (FGFR4ed) for structural studies. We show that baculovirus-insect cell-expressed FGFR4ed is glycosylated on three (N88, N234, and N266) of the six possible N-glycosylation sites but is not O-glycosylated. The deglycosylated triple mutant was expressed and had binding properties similar to those of glycosylated FGFR4ed, but was still heterogeneous. Large am…

Peptide-mediated interference with baculovirus transduction

Baculovirus represents a multifunctional platform with potential for biomedical applications including disease therapies. The importance of F3, a tumor-homing peptide, in baculovirus transduction was previously recognized by the ability of F3 to augment viral binding and gene delivery to human cancer cells following display on the viral envelope. Here, F3 was utilized as a molecular tool to expand understanding of the poorly characterized baculovirus-mammalian cell interactions. Baculovirus-mediated transduction of HepG2 hepatocarcinoma cells was strongly inhibited by coincubating the virus with synthetic F3 or following incorporation of F3 into viral nucleocapsid by genetic engineering, th…

Disassembly of structurally modified viral nanoparticles: characterization by fluorescence correlation spectroscopy.

Abstract Analysis of the breakdown products of engineered viral particles can give useful information on the particle structure. We used various methods to breakdown both a recombinant enveloped virus and virus-like particles (VLPs) from two non-enveloped viruses and analysed the resulting subunits by fluorescence correlation spectroscopy (FCS). Analysis of the enveloped baculovirus, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), displaying the green fluorescent protein (GFP) fused to its envelope protein gp64 was performed in the presence and absence of 5 mM SDS and 25 mM DTT. Without treatment, the viral particle showed a diffusion time of 3.3 ms. In the presence of SDS…

Maturation of IgG avidity to individual rubella virus structural proteins.

Background: the structural proteins of rubella virus, the capsid protein C and the envelope glycoproteins E1 and E2 were produced in lepidopteran insect cells using baculovirus expression vectors. The C-terminal ends of the corresponding proteins were fused to a polyhistidine tag for easy and gentle purification by metal ion affinity chromatography. Objectives: to investigate the maturation of natural and vaccinal IgG avidity against individual authentic and recombinant rubella virus (RV) structural proteins. Study design the analysis was carried out using a modified immunoblotting technique where the purified baculovirus-expressed proteins were compared with authentic rubella virus protein…

Method and apparatus using selected superparamagnetic labels for rapid quantification of immunochromatographic tests

Mika PA Laitinen1, Jari Salmela2, Leona Gilbert1, Risto Kaivola1, Topi Tikkala2, Christian Oker-Blom1, Jukka Pekola3, Matti Vuento11Department of Biological and Environmental Science; 2Department of Physics, University of Jyväskylä, Jyväskylä, Finland; 3Low Temperature Laboratory, Helsinki University of Technology, Helsinki, FinlandAbstract: A rapid method and instrumentation for quantification of immunochromatographic tests (ICT) are described. The principle and performance of the method was demonstrated by measuring the levels of human chorionic gonadotropin (hCG) present in urine. The test format was a sandwich assay using two distinct monoclonal antib…

Hydrophobin (HFBI): a potential fusion partner for one-step purification of recombinant proteins from insect cells

Hydrophobins play an important role in binding and assembly of fungal surface structures as well as in medium-air interactions. These, hydrophobic properties provide interesting possibilities when purification of macromolecules is concerned. In aqueous micellar two-phase systems, based on surfactants, the water soluble hydrophobins are concentrated inside micellar structures and, thus, distributed to defined aqueous phases. This, one-step purification is attractive particularly when large-scale production of recombinant proteins is concerned. In the present study the hydrophobin HFBI of Trichoderma reesei was expressed as an N-terminal fusion with chicken avidin in baculovirus infected inse…

Developments in the use of baculoviruses for the surface display of complex eukaryotic proteins

The ability to couple genotype to phenotype has proven to be of immense value in systems such as phage display and has allowed genes encoding novel functions to be selected directly from complex libraries. However, the complexity of many eukaryotic proteins places a severe constraint on successful display in Escherichia coli. This restriction could be resolved if a eukaryotic virus could be similarly engineered for display purposes. Preliminary data have suggested that the baculovirus Autographa californica, a multiple nuclear polyhedrosis virus (AcMNPV) is a candidate for eukaryotic virus display because the insertion of peptides into the native virus coat protein, or the expression of for…

Expression and purification of polyhistidine-tagged firefly luciferase in insect cells

The coleopteran firefly, Photinus pyralis, luciferase was produced in lepidopteran Trichoplusia ni insect cells using a baculovirus expression vector. The recombinant protein was equipped with a polyhistidine affinity tag at the carboxyl terminus and purified by immobilized metal-ion affinity chromatography in combination with an expanded bed adsorption system. This approach enabled an efficient, one-step purification protocol of a genetically modified luciferase with properties similar to those of the authentic counterpart. According to light emission measurements, the final yield of highly purified protein was 23 mg l−1 of cell culture. In addition, no specific interaction of interfering …

Interaction of Neuronal Calcium Sensor-1 (NCS-1) with Phosphatidylinositol 4-Kinase β Stimulates Lipid Kinase Activity and Affects Membrane Trafficking in COS-7 Cells

Phosphatidylinositol 4-kinases (PI4K) catalyze the first step in the synthesis of phosphatidylinositol 4,5-bisphosphate, an important lipid regulator of several cellular functions. Here we show that the Ca(2+)-binding protein, neuronal calcium sensor-1 (NCS-1), can physically associate with the type III PI4Kbeta with functional consequences affecting the kinase. Recombinant PI4Kbeta, but not its glutathione S-transferase-fused form, showed enhanced PI kinase activity when incubated with recombinant NCS-1, but only if the latter was myristoylated. Similarly, in vitro translated NCS-1, but not its myristoylation-defective mutant, was found associated with recombinant- or in vitro translated P…

Production of biologically active recombinant avidin in baculovirus-infected insect cells

Abstract An efficient lepidopteran insect cell system was established for the expression of a recombinant form of chicken egg-white avidin. The gene product was obtained in both secreted and intracellular forms, and biologically active recombinant avidin was isolated using affinity chromatography on an iminobiotin–agarose column. Similar to the known quaternary structure of the native egg-white protein, the purified recombinant protein was glycosylated and assembled mainly into tetramers. Like native avidin, the recombinant tetramer also exhibited a high level of thermostability, and was further stabilized upon binding biotin. The biotin-binding and structural properties of the recombinant …

Purification and analysis of polyhistidine-tagged human parvovirus B19 VP1 and VP2 expressed in insect cells

Human parvovirus B19 is an autonomously replicating human pathogen with a specific tropism for human erythroid progenitor cells. There is an interest in producing empty nucleocapsids of B19 as they can be used as tools in molecular biology and diagnostics. Native B19 virus particles are formed from two structural viral proteins, VP1 and VP2. The VP2 protein alone is able to self assemble and consequently form virus-like particles (VLPs) in heterologous expression systems. Purification of recombinant VLPs has been conducted using various traditional methods. These include laborious and time-consuming, e.g. cesium chloride or sucrose gradient ultracentrifugation steps, allowing limited workin…

T helper cell-mediated interferon-gamma expression after human parvovirus B19 infection: persisting VP2-specific and transient VP1u-specific activity.

SummaryHuman parvovirus B19 is a small non-enveloped DNA virus with an icosahedral capsid consisting of proteins of only two species, the major protein VP2 and the minor protein VP1. VP2 is contained within VP1, which has an additional unique portion (VP1u) of 227 amino acids. We determined the ability of eukaryotically expressed parvovirus B19 virus-like particles consisting of VP1 and VP2 in the ratio recommended for vaccine use, or of VP2 alone, to stimulate, in an HLA class II restricted manner, peripheral blood mononuclear cells (PBMC) to proliferate and to secrete interferon gamma (IFN-γ) and interleukin (IL)-10 cytokines among recently and remotely B19 infected subjects. PBMC reactiv…

Endosomal escape of canine parvovirus is assisted by membrane fluidization

Baculovirus display strategies: Emerging tools for eukaryotic libraries and gene delivery

Recombinant baculoviruses have been extensively used as vectors for abundant expression of a large variety of foreign proteins in insect cell cultures. The appeal of the system lies essentially in easy cloning techniques and virus propagation combined with the eukaryotic post-translational modification machinery of the insect cell. Recently, a novel molecular biology tool was established by the development of baculovirus surface display, using different strategies for presentation of foreign peptides and proteins on the surface of budded virions. This eukaryotic display system enables presentation of large complex proteins on the surface of baculovirus particles and has thereby become a ver…

Enhanced baculovirus-mediated transduction of human cancer cells by tumor-homing peptides.

ABSTRACT Tumor cells and vasculature offer specific targets for the selective delivery of therapeutic genes. To achieve tumor-specific gene transfer, baculovirus tropism was manipulated by viral envelope modification using baculovirus display technology. LyP-1, F3, and CGKRK tumor-homing peptides, originally identified by in vivo screening of phage display libraries, were fused to the transmembrane anchor of vesicular stomatitis virus G protein and displayed on the baculoviral surface. The fusion proteins were successfully incorporated into budded virions, which showed two- to fivefold-improved binding to human breast carcinoma (MDA-MB-435) and hepatocarcinoma (HepG2) cells. The LyP-1 pepti…

Specific Binding of Baculoviruses Displaying gp64 Fusion Proteins to Mammalian Cells

Viral vectors displaying specific ligand binding moieties have raised an increasing interest in the area of targeted gene therapy. In this report, we describe baculovirus vectors displaying either a functional single chain antibody fragment (scFv) specific for the carcinoembryonic antigen (CEA) or the synthetic IgG binding domains (ZZ) derived from protein A of Staphylococcus aureus. In addition, the vectors were engineered to incorporate a reporter gene encoding the enhanced green fluorescent protein (EGFP) under the transcriptional regulation of the cytomegalovirus (CMV) IE promoter. Display of the targeting moieties on the viral surface was achieved through fusion to the N-terminus of gp…