Biology Reference
In-Depth Information
(Yamanishi and Rapp 1977; Ihara et al. 1978). Only few HCMV
ts
mutants could
be mapped and associated with open reading frames (ORFs) (Dion et al. 1990;
Ihara et al. 1994). The analysis of MCMV
ts
mutants has mainly centered on
direct in vivo studies using mutants without mapping (Akel et al. 1993; Akel and
Sweet 1993; Bevan et al. 1996; Gill et al. 2000). To our knowledge, none of the
ts
mutations of MCMV has so far been associated with a specific genetic locus.
Recently, targeted
ts
mutants for the HCMV IE 2 gene were constructed using
BAC technology (Heider et al. 2002).
Reverse Genetics
The ability to clone viral DNA fragments, to propagate and manipulate those
recombinant plasmids in
E. coli
, opened several opportunities for the analysis
of isolated viral genes. Subcloned viral genes, after mutation and functional
analysis in vitro, could be reintroduced into the viral genome to investigate the
phenotype in the genomic context (reverse genetics). First, the herpes simplex
virus (HSV) genome was used to construct site-directed genome mutants
(Mocarski et al. 1980; Smiley 1980; Post and Roizman 1981). After the availa-
bility of sequences, site-directed mutagenesis also became an option for CMVs
(Spaete and Mocarski 1987; Manning and Mocarski 1988). In principle, a
marker gene is introduced into the viral genome by homologous recombination
in infected cells thereby disrupting or deleting a viral gene (Fig. 1a). The
recombination is under the control of the cellular recombination machinery.
It is a rare event and the
wt
virus dominates the resulting progeny pool.
Therefore, labeling or even positive selection of the mutants is essential for their
isolation by markers such as the β-galactosidase (Spaete and Mocarski 1987;
Manning and Mocarski 1988), the neomycin resistance gene (Wolff et al. 1993),
and the xanthine guanine phosphoribosyltransferase (gpt) gene (Mulligan and
Berg 1981; Greaves et al. 1995).
The advent of cosmid vectors with a capacity to maintain larger DNA fragments
(20-50 kbp) in
E. coli
led to cloning of the HSV genome as a set of overlapping
cosmid clones (van Zijl et al. 1988). Infectious
wt
virus is reconstituted after
co-transfection of the overlapping cosmid set into permissive cells via multiple
homologous recombination (Fig. 1b). For mutagenesis, the genetic change is
introduced into one of the cosmid fragments. The mutant virus is reconstituted by
co-transfection of the mutated cosmid clone with the other cosmids. HSV cloning
was followed by cosmid-based reconstruction of HCMV and MCMV (Kemble
et al. 1996; Ehsani et al. 2000). The utility of cosmids for generation of mutant
CMVs was demonstrated by generation of 17 different UL54 mutants of HCMV
(Cihlar et al. 1998). The huge advantage of the cosmid approach over the direct
recombination methods is the absence of the
wt
genome. The size of the cloned
viral fragment to be mutated allows standard in vitro mutagenesis techniques
applicable for plasmids. However, the genetic instability of the system during
