FIG. 2.

Construction of the MHV-68 BAC genome and structural analysis of reconstituted virus genomes. (A) The BAC cloned genome was generated in eukaryotic cells by homologous recombination of the MHV-68 DNA with the recombination plasmid pHA2. The recombination plasmid contained 1.5 kbp of flanking homologous sequence (shaded box) as well as the BAC vector, the gpt gene, and the gfp gene, flanked by loxP sites. Electroporation of the circular BAC cloned genome RγHV68A98.01 into E. coli generated the MHV-68 BAC-plasmid pHA3. Integration of the BAC vector into the linear recombinant virus genome resulted in a new EcoRI fragment of 7.4 kbp which is indicated by an arrow. An additional EcoRI fragment of approximately 18 kbp in the BAC plasmid resulted from the fusion of the terminal EcoRI fragments (containing the terminal repeats of the virus genome). P, probe. (B) Structural analysis of BAC plasmids and of reconstituted virus genomes by ethidium bromide-stained agarose gel analysis of EcoRI-digested DNA. The lanes show MHV-68 WT DNA isolated from infected cells (lane 1), MHV-68 BAC plasmid pHA3 DNA isolated from E. coli (lane 2), reconstituted MHV-68 BAC virus RγHV68A98.01 DNA isolated from infected cells (lane 3), and reconstituted MHV-68 BAC virus RγHV68A98.02 DNA (with the BAC vector excised by recombinase Cre) isolated from infected cells (lane 4). The upper arrowhead indicates an additional 18-kbp band present only in lane 2, and the lower arrowhead indicates a 7.4-kbp fragment resulting in a double band in lanes 2 and 3. (C) Southern blot analysis of the gel shown in panel B using a DIG-labeled probe (indicated in panel A). Lanes 3 and 4 were from a longer exposure than lanes 1 and 2. The arrowhead indicates the additional 18-kbp band present only in lane 2. Marker (M) sizes (in kilobase pairs) are indicated on the left.