Reverse Genetics Modification of Cytomegalovirus Antigenicity and Immunogenicity by CD8 T-Cell Epitope Deletion and Insertion
Figure 1
Method of herpesvirus point mutagenesis. (a) Site-directed mutagenesis by overlap extension. The dsDNA and primers are represented by double lines and arrows, respectively. The direction of the arrows is indicating the 5′-to-3′ orientation of primers, which are denoted by capital letters. The site of mutagenesis is marked with red symbol X. PCR amplificates yielded by PCR1 and PCR 2 served as templates in the combination PCR (PCR3) performed with primers A and D to generate the final amplificate that includes the intended point mutation. (b) Shuttle-plasmid allelic exchange in Escherichia coli. A shuttle plasmid harbouring the point mutation (red box) flanked by homologous viral sequences (green and yellow boxes) is transformed into bacteria that contain the CMV BAC. The first homologous recombination leads to formation of cointegrates due to recombination via one homology arm. Cointegrates are selected by chloramphenicol (Cm) and kanamycin (Kn). Nonintegrated shuttle plasmids are removed at 43°C. In the second recombination step, cointegrates are resolved to generate either a WT viral BAC (via yellow box) or a mutant viral BAC (via green box). Clones still containing cointegrates are eliminated by sucrose counterselection against SacB (gray box), and finally, bacterial clones containing either mutated or WT CMV BACs are isolated (see [61, 62], for detailed description of the method, see [82]). (c) DNA of selected bacterial clones was tested for genomic structural integrity, and the presence of the mutation was confirmed by sequencing. Reconstitution of BAC-derived recombinant virus was achieved by transfection of mutated BAC DNA into permissive eukaryotic cells. After at least four viral passages and a second round of plaque purification, PCRs were performed to verify the absence of BAC vector sequences [8].
