FIG. 1.

Construction of a CXCL10-expressing recombinant of MHV-A59. (A) Construction of a transcription vector for synthesis of donor RNA. Plasmid pCXCL10A was derived in two steps from parent plasmid pMH54 via an intermediate construct, pCXCL10int; the net change was the removal of 267 nucleotides of MHV sequence from pMH54 and its replacement with 313 nucleotides of CXCL10 sequence in pCXCL10A. Restriction sites shown are those relevant to plasmid construction, runoff in vitro transcription, or recombinant analysis. The expanded region of sequence shows the context of the inserted CXCL10 ORF. The start and stop codons of the CXCL10 ORF and the stop codon of the upstream S ORF are boxed. The gene 4 transcription regulating sequence is underlined. Arrowheads mark the boundaries between MHV sequence and the CXCL10 insert. (B) Scheme for generation of MHV-A59 CXCL10 recombinants by targeted recombination between the parental virus, thermolabile N gene deletion mutant Alb4, and donor RNA transcribed from plasmid pCXCL10A. A single crossover upstream of the gene 4 region generated recombinants that had simultaneously acquired the CXCL10 insertion and the wild-type copy of the N gene. The same strategy, with donor RNA transcribed from pMH54, was used to generate an isogenic wild-type control recombinant. At the bottom are shown the primer pairs used in reverse transcription-PCR analysis of recombinant viral genomes.