Tn7-mediated introduction of DNA sequences into bacmid-cloned cytomegalovirus genomes for rapid recombinant virus construction.
Our basic understanding of how viruses infect, replicate, and cause disease has been largely derived from genetic approaches in which viral sequences have been mutated and the consequences of those mutations determined. In the herpesvirus field, deletions or insertions that preclude expression of specific viral proteins or remove critical cis elements have been invaluable in identifying their overall functions. We are now ready to move to a new level of detail-mapping functional domains within viral proteins or defining cis elements to the nucleotide level. This level of detail will require mutagenesis on a new scale, with recombinant viruses containing mutations within a given locus perhaps numbering in the hundreds. Mutagenesis on this scale would be greatly facilitated by more rapid methods of recombinant virus construction. In this report, we adapted a technology employing Tn7-mediated site-specific transposition [J. Virol. 67 (1993) 4566] as a rapid and highly reliable method to introduce novel sequences into bacmid-cloned herpesvirus genomes. We show that recombinant viruses can be rapidly created and that a deletion of the human cytomegalovirus (HCMV) essential gene ie2 can be complemented in cis by reintroduction, via transposition, of an ie2 cDNA; detailed mutagenesis of the complementing ie2 gene can now follow. Tn7-mediated transposition should accelerate greatly the pace at which recombinant herpesviruses can be constructed and, thus, facilitate the use of recombinant viruses for detailed mutagenic studies of both cis- and trans-acting genetic elements.
Hahn
Gabriele
G
Max von Pettenkofer Institut für Virologie, Ludwig-Maximilians-Universität München Pettenkoferstrasse 9A, 80336, München, Germany.
Jarosch
Margit
M
Wang
Jian Ben
JB
Berbes
Carlos
C
McVoy
Michael A
MA
D002478:Cells, Cultured; D022202:Chromosomes, Artificial, Bacterial; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000235:genetics* / Q000472:pathogenicity*; D004251:DNA Transposable Elements / Q000187:drug effects*; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005347:Fibroblasts; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D016679:Genome, Viral; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D013997:Time Factors; D015534:Trans-Activators
D004251:DNA Transposable Elements; C069186:IE2 protein, Cytomegalovirus; D017874:Immediate-Early Proteins; D015534:Trans-Activators