Abstract
Inactivation of gene products by dominant negative mutants is a valuable tool to assign functions to yet uncharacterized proteins, to map protein-protein interactions or to dissect physiological pathways. Detailed functional and structural knowledge about the target protein would allow the construction of inhibitory mutants by targeted mutagenesis. Yet, such data are limited for the majority of viral proteins, so that the target gene needs to be subjected to random mutagenesis to identify suitable mutants. However, for cytomegaloviruses this requires a two-step screening approach, which is time-consuming and labor-intensive. Here, we report the establishment of a high-throughput suitable screening system for the identification of inhibitory alleles of essential genes of the murine cytomegalovirus (MCMV). In this screen, the site-specific recombination of a specifically modified MCMV genome was transferred from the bacterial background to permissive host cells, thereby combining the genetic engineering and the rescue test in one step. Using a reference set of characterized pM53 mutants it was shown that the novel system is applicable to identify non-complementing as well as inhibitory mutants in a high-throughput suitable setup. The new cis-complementation assay was also applied to a basic genetic characterization of pM99, which was identified as essential for MCMV growth. We believe that the here described novel genetic screening approach can be adapted for the genetic characterization of essential genes of any large DNA viruses.
Highlights
Irradiation or chemicals were used to increase the mutation rate during replication, and interesting phenotypes were subsequently investigated regarding the causative genetic change [1]
The disadvantages of uncontrollable cellular recombination was overcome, when full length herpesvirus genomes were cloned as infectious bacterial artificial chromosomes (BAC), which was pioneered for the murine cytomegalovirus (MCMV) [4], and subsequently adapted to numerous herpesviruses such as HSV-1, PrV, HCMV, EBV and MHV68 [5,6,7,8,9], and to adenoviruses [10]
This BAC gives rise to the virus MCMV-D1-16-FLP recombination target (FRT) after transfection of permissive cells, which replicates in tissue culture with wt characteristics [19]
Summary
Irradiation or chemicals were used to increase the mutation rate during replication, and interesting phenotypes were subsequently investigated regarding the causative genetic change [1]. The establishment of genetic systems that permit directed mutagenesis of the gene of interest in its genomic context facilitated the generation of virus mutants in order to analyze resulting phenotypic alterations (reviewed in [2]). The modified DNA is transfected subsequently into permissive eukaryotic cells to reconstitute infectious virus, which results in pure, clonal viral populations, and even allows the production of attenuated mutants. The study of null- or nonfunctional mutants of essential genes requires functional analysis by trans-complementation or the application of dominant negative (DN) mutants (reviewed in [11,12])
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
