Cloning and mutagenesis of the murine gammaherpesvirus 68 genome as an infectious bacterial artificial chromosome

Gammaherpesviruses cause important infections of humans, in particular in i mmunocompromised patients. Recently, murine gammaherpesvirus 68 (MHV-68) in fection of mice has been developed as a small animal model of gammaherpesvi rus pathogenesis. Efficient generation of mutants of MHV-68 would significa ntly contribute to the understanding of viral gene functions in virus-host interaction, thereby further enhancing the potential of this model. To this end, we cloned the MHV-68 genome as a bacterial artificial chromosome (BAC ) in Escherichia coli. During propagation in E. coli, spontaneous recombina tion events within the internal and terminal repeats of the cloned MHV-68 g enome, affecting the copy number of the repeats, were occasionally observed . The gene for the green fluorescent protein was incorporated into the clon ed BAC for identification of infected cells. BAC vector sequences were flan ked by loxP sites to allow the excision of these sequences using recombinas e Cre and to allow the generation of recombinant viruses with wild-type gen ome properties. Infectious virus was reconstituted from the BAG-cloned MHV- 68. Grow th of the BAG-derived virus in cell culture was indistinguishable from that of wild-type MHV-68. To assess the feasibility of mutagenesis of the cloned MHV-68 genome, a mutant virus with a deletion of open reading fr ame 4 was generated. Genetically modified MHV-68 can now be analyzed in fun ctionally modified mouse strains to assess the role of gammaherpesvirus gen es in virus-host interaction and pathogenesis.