Construction and Characterization of an Infectious Murine Gammaherpesivrus-68 Bacterial Artificial Chromosome

Figure 1

The genomic structures of the MHV-68 recombinant viruses and the MHV-68 BAC plasmid. (a) Schematic diagram of the construction of the MHV-68 BAC virus. Shown is the left end of the viral genome encompassing terminal repeats and ORFs M1–M3. The BAC vector sequence and a puromycin expression cassette was recombined into the locus where the EGFP expression cassette was inserted in MHV-68(EGFP). MHV-68(BAC) was selected for the loss of the EGFP expression. The pMHV-68(BAC) plasmid was obtained by electroporating the circular genome of MHV-68(BAC) into DH10B cells. Following transfection of pMHV-68(BAC) into BHK-21 cells, the reconstituted virus, pMHV-68(BAC)v, was generated. The structures of pMHV-68(BAC) and pMHV-68(BAC)v are identical to MHV-68(BAC), except that pMHV-68(BAC) is circular with termini fused. To remove the BAC insert flanking by loxP sites, pMHV-68(BAC)v was co-transfected with a Cre recombinase expression plasmid and MHV-68(loxP) was isolated. The sites of HindIII are indicated with H and the ones of EcoRI were indicated with E. (b) Restriction enzyme analysis of the genome of MHV-68 recombinant viruses and the MHV-68 BAC plasmid. Each DNA sample was digested with either EcoRI or HindIII. Lane 1 is virion DNA of wild-type MHV-68; lane 2 is virion DNA of MHV-68(BAC); lane 3 is the plasmid DNA of pMHV-68(BAC); lane 4 is virion DNA of pMHV-68(BAC)v; lane 5 is virion DNA of MHV-68(loxP) and lane 6 is virion DNA of MHV-68(EGFP). The wild-type fragments which were eliminated due to the insertion of the BAC sequence were marked with asterisks. The fragments marked with a, b, d, and e, were generated because of the BAC insert. The fragments marked with c and f, resulted from the removal of BAC, 200 bp larger than the fragments indicated with asterisks. The fragments marked with F were from the fused termini of the circular pMHV-68(BAC). On the bottom of each agarose gel picture is the corresponding Southern blot using the probe spanning nt 51–6298. The laddering patterns in EcoRI-digested samples were due to various number of repeats on the linear virion DNA but not present in pMHV-68(BAC) (lane 3).