ABSTRACT
On the basis of the B lymphotropic Epstein-Barr virus (EBV), we have constructed a virus-free packaging cell line that allows encapsidation of plasmids into herpesvirus particles. This cell line harbors an EBV mutant whose packaging signals have been deleted. The gene vectors, which can encompass very large, contiguous pieces of foreign DNA, carry all cis-acting elements involved in amplification and encapsidation into virus-like particles as well as those essential for extrachromosomal maintenance in the recipient cell. Although this first-generation packaging cell line suffers from unwanted recombination between the helper virus genome and gene vector DNAs, this approach opens the way to delivery and stable maintenance of any transgene in human B cells.
Subject(s)
Cell Line , Gene Transfer Techniques , Genetic Vectors , Herpesvirus 4, Human , B-Lymphocytes , HumansABSTRACT
With current techniques, genetic alterations of herpesviruses are difficult to perform, mostly because of the large size of their genomes. To solve this problem, we have designed a system that allows the cloning of any gamma-herpesvirus in Escherichia coli onto an F factor-derived plasmid. Immortalized B cell lines were readily established with recombinant Epstein-Barr virus (EBV), demonstrating that the F factor-cloned EBV genome has all the characteristics of wild-type EBV. Because any genetic modification is possible in E. coli, this experimental approach opens the way to the genetic analysis of all EBV functions. Moreover, it is now feasible to generate attenuated EBV strains in vitro such that vaccine strains can be designed. Because we incorporated the genes for hygromycin resistance and green fluorescent protein onto the E. coli cloned EBV genome, the still open question of the EBV target cells other than B lymphocytes will be addressed.
