Development of methods that facilitate generation of recombinant herpesviruses using BAC system

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In recent years, several laboratories have reported on the cloning of herpesvirus genomes as bacterial artificial chromosomes (BACs) in E. coli and on procedures to manipulate these genomes using the bacterial recombination machinery. However, the herpesvirus-BACs reported so far are either replication-incompetent or infectious with a deletion of one or more viral genes due to the BAC vector insertion. As a multi-purpose clone for use in the research of herpesviruses, we attempted to generate infectious herpes simplex virus (HSV) -BACs containing the full genome of HSV-1 without any loss of^viral genes. Our results were as follows. (i)E. coli (YEbac102) harboring the full-length HSV-1 genome (pYEbac102) in which a BAG, flanked by loxP sites were inserted into the intergenic region between U_L3 and U_L4 was constructed, (ii) pYEbac102 was an infectious molecular clone, given that its transfection into rabbit skin cells resulted in production of infectious virus (YK304). (iii)' The BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus YK304 by co-infection of Vero cells with YK304 and a recombinant adenovirus AxCANCre expressing Cre recombinase. (iv) As far as was examined, the reconstituted viruses from pYEbac102 could not be phenotypically differentiated from wild-type viruses in vitro and in vivo. Thus the viruses grew as well in Vero cells as did the wild-type virus and exhibited wild-type virulency in mice on intracerebral inoculation. (v) The infectious molecular clone pYEbac102 is in fact useful for mutagenesis of the HSV-1 genome by bacterial genetics and a recombinant virus carrying amino acid substitutions in both copies of the αO gene was generated. pYEbac102 will be multi-applicable to the rapid generation of genetically engineered HSV-1 recombinants in basic research into HSV-1 and in the development of HSV vectors in human therapy.