Molecular Cloning of the Guinea Pig Cytomegalovirus (GPCMV) Genome as an Infectious Bacterial Artificial Chromosome (BAC) in Escherichia coli

Abstract

Since cytomegalovirus (CMV) infection is highly species-specific, it is necessary to study animal cytomegaloviruses to assess viral factors which contribute to pathogenesis. The generation of recombinant viruses carrying reporter genes would provide useful tools for studying the genetics of CMV pathogenicity in vivo. We evaluated whether the guinea pig cytomegalovirus (GPCMV) was amenable to such manipulation. Metabolic selection using the guanosylphosphoribosityl transferase (gpt) gene facilitated recovery of a recombinant virus, vAM403, containing a gpt/green fluorescent protein (eGFP) cassette 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 plasmid containing a gpt/eGFP cassette and the chloramphenicol resistance marker was introduced into HindIII “N” to generate another GPCMV recombinant, vAMBGPCMV. Circular viral DNA isolated from vAMBGPCMV-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 plasmid DNA confirmed that the BAC clone was infectious. A novel strategy based on a unique PmeI site was devised to quickly modify the BAC GPCMV plasmid. Recombinants retained the capability to replicate and express reporter genes in guinea pigs, suggesting that these viruses will be useful for in vivo pathogenesis studies.