Biology Reference
In-Depth Information
Introduction
Animal viruses use a small set of genes to profoundly affect functions of
complex hosts. The goal of virus genetics is to understand virus-host interac-
tions at the molecular level. Collection or construction of virus mutants is
obligatory for this goal. Until recently, the manipulation of herpesvirus genomes
was bound to construction of mutants by homologous recombination in infected
cells. Bacterial artificial chromosomes (BACs) are large, circular single-copy
episomes of Escherichia coli which are suited for maintenance of very large
foreign DNA fragments including genomes of large DNA viruses, as first dem-
onstrated by Luckow and colleagues for baculovirus (Luckow et al. 1993). In
1997, we pioneered the BAC-based genetic analysis of herpesviruses with the
first example of the cloning and mutagenesis of infectious mouse CMV
(MCMV) genome in E. coli (Messerle et al. 1997). BAC technology has become
a general approach in herpesvirus genetics and beyond for analysis of large
and/or unstable viral genomes (Almazan et al. 2000; Ruzsics et al. 2006). This
review discusses the different strategies for generation of recombinant herpes-
viruses and shows the potential of BAC-based herpesvirus genetics. Examples
are taken mainly from human CMV (HCMV) and MCMV; reference is also
given to work on other herpesviruses.
CMV Genetics in Cells
Forward (Classical) Genetics
The objects of viral genetics are the mutant alleles, where changes in genetic material
result in phenotypic alterations that can be analyzed. The frequency of spontaneous
mutations in the DNA genomes of herpesviruses ranges between 10 -8 and 10 -11 per
incorporated nucleotide. Therefore, mutagens have been used to increase the rate of
mutations during virus replication by a procedure called in vivo mutagenesis
(Schaffer 1975; Schaffer et al. 1984). The characterization of conditional alleles,
such as temperature sensitive ( ts ) mutations, has been favored, because both isola-
tion of mutants and their analysis required operational viability. Ts mutants are a
result of a missense point mutation that alters the primary amino acid sequence of
the encoded protein, leading to a loss of function only at a higher (restrictive)
temperature. The demanding step is the genetic mapping of the causative muta-
tions. The methods for genetic mapping of ts mutations, such as cross-complemen-
tation and marker rescue assays, are time-consuming and work-intense. Yet, until
recently, ts mutants were the only generally applicable tools for studying null
phenotypes of essential herpesvirus genes.
Chemical mutagenesis has also been applied to HCMV and MCMV genetics.
Early work on HCMV led to the classification of complementation groups
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