Mutagenesis of the Cytomegalovirus Genome - Human Cytomegalovirus - page 49

not give rise to progeny (Brune et al. 2001b; Menard et al. 2003; Zimmermann
et al. 2005). In its random approach, it is most comparable to the classical herpesvirus
genetics by chemical mutagenesis. The advantage over the older method is the
immediate access to the mutated position. However, Tn mutants identify nonessen-
tial gene functions, whereas ts mutants characterize essential genes. Interestingly,
E. coli carrying the infectious herpesvirus BAC can also reconstitute the infectious
virus in the natural host. However, only high bacterial load and the parenteral route
gave rise to a barely detectable MCMV progeny in vivo (Cicin-Sain et al. 2003).
Mutants, Revertants and the Mutation-Phenotype Connection
The experimental mutations but also spontaneous mutations elsewhere in the
genome - or both - may characterize a phenotype. To date, the size of herpesvirus
genomes precludes sequencing as a method to completely exclude the contribution
of unwanted mutations. Restriction pattern and Southern blot analysis can exclude
only gross genomic alterations. Therefore, different experimental approaches have
been developed to confirm the observed mutation/phenotype correlation (Fig. 5).
The local genetic change is considered as the causative principle when at
least two independent mutants are evaluated and resulted in the same pheno-
type. The same conclusion is drawn when a revertant is associated with the wt
phenotype. Both controls support the direct correlation between the targeted
site and the observed phenotype. In the ts mutagenesis, independent mutants of
the same locus confirm the mutation-phenotype connection. CMV-BAC mutants
permit both construction of independent mutants of the same locus and genera-
tion of a revertant to the wt sequence. Beside the shuttle plasmid method, the
recent developments in linear fragment mutagenesis provide convenient access
to both kinds of controls.
The genotype-phenotype connection, however, cannot answer the question of
whether the phenotype is caused by the mutation of the intended gene of interest,
or is a consequence of polar effects on other genetic features. Traditionally, a link-
age between a specific mutation and a gene product is proven when the principle of
the wt phenotype can be restored by providing the wt gene product in trans. Trans-
complementation of CMV gene products is a plausible approach but is associated
with major technical difficulties because of the slow virus growth and the limited
access of suitable cell lines for genetic engineering. Toxicity of the viral proteins
will also inhibit generation trans- complementing cell lines.
Recently, a trans- complementation of an HCMV gene product by adenovirus
mediated transient transduction has been reported which provides a promising
alternative approach to deliver CMV genes to infected cells (Murphy et al. 2000).
Cis -complementation via reinserting the deleted gene product at an ectopic position
into the viral genome is another option (Borst et al. 2001). An FRT site is inserted at a
position into the genome where it does not affect the wt properties. This wt -like viral
BAC is used for mutagenesis and the induced phenotypes can be analyzed and