Generation and precise modification of a herpesvirus saimiri bacterial artificial chromosome demonstrates that the terminal repeats are required for both virus production and episomal persistence.
Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus, and shares considerable homology with the human gammaherpesviruses Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus. The generation of herpesvirus mutants is a key facet in the study of virus biology. The use of F-factor-based bacterial artificial chromosomes (BACs) to clone and modify the genomes of herpesviruses has enhanced the variety, precision and simplicity of mutant production. Here we describe the cloning of the genome of HVS non-transforming strain A11-S4 into a BAC. The cloning of the BAC elements disrupts open reading frame (ORF) 15 but the HVS-BAC can still replicate at levels similar to wild-type virus, and can persistently infect fibroblasts. The HVS-BAC was modified by RecA-mediated recombination initially to substitute reporter genes and also to delete the terminal repeats (TR). After deletion of the TR, the HVS-BAC fails to enter a productive virus lytic cycle, and cannot establish a persistent episomal infection when transfected into fibroblast cell lines. This shows that while ORF 15 is dispensable for virus function in vitro, the TR is required for both virus latency and lytic virus production. In addition, the HVS-BAC promises to be a valuable tool that can be used for the routine and precise production and analysis of viral mutants to further explore gammaherpesvirus biology.
White
Robert E
RE
School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Calderwood
Michael A
MA
School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Whitehouse
Adrian
A
School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
D000818:Animals; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D005822:Genetic Vectors; D016679:Genome, Viral; D006567:Herpesvirus 2, Saimiriine / Q000235:genetics*; D006801:Humans; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D011938:Rec A Recombinases / Q000378:metabolism; D011995:Recombination, Genetic; D020079:Terminal Repeat Sequences / Q000502:physiology*; D017735:Virus Latency; D014779:Virus Replication
D011938:Rec A Recombinases