Mutagenesis of the Cytomegalovirus Genome - Human Cytomegalovirus - page 50

Fig. 5 Different approaches for confirmation of the mutation-phenotype connection. a Cis -
complementation in cells allows reversion of the mutation to the wt sequence. By transfection of
cells with the mutant BAC genome ( I ) or infection with the mutant virus ( II ) and co-transfection of
a DNA fragment carrying the wild type (wt) sequence and appropriate viral homologies, the mutation
( M ) can be reverted to the wt sequence. Since revertant and mutant viruses need further separation,
this approach only works efficiently if one can select for the revertant, e.g., if it has a growth advan-
tage over the mutant virus. b Cis -complementation of viral BACs in E. coli is best performed by
shuttle plasmid mutagenesis. The shuttle plasmid carrying the wt sequence and appropriate homolo-
gies is introduced into E. coli carrying the mutant BAC plasmid. By RecA-mediated homologous
recombination, the wt sequence is inserted at the mutation site without leaving any operational
sequences. After transfection of the revertant BAC genome into permissive cells, a homogenous
revertant population is gained without any further need for selection against mutant viruses. c Protein
trans- complementation in cells. Cells that express the viral wt gene product permanently ( I ) or
transiently by an additional expression vector ( II ) are superinfected with the mutant virus. This
allows transient complementation of the mutant phenotype if the expression times and levels of
the wt gene product are appropriate. d