Viral bacterial artificial chromosomes: generation, mutagenesis, and removal of mini-F sequences

Abstract

Maintenance and manipulation of large DNA and RNA virus genomes had presented an obstacle for virological research. BAC vectors provided a solution to both problems as they can harbor large DNA sequences and can efficiently be modified using well-established mutagenesis techniques in Escherichia coli. Numerous DNA virus genomes of herpesvirus and pox virus were cloned into mini-F vectors. In addition, several reverse genetic systems for RNA viruses such as members of Coronaviridae and Flaviviridae could be established based on BAC constructs. Transfection into susceptible eukaryotic cells of virus DNA cloned as a BAC allows reconstitution of recombinant viruses. In this paper, we provide an overview on the strategies that can be used for the generation of virus BAC vectors and also on systems that are currently available for various virus species. Furthermore, we address common mutagenesis techniques that allow modification of BACs from single-nucleotide substitutions to deletion of viral genes or insertion of foreign sequences. Finally, we review the reconstitution of viruses from BAC vectors and the removal of the bacterial sequences from the virus genome during this process.

Figure 1

Schematic illustration of shuttle mutagenesis. In a first step, a shuttle plasmid is inserted into the target sequence via RecA-mediated recombination of homologous sequences. Replication of the shuttle plasmid containing a temperature sensitive origin (oriTS) is repressed by a temperature increase to 42°C. Positive co-integrates are selected with corresponding antibiotics. In a second step, vector sequences are excised from co-integrates by another recombination. Negative selection markers can be used to select BAC constructs that lost the shuttle plasmid [28, 64]. Dotted lines symbolize recombination events.

Figure 2

Overview of strategies for the Red-mediated deletion of sequences. Boxes of same color represent identical sequences.

Figure 3

Overview of techniques that facilitate the insertion of a sequence of interest (soi) into a target site. Boxes of same color represent identical sequences.

Figure 4

Overview of strategies that allow sequence editing of the target sequences. Boxes of same color represent identical sequences.

Figure 5

Overview of strategies that allow mini-F removal upon virus reconstitution. Positive selection marker is portion of the BAC vector backbone. Boxes of same color represent identical sequences.