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. 2006 Aug 23;80(21):10436–10456. doi: 10.1128/JVI.01248-06

FIG. 1.

FIG. 1.

Construction and verification of recombinant viruses. (A) Maps of the mutagenesis, drawn to scale. The HindIII physical map of the mCMV Smith strain genome is shown at the top with the ie1/3 transcription unit expanded to reveal the location of the authentic antigenic IE1 peptide 168-YPHFMPTNL-176 coding region (WT/Revertant). By means of site-directed mutagenesis, the C-terminal MHC class I anchor residue Leu of the IE1 peptide was point mutated to Ala by mutating the Leu codon CTA into the Ala codon GCA (Mutant). Authentic revertant and wobble revertant (*) were generated by back-mutation of Ala codon GCA into Leu and Leu* codons CTA and CTT. The 6,523-bp BssHII/HpaI fragment comprises the ie1/3 transcription unit with the authentic or mutated IE1 peptide-coding region used in the shuttle plasmids for recombination. (B) Structural analysis of BAC plasmids. Purified DNA of BAC plasmids pSM3fr (lanes 1), C3XIE1Leu (lanes 2), C3XIE1Ala (lanes 3), and C3XIE1Leu* (lanes 4) was subjected to cleavage by EcoRI, HindIII, and XbaI, and fragments were analyzed by agarose gel electrophoresis and staining with ethidium bromide. Lanes M show the size markers. (C) Sequence analysis of mutated BAC plasmids. The fidelity of the sequences is shown between n181,011 and 181,028 for the BAC plasmids C3XIE1Ala (mutant L176A), C3XIE1Leu (revertant A176L), and C3XIE1Leu* (wobble revertant A176L*).