Gilbert, L., Toivola, J., Lehtomäki, E., Käpylä, P., Vuento, M., and Oker-Blom, C.

Canine parvoviruses (CPV) are small autonomous, non-enveloped T = 1 icosahedral particles that are composed of viral protein (VP) VP1, VP2 and VP3. VP2 is an N-terminal truncated form of VP1 that could be further proteolytically cleaved to VP3. Earlier studies have shown that VP1 and VP3 are unnecessary for capsid formation and VP2 alone is sufficient for capsid formation, but its N-terminal spatial position has not been assigned due to its disordered structure. We have hypothesized that an insert of enhanced green fluorescent protein (EGFP) at the amino end of VP2 could be fused without altering the auto assembly nature of VP2. To investigate the possibility to develop virus like particles (VLPs) from fluorescent chimeric VP2 proteins, a baculovirus expression vector system (BEVS) was utilized. Immuno-blot analysis of purified fluorescent VLPs indicated that the chimeric proteins were expressed. Confocal and electron microscopy indicated that VP2 could facilitate partnership at its amino terminal that does not disrupt its capsid formation. Similarly, electron microscopy and fluorescence correlation spectroscopy (FCS) studies attest that EGFP is on the VLP. Here we have developed protocols to develop nano-virions that carry a heterologous entity that could be utilized as a visualization tool to elucidate the enigma surrounding canine parvovirus viral infections.

Nuclear and cytosolic localization of VP1-EGFP in BHK cells.
(DIC Differential Interface Contrast)

Nuclear and cytosolic localization of VP1 in NLFK cells.

Cytosolic localization of VP2 in Sf9 cells.

Heli Matilainen, Reetta Riikonen, Taija Saloniemi, Timo Hyypiä, Jyrki Heino and Christian Oker-Blom.

The use of baculovirus vectors shows promise as a new tool for gene delivery into mammalian cells. These insect viruses have been shown to enter different mammalian cell lines and gene transfer with wild-type baculovirus has also been demonstrated in vivo. To enhance mammalian cell transduction and specificity, different protein motifs have been displayed on the viral surface to serve as ligands for cell-specific receptor molecules. In this study, we generated several recombinant baculovirus vectors displaying an integrin specific motif, RGD, on the viral surface. The RGD motifs within the C-terminus of coxsackievirus A9 (CAV9) or human parechovirus 1 (HPEV1) VP1 protein were fused to the N-terminus of the major envelope glycoprotein, gp64, of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Recombinant RGD presenting viruses seemed to bind the A549 (human lung carcinoma) cell surface, known to contain RGD binding alpha(v)beta3-integrin ( v 3), more efficiently as compared to wild type (WT) baculoviruses. In addition, the binding pattern of the RGD display baculoviruses showed extensive clustering, which was not seen with the wild type baculovirus. This might represent clustering of the V 3 integrin molecules on the surface of these cells induced by binding of the RGD displaying baculoviruses. In conclusion, these results suggest that the RGD motif is functional on the surface of these viruses and thereby may enhance the transduction efficiency of v 3 integrin expressing mammalian cells.

Kirsi Ojala

Baculoviruses are enveloped, double stranded DNA viruses which infect mainly insects such as those of the order Lepidoptera. Baculoviruses are pathogenic to insects but are very species specific and non-pathogenic to humans, since no virus replication in mammalian cells can be detected. Most of the baculoviral studies employ Autographa californica multiple nucleopolyhedrovirus virus (AcNPV) which expresses an important envelope glycoprotein, gp64, on the surface of budded form of the virus. Foreign peptides and proteins can be fused to this protein and thus expressed, i.e. displayed on the virus surface. As a eukaryotic system, post-transcriptional modification of the expressed proteins can be achieved using the baculovirus-insect cell system. This is not possible e.g. in phage display system. The modifications enable the surface expression of complex, eukaryotic proteins which are processed and folded correctly.
In this research, various baculoviral display vectors have been developed and tested for targeting and gene transfer to mammalian cells. For efficient targeting, the proteins that are displayed are chosen so that they bind to the plasma membrane of desired cell types and in this way the viruses are internalized. We have developed numerous recombinant baculovirus display vectors which have different proteins displayed on their surface and have also a fluorescent expression cassette inserted into the genome. This cassette enables detection of transduced mammalian cells since the fluorescent marker gene, enhanced green fluorescent protein (EGFP), is under transcriptional regulation of cytomegalovirus immediate early (CMV-IE) promoter. The displayed proteins include rubella virus (RV) envelope proteins E1 and E2, single-chain antibody (scFv) specific for carcinoembryonic antigen (CEA) and synthetic IgG -binding domains (ZZ domains) of protein A. These recombinant viruses are studied for their binding and transduction efficiency in mammalian cell lines such as BHK, PC-3 and CHO. Especially the viruses displaying scFvCEA or ZZ domains have proven to be functional in binding assays. The scFvCEA displaying virus can be targeted to cells that contain CEA on their surface. This antigen is especially expressed on the surface of many tumor cells. In contrast, the ZZ displaying virus binds to IgG class molecules and can therefore be targeted to any cell type if an antibody specific for a protein on the cell membrane is available. Since the proteins are in most cases expressed as a fusion to a second copy of the gp64, only few fusion proteins are displayed on the virus surface. For more efficient display, a novel vector was developed in which the ZZ domains were fused to a transmembrane domain of vesicular stomatitis virus (VSV) G protein. This led to enhanced display of the ZZ domains and improved binding properties compared to the gp64 fusion. This baculoviral vector is currently under investigation for targeting to mammalian cells.
The goal of this research is to develop viral vectors for gene therapy applications. Since baculoviruses are still quite new tools in the gene therapy field, the research gives significant new information about the efficiency and safety of baculoviruses as gene delivery vehicles. The use of gene therapy as an alternative method for treatment of inherited and acquired diseases, such as cancer and cardiovascular diseases, increases rapidly and for this reason the development of novel vectors is important.