Genetic engineering of herpes simplex virus and vector genomes carrying IoxP sites in cells expressing Cre recombinase

The prokaryotic Cre-IoxP recombination system Is a powerful tool that enabl es in vitro and in vivo site-specific manipulations of the genome of eukary otic cells as well as of DNA viruses and their derived vectors. This system , however, has not yet been exploited in the context of herpes simplex viru s type 1 (HSV-1) infected cells, perhaps because this virus encodes several functions that induce a strong shutoff of cellular protein synthesis, a fa ct that could preclude expression of cellular-encoded Cre recombinase. In t he present study, we show that efficient site-specific recombination can ta ke place in cell lines expressing Cre, even in the context of HSV-1 infecti on, as evidenced by the engineering of an HSV-1 recombinant virus and sever al viral vectors carrying one or two IoxP sequences. More precisely, we hav e used this system to induce an irreversible switch in the expression of a viral complex transcription unit encoding two different open reading frames and allowing consecutive expression of two reporter genes. Furthermore Cre recombinations were also used to induce the decatenation of the genomic co ncatemers harbored by amplicon particles upon infection of cells under nonr eplicative conditions, thus enabling the rescue of many independent plasmid s corresponding to the original amplicon plasmid used to generate the vecto rs. Thus the Cre-IoxP recombination system can successfully be used for eng ineering the genome of HSV-1 or HSV-1-based Vectors in cultured cells. (C) 2000 Academic Press.