Molecular cloning of the guinea pig cytomegalovirus (GPCMV) genome as an infectious bacterial artificial chromosome (BAC) in Escherichia coli

Since cytomegalovirus (CMV) infection is highly species-specific, it is nec essary to study animal cytomegaloviruses to assess viral factors which cont ribute to pathogenesis. The generation of recombinant viruses carrying repo rter genes would provide useful tools for studying the genetics of CMV path ogenicity in vivo. We evaluated whether the guinea pig cytomegalovirus (GPC MV) was amenable to such manipulation, Metabolic selection using the guanos ylphosphoribosityl transferase (gpt) gene facilitated recovery of a recombi nant virus, vAM403, containing a gpt/green fluorescent protein (eGFP) casse tte introduced into the HindIII "N" region of the viral genome, This virus had replication kinetics identical to wild-type virus, We next attempted to clone the GPCMV genome as a bacterial artificial chromosome (BAC). A BAC p lasmid containing a gpt/eGFP cassette and the chloramphenicol resistance ma rker was introduced into HindIII "N" to generate another GPCMV recombinant, vAMBG-PCMV, Circular viral DNA isolated from vAMBG-PCMV-infected cells was used to transform Escherichia coli. Restriction profiles revealed that the GPCMV genome had been cloned as a BAC plasmid, and transfection of BAC pla smid DNA confirmed that the BAC clone was infectious, A novel strategy base d on a unique PmeI site was devised to quickly modify the BAC GPCMV plasmid , Recombinants retained the capability to replicate and express reporter ge nes in guinea pigs, suggesting that these viruses will be useful for in viv o pathogenesis studies, (C) 2001 Academic Press.