Polyoma Virus Pseudocapsids as Efficient Carriers of Heterologous DNA into Mammalian Cells

Abstract

Polyoma virus VP1 pseudocapsids, generated from a recombinant baculovirus, have been successfully used to transfer exogenous DNA stably into rodent (rat-2) cells. To evaluate the efficiency and biological usefulness of this route for introducing heterologous DNA into cells, the gene for a transforming deletion mutant of the middle T antigen of polyoma virus, dl8 MT, was used initially. Whereas the amount of DNA packaged together with pseudocapsids was found to be variable (2-30%), even at low efficiency its transfer as biologically functional information was high. The dl8 MT gene was stably transferred and integrated in low copy numbers into the host chromosome. Transformed cell lines (derived from single foci) were shown to produce high levels of the corresponding mutant protein, which was active in an in vitro protein kinase assay. In comparisons with the calcium phosphate DNA coprecipitation procedure (or lipofectin route), the VP1 pseudocapsid approach was shown to have many advantages in terms of maintenance of DNA fidelity and increased efficiency of gene expression. This system was also assessed for its ability to transfer into and express the chloramphenicol acetyl transferase (CAT) gene in a human liver cell line. Here again, the assay for functional CAT expression showed the pseudocapsid transfer procedure to compare favorably with lipofectin transfer. In another transient assay, a low-level endogenously expressed gene, p43, was complexed with pseudocapsids and transferred into human embryo lung fibroblasts, thereby increasing the expression levels. The ease of production of VP1 pseudocapsids, coupled with their efficient transfer of biologically useful information, should make this route of gene delivery an attractive proposition for further exploration with regard to gene therapy.

Role of sialic acid-containing molecules and the α4β1 integrin receptor in the early steps of polyomavirus infection

“…Moreover, according to this hypothesis, branched SA-containing molecules, also designated by Bauer et al (1999) as pseudoreceptors, could represent glycoproteins that allow MPyV cell attachment but not secondary post-attachment interactions with the protein moiety required for virus internalization. Further investigation in this direction is required to develop tissue-restricted MPyV VLP-based vectors for gene therapy (Forstovà et al, 1995;Soeda et al, 1998;Krauzewicz & Griffin, 2000).…”

“…Initial transfection lysates were used to subsequently infect cells in order to increase baculovirus titres. Production and purification of VLPs were by CsCl and sucrose gradients, as described previously (Forstovà et al, 1995).…”

The major capsid protein, VP1, of human JC virus expressed in Escherichia coli is able to self-assemble into a capsid-like particle and deliver exogenous DNA into human kidney cells.

“…Murine polyomavirus VP1 is able to self-assemble into a capsid-like particle when expressed in insect cells (Forstova et al, 1993 ;Gillock et al, 1997 ;Montross et al, 1991). Forstova et al (1995) demonstrated that murine polyomavirus VP1 pseudocapsids generated from a recombinant baculovirus are able to transfer heterologous DNA efficiently into mammalian cells. More recently, SV40 pseudovirions self-assembled in insect cells have also been shown to be able to introduce foreign genes into mammalian cells (Sandalon et al, 1997).…”

“…Procedures for packaging exogenous DNA into pseudocapsid particles were described by Forstova et al (1995). Briefly, 40 µg pseudocapsid protein was mixed with 3 µg pEGFP-C1 plasmid DNA (Clontech) in 150 mM NaCl, 10 mM Tris-HCl, pH 7n4, 0n01 mM CaCl # .…”

H1 and HMG17 extracted from calf thymus nuclei are efficient DNA carriers in gene transfer

“…While polylysine-ligand conjugates for tar-5.5V exhibit the ability to package DNA into transfectiongeting surface receptors of the host cell in complex with active protein-DNA complexes. These protein fractions DNA via the endocytotic pathway 3 or viral capsid prowere obtained by extraction of nuclei with 5% perchloric teins 4 have been used successfully in gene transfer, acid and subsequent gradual acetone precipitation of nuclear proteins have received little attention. The nonhithese extracts.…”