Extracellular self-assembly of virus-like particles from secreted recombinant polyoma virus major coat protein.

Mouse polyoma virus major coat protein (VP1) expressed from a recombinant baculovirus is efficiently transported to infected cell nuclei and assembles into protein nanospheres morphologically similar to natural capsids. The nanospheres readily combine with plasmid DNA to form a hybrid gene therapy agent known as virus-like particles (VLPs). To facilitate large-scale production of VLPs free from cellular contaminants, the use of stable Drosophila cell lines expressing either wild-type protein, or VP1 tagged with a secretion signal for targeting to the extracellular medium, was investigated. Both wild-type and tagged VP1 expressed at 2-4 mg VP1/litre of culture. As expected, the wild-type protein self-assembled into VLPs. The tagged VP1 was efficiently secreted to the extracellular medium but was also glycosylated, unlike wild-type VP1. Despite this fact, a small fraction of the recombinant secreted protein assembled into VLP-like structures that had altered disulphide bonding, but were still biologically active. These results demonstrate the considerable tolerance in the nanosphere assembly to structural (i.e. aberrant glycosylation) and environmental (i.e. extracellular medium vs. nuclear milieu) changes. Thus, with modifications to improve nanosphere assembly, the secretion method could be adapted to large-scale preparation of VLPs, providing significant advantages over current methods of production of the vector.

Cell fractionation in non-ionic detergent distinguishes sub-populations of polyoma virus middle T antigen and reveals a novel form.

The transforming function of polyoma virus, middle T antigen (MT), interacts with several cellular enzymes, essential to its oncogenic activity. We have used cell fractionation to study the various MT/cellular protein complexes. We demonstrate that MT can be separated into three sub-species, dependent upon extraction in two buffers that we designate A and B: Antigen extracted from whole cells by both buffers (called MT1) is associated with most of the phosphorylated phosphatidyl-inositol kinase 85 kD subunit, pp85, and protein phosphatase 2A. Antigen (MT2), associated with the greater portion of pp60c-src, is extracted by buffer B, but not buffer A. A third population (MT3), resistant to extraction by either buffer, is not detectably associated with protein phosphatase 2A or pp85. It is, however, associated with a low level kinase activity. The interaction between pp60c-src and MT appears to influence the formation of both MT2 and MT3. MT2 fractionates with the cellular microtubule network, but does not appear to be directly associated with it. MT3, a previously undescribed population, comprises about one third of MT in wild type antigen-containing cells. It is missing in mutants incapable of interacting with pp60c-src, but exists in the absence of an interaction with pp85. We suggest that MT3 may be an intermediate in, or product of, one of the MT/pp60c-src signalling pathways, distinct from that involving pp85.

Regulation of cytoskeletal association by a basic amino acid motif in polyoma virus middle T antigen.

The subcellular localization of many oncogenic proteins is thought to be important for their function. In the case of the middle T antigen of the DNA tumour virus, polyoma, localization to membranes in a specific manner is essential for its cellular transforming activity. To investigate factors that influence this localization, heterologous membrane targetting sequences were substituted for the middle T antigen transmembrane domain and the properties of the resulting proteins studied. Whereas C-terminal lipid modification derived from the H-ras CaaX box restored oncogenic activity to non-transforming truncated middle T antigen species, N-terminal myristylation from pp60c-src did not. Furthermore, a region, rich in basic amino acids and adjacent to the middle T transmembrane domain, was found to mediate association with detergent-insoluble cytoskeleton. Co-operation between the basic motif and neighbouring membrane binding domains resulted in specific localization of proteins to particular membrane sites, characterized by the association with subcellular structures, likely to be cytoskeletal in nature. These results demonstrate that the cellular localization of MT is regulated by at least two determinants, a transmembrane sequence which confers membrane binding and a basic motif which specifies a particular site within the membrane.

Medullary thyroid carcinomas in transgenic mice expressing a Polyoma carboxyl-terminal truncated middle-T and wild type small-T antigens.

Medullary thyroid carcinoma (MTC) is a rare human tumor affecting the calcitonin-secreting c-cells of the thyroid. Here we report that two independent strains of transgenic mice expressing a Polyomavirus (Py) truncated middle-T antigen (deltaMT), consisting of the amino-terminal 304 amino acids, and the full length Py small-T antigen, developed multifocal bilateral MTCs with 100% penetrance. Occasionally one strain also developed mammary and bone tumors. Furthermore, offspring from both transgenic lines displayed pronounced waviness of the whiskers and fur, previously associated with defective epidermal growth factor receptor signaling. Transgene transcription, driven by the homologous early promoter/enhancer, and the corresponding translation products were detected in tumors and in many other organs which did not develop pathologies. The subcellular distribution of deltaMT and its interactions with the adapter proteins of the SHC family have also been analysed. Our study describes a novel murine model of MTC and provides evidence that the N-terminal 304 amino acid fragment of Py middle-T antigen, possibly in co-operation with small-T antigen, acts as a potent oncogene in c-cells of the thyroid.

Polyoma virus pseudocapsids as efficient carriers of heterologous DNA into mammalian cells.

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.

Studying the interaction of polyoma virus middle T antigen with cellular proteins.

The oncogenic mouse polyoma virus encodes six proteins, two of which (the large and middle T antigens), when expressed together in primary rodent cells, generate an alteration of growth patterns commonly known as cellular transformation. The transformed cells grow with an unlimited life span and when introduced into immunocompromised mice rapidly form tumours. The large T gene product confers an indefinite growth potential on primary cells; however, the middle T gene product has been identified as bringing about the changes which ultimately allow the cell to form tumours. The 55 kDa middle T antigen has been shown to associate with a number of cellular enzymes involved in regulation of growth factor signalling pathways, all of which were identified as being components of the immunocomplexes that can be isolated from transformed cells using middle T antigen specific antibodies. Two-dimensional gels have assisted the search for less prominent species present in these complexes. These methods represents one approach to investigating associating proteins, and as such, select for those interactions that are stable under the conditions used. In order to explore the possibility that middle T antigen could form complexes with other cellular proteins given different conditions, recombinant middle T antigen was used in a series of "filter overlay" experiments.

Recombination resulting in unusual features in the polyomavirus genome isolated from a murine tumor cell line.

Polyomavirus-induced tumor formation in the adult natural mouse host has been investigated. Tumors were produced in nude mice with the transformation-defective mutant strain NG18 after a long latency period by apparent activation of a cryptic endogenous transforming viral function. A tumor cell line, designated ScB, was established and characterized. Cells from this morphologically distinct line were unusual in that they grew in soft agar but did not form foci. They were highly tumorigenic. They had a 3.1-kilobase major viral transcript that hybridized to probes derived from regions encoding both the T antigens and the structural proteins. ScB cells expressed polyomavirus small T antigen, a slightly altered middle T antigen, and a truncated large T antigen but no capsid proteins. Middle T antigen preserved its interactions with host proteins of 60 and 37 kilodaltons and with c-src. Analysis of cDNA and genomic clones indicated that the stable viral insert in the ScB genome contained multiple copies of the viral B-enhancer. The genome contained two intragenic inversions which created novel early- to late-strand switches. A simple model for the generation of one inversion is proposed that involves the juxtaposition of two stem-loop structures at an illegitimate recombination site; the location of the inverted segment within the integrated sequence permits use of the viral late polyadenylation signal in early-region transcripts, as confirmed by DNA sequence. A repetitive sequence may facilitate recombination at the other inversion site. Both the biological consequences of the observed rearrangements and the structure of the integrated viral DNA suggest that the recombination events are nonrandom.

Expression of biologically active middle T antigen of polyoma virus from recombinant baculoviruses.

Two different recombinant baculoviruses have been generated for expressing the middle T antigen (MT) of polyoma virus in insect (Sf9) cells. One (pAcI-PyMT) produces moderate levels of MT and the other (pVL-PyMT) high levels. Indirect immunofluorescence and cellular fractionation studies with pAcI-PyMT infected Sf9 cells give results similar to those observed with wild type polyoma virus infected mouse cells, and show MT to be mainly associated with cytoplasmic membranes in the insect cell. In the latter, a sub-population of MT is phosphorylated in in vitro protein kinase assays. The yields of MT from pVL-PyMT infected cells are high enough to suggest that this protein can now be produced by this method in sufficient amounts for definitive biochemical and crystallographic analyses.

Enhancement by polylysine of transient, but not stable, expression of genes carried into cells by polyoma VP1 pseudocapsids.

Gene transfer to provide long-term expression of a therapeutic product, without introducing unwelcome genetic information, is a goal being sought for therapy of both hereditary and acquired diseases. Polyoma virus pseudocapsids, generated from a VP1-expressing recombinant baculovirus, lack viral DNA and have been successfully used to introduce small exogenous genes stably into cells in vitro by a process designated 'pseudofection'; although pseudocapsids protect only about 3 kbp of exogenous DNA, low efficiency transfer of a larger fragment (6.2 kbp) has been observed. Here, expression of a 7.2 kbp plasmid (pCMV beta) encoding the beta-galactosidase gene was assessed to monitor not only efficiency, but the ability of pseudocapsids to transfer larger-sized DNA on their own, or in the presence of the polycation, poly-L-lysine, added to protect nonencapsidated DNA. When complexed to pseudocapsids only, the efficiency of expression of the transferred beta-galactosidase gene (in human or rodent cells), although low, appeared to stabilise with time. In the presence of polylysine, unencapsidated DNA was shown to be protected against DNase activity, but electron microscopy (EM) revealed the formation of large mixed aggregates. The addition of pseudocapsids to these aggregates, and measurement of mobilities of the complexes in CsCl equilibrum centrifugation, indicated that they contained negligible amounts of VP1. For subsequent pseudofection experiments, DNA was complexed first with pseudocapsids, then polylysine was added. The latter did not appear to displace pseudocapsids from DNA, and was found to increase the efficiency of short-term expression both in in vitro and in vivo experiments. Gene expression, analysed histochemically or by the polymerase chain reaction, revealed transcriptional activity of the input gene, with expression first diminishing, then stabilising over time. The presence of pseudocapsids, in complexes with DNA with or without polylysine, allowed for stable and persistent gene expression.

Sustained ex vivo and in vivo transfer of a reporter gene using polyoma virus pseudocapsids.

Properties of a virus-like artificial gene delivery vehicle, synthesised from recombinant major coat protein of mouse polyoma virus, have been explored. The protein, VP1, self assembles into protein spheres, or 'pseudocapsids, which can bind and transfer DNA into cells in vitro and in vivo. Here, the ability of pseudocapsids to carry DNA into a complex cell system (ex vivo organ cultures of rabbit cornea) or whole animals (mice) has been assessed. Evidence from histochemical and PCR experiments indicate that pseudocapsids stimulate uptake and stable maintenance of marker DNA in nondividing corneal cells as efficiently as a recombinant adenovirus. In athymic and immunocompetent mice, gene transmission occurs with no apparent adverse effects on the animals. In the presence of pseudocapsids, the marker gene was transferred to a range of organs, including the brains of animals, following peripheral or intranasal administration. In immunocompetent mice, significant long-term transcriptional expression (at least 22 weeks) was observed with pseudocapsids, a period significantly longer than observed with DNA alone (several weeks only), again with no obvious adverse effects. This study demonstrates that pseudocapsids from the murine virus, polyoma, constitute a novel transfer agent for long-term gene therapeutic applications in tissues or whole animals.

Persistence and tissue distribution of DNA in normal and immunodeficient mice inoculated with polyomavirus VP1 pseudocapsid complexes or polyomavirus.

Introduction of DNA into normal and immunodeficient mice, alone or in complex with VP1 pseudocapsids, has been compared to DNA transfer by viral infection. Similar to natural infection and in contrast to plasmid alone, VP1 pseudocapsids efficiently introduced DNA, which remained for months in normal mice and possibly longer in B- and T-cell-deficient mice.

Virus-like gene transfer into cells mediated by polyoma virus pseudocapsids.

Mouse polyoma virus-like particles (or pseudocapsids) are composed solely of recombinant viral coat protein. They can interact with DNA and transport it to cells, resulting in gene expression both in tissue culture and in mice. We demonstrate that DNA transfer in vitro depends on partial packaging of DNA within the virus-like capsid. Cell surface sialic acid residues and an intact microtubule network, required for viral infectivity, are also necessary for pseudocapsid-mediated gene expression from heterologous DNA. Thus, gene delivery in this system requires pathways utilised by polyoma virions, rather than proceeding via the 'nonspecific' endosomal route typical of nonviral systems such as liposomes or calcium phosphate precipitates. Despite the fact that all cells appear to internalise pseudocapsid/DNA complexes, only a proportion show productive gene delivery. Bulk internalisation of complexes is dependent on actin fibres, but not cell surface sialic acid or microtubules, indicating that a second transport pathway exists for pseudocapsids which is nonproductive for gene transfer. The model suggested by these data demonstrates the virus-like properties of the pseudocapsid system, and provides a basis for further development to produce a highly effective gene delivery vehicle. Gene Therapy (2000) 7, 2122-2131.

Cooperation of structural proteins during late events in the life cycle of polyomavirus.

The polyomavirus minor late capsid antigen, VP2, is myristylated on its N-terminal glycine, this modification being required for efficient infection of mouse cells. To study further the functions of this antigen, as well as those of the other minor late antigen, VP3, recombinant baculoviruses carrying genes for VP1, VP2, and VP3 have been constructed and the corresponding proteins have been synthesized in insect cells. A monoclonal antibody recognizing VP1, alpha-PyVP1-A, and two monoclonal antibodies against the common region of VP2 and VP3, alpha-PyVP2/3-A and alpha-PyVP2/3-B, have been generated. Reactions of antibodies with antigens were characterized by indirect immunofluorescence, immunoprecipitation, and immunoblot analysis. Immunofluorescent staining of mouse cells infected with polyomavirus showed all antigens to be localized in nuclei. When the late polyomavirus proteins were expressed separately in insect cells, however, only VP1 was efficiently transported into the nucleus; VP2 was localized discretely around the outside of the nucleus, and VP3 exhibited a diffused staining pattern in the cytoplasm. Coexpression of VP2, or VP3, with VP1 restored nuclear localization. Immunoprecipitation of infected mouse cells with either anti-VP1 or anti-VP2/3 antibodies precipitated complexes containing all three species, consistent with the notion that VP1 is necessary for efficient transport of VP2 and VP3 into the nucleus. Purified empty capsid-like particles, formed in nuclei of insect cells coinfected with all three baculoviruses, contained VP2 and VP3 proteins in amounts comparable to those found in empty capsids purified from mouse cells infected with wild-type polyomavirus. Two-dimensional gel analysis of VP1 species revealed that coexpression with VP2 affects posttranslational modification of VP1.

Myristylated polyomavirus VP2: role in the life cycle of the virus.

The double-stranded genome of the small DNA tumor virus, polyomavirus, is enclosed in a capsid composed of a major protein, VP1, which associates as pentameric capsomeres into an icosahedral structure, and two minor proteins, VP2 and VP3, whose functions and positions within the structure are unknown. The N-terminal glycine of the VP2 coat protein has been shown to be cotranslationally acylated with myristic acid. To study the function of this modification and the role of VP2 in the life cycle of polyomavirus, the N-terminal glycine, critical to the myristylation consensus sequence, has been altered to a glutamic acid or a valine residue by site-directed oligonucleotide mutagenesis. The glycine----glutamic acid mutant DNA has been further studied. When transfected into cells permissive for the polyomavirus full lytic life cycle, this mutant DNA replicated at levels comparable to those of wild-type viral DNA, and small amounts of nonrevertant (mutant) virus could be harvested from the cultures. The virus particles viewed by electron microscopy appeared slightly distorted, but the ratio of full to empty particles was similar to that produced in a wild-type viral infection. Mutant virus was capable of reinfecting permissive cells but with a considerably reduced efficiency.

Scanning surface confocal microscopy for simultaneous topographical and fluorescence imaging: application to single virus-like particle entry into a cell.

We have developed a method for simultaneous recording of high-resolution topography and cell surface fluorescence in a single scan which we call scanning surface confocal microscopy. The resolution of the system allows imaging of individual fluorescent particles in the nanometer range on fixed or live cells. We used this technique to record the interaction of single virus-like particles with the cell surface and demonstrated that single particles sink into the membrane in invaginations reminiscent of caveolae or pinocytic vesicles. This method provides a technique for elucidating the interaction of individual viruses and other nanoparticles, such as gene therapy vectors, with target cells. Furthermore, this technique should find widespread application for studying the relationship of fluorescently tagged molecules with components of the cell plasma membrane.