Identification and Analysis of Dominant Negative Mutants

The function of nonessential genes is studied by gene deletion and by loss-of-function mutants. By ris-complementation assays functionally important sites of essential genes can be mapped. Unfortunately, a major target of genetics, the null phenotype of an essential gene is generally hard to come by. This requires the cumbersome establishment and optimization of a trans-complementation system for each gene under study. Here, we try to develop a systematic approach. Dominant negative (DN) mutants are special null mutants that induce the null phenotype even in the presence of the wt allele. DN mutants of cellular genes have been proven to be a valuable for genetic analysis of complex pathways (Herskowitz 1987). Knowledge of protein structure, protein functions or sequence motifs aid the design of DN mutants (Crowder and Kirkegaard 2005). Unfortunately, the information on the majority of herpesvirus proteins is too limited for knowledge based construction of DN mutants.

Therefore, we set up a random approach to isolate DN mutants of CMV genes. The Tn7-based linker-scanning mutagenesis introduces 5-aa insertions into coding sequences and provides a comprehensive set of subtle insertion mutants of the ORF. Nonfunctional mutants are selected from a library by a ds-complementation screen as described above. These mutants can then be introduced into the wt MCMV genome. This allows testing their inhibitory potential (Fig. 7a). If the mutant interferes with the function of the essential wt allele, virus reconstitution is inhibited. As we showed for both M50 (Rupp et al. 2007) and the M53 (Z. Ruzsics and U.H. Koszinowski, unpublished data), such mutants represent only a small proportion of the null mutants but can be isolated by a standardized procedure.

Transfection of the viral nucleic acid is error-prone and not only a DN function, but also unrelated effects may prevent virus reconstitution. Conditional expression of the inhibitory mutants in the context of the wt genome should allow virus reconstitution in the off state and should induce the null phenotype when turned on (Fig. 7b). We constructed a regulated expression system for MCMV (Rupp et al. 2005) in which the constitutive viral expression of the TetR blocks the transcription of the

Fig. 7 Screening for and characterization of dominant-negative mutants of essential viral genes. a Screening for inhibitory mutants. An essential viral gene, the target gene (gray box, T), is sub-cloned and subjected to a random and comprehensive mutagenesis in vitro leading to a mutant library M1, M2, . . . Mn (small black boxes indicate mutations). Mutated ORFs are placed under the control of a strong constitutive promoter into an insertion plasmid containing an FRT site (open box with gray triangle). The insertion plasmids can only be maintained in a special E. coli strain. Normal E. coli (open boxes) carrying an FRT site-labeled viral bacterial artificial chromosome (BAC) and a temperature-sensitive plasmid-expressing FLP recombinase (FLP) are transformed with the insertion plasmids carrying different mutants one by one. The FLP recombinase mediates site-specific recombination between the FRT sites in the BAC and the insertion plasmids. This recombinants can then be isolated under combined antibiotic selection for both the BACs and the insertion plasmid. The FLP-expressing helper plasmid is removed by elevated temperature. Then BAC DNA is prepared and permissive cells are transfected with each construct. The mutants that are able to inhibit the virus reconstitution can be selected on the basis of the inability of plaque formation upon transfection. b Validation of dominant negative mutants by conditional gene expression. The inhibitory mutants are subcloned under the control of a promoter regulated by the TetR (black box) into an insertion plasmid with an FRT site. These constructs are delivered into the viral BAC as described above. Then permissive cells are transfected with the recombinants in order to reconstitute viruses carrying the regulation cassettes for the inhibitory mutants. The inhibitory mutants are not expressed during reconstitution because in the absence of doxycycline (- Dox), the consti-tutively expressed TetR blocks their transcription. The inhibitory function of the mutants can be analyzed upon doxycycline administration (+ Dox), which leads to the expression of the inhibitory mutant by releasing the expression cassette from the TetR regulation regulated gene and induction by doxycycline exposes the viral replication program to the DN mutant. This system allowed us detailed quantitative and qualitative analysis of the effect of DN mutants of both M50 and M53. In addition, the result of the random screen on MCMV M50 aided the construction of a DN mutant of the homolog in HCMV (UL50) (Rupp et al. 2007). We believe that this systematic approach will facilitate the functional analysis of essential CMV genes.

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