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.
SUMMARYEpstein-Barr virus (EBV) has the capacity to immortalize a subpopulation of resting B lymphocytes. Lymphoblastoid cell lines (LCL) established in this way carry the latent EBV genome as multiple copies of an extrachromasomal episome. Viral gene expression in LCLs is highly restricted; products identified correspond to a membrane protein (latent membrane protein; LMP), a nuclear antigen complex (Epstein-Barr nuclear antigens; EBNAs 1 to 6), two small RNA species (EBERs 1 and 2) and RNA species thought to encode a second membrane-associated polypeptide designated terminal protein (TP). Here we have investigated the temporal sequence of expression of the characterized 'latent' proteins during the initiation of immortalization when resting B cells are stimulated to enter and traverse the cell cycle. The analysis has been carried out on prolymphocytic leukaemia cells infected in vitro with either the immortalizing B95-8 strain of virus or the non-immortalizing P3HR1 strain. The results reveal that following B95-8 infection, a sequence of EBV expression is initiated within approximately 8 h with the synthesis of detectable levels of EBNA 2 shortly followed by EBNAs 1, 3, 4, 5 and 6. There is then a delay of approximately 40 h until the expression of LMP completes the latent pattern of proteins found in LCLs. P3HR1 infection, however, produces only transient expression of some EBNA species in a small percentage of cells after approximately 48 h. These observations suggest the failure of P3HR1 virus to immortalize may not be due solely to the absence of EBNA 2 expression and that cellular and/or virus-mediated events occur after EBNA synthesis which then facilitate efficient LMP expression and immortalization.
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 pseudocapsidmediated 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 lipo-
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