Construction of an Excisable Bacterial Artificial Chromosome Containing a Full-Length Infectious Clone of Herpes Simplex Virus Type 1: Viruses Reconstituted from the Clone Exhibit Wild-Type Properties In Vitro and In Vivo
Open Access
- 15 January 2003
- journal article
- Published by American Society for Microbiology in Journal of Virology
- Vol. 77 (2) , 1382-1391
- https://doi.org/10.1128/jvi.77.2.1382-1391.2003
Abstract
In recent years, several laboratories have reported on the cloning of herpes simplex virus type 1 (HSV-1) genomes as bacterial artificial chromosomes (BACs) in Escherichia coli and on procedures to manipulate these genomes by using the bacterial recombination machinery. However, the HSV-BACs reported so far are either replication incompetent or infectious, with a deletion of one or more viral genes due to the BAC vector insertion. For use as a multipurpose clone in research on HSV-1, we attempted to generate infectious HSV-BACs containing the full genome of HSV-1 without any loss of viral genes. Our results were as follows. (i) E. coli (YEbac102) harboring the full-length HSV-1 genome (pYEbac102) in which a BAC flanked by loxP sites was inserted into the intergenic region between UL3 and UL4 was constructed. (ii) pYEbac102 was an infectious molecular clone, given that its transfection into rabbit skin cells resulted in production of infectious virus (YK304). (iii) The BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus YK304 by coinfection of Vero cells with YK304 and a recombinant adenovirus, AxCANCre, expressing Cre recombinase. (iv) As far as was examined, the reconstituted viruses from pYEbac102 could not be phenotypically differentiated from wild-type viruses in vitro and in vivo. Thus, the viruses grew as well in Vero cells as did the wild-type virus and exhibited wild-type virulence in mice on intracerebral inoculation. (v) The infectious molecular clone pYEbac102 is in fact useful for mutagenesis of the HSV-1 genome by bacterial genetics, and a recombinant virus carrying amino acid substitutions in both copies of the α0 gene was generated. pYEbac102 will have multiple applications to the rapid generation of genetically engineered HSV-1 recombinants in basic research into HSV-1 and in the development of HSV vectors in human therapy.Keywords
This publication has 66 references indexed in Scilit:
- Selective extraction of polyoma DNA from infected mouse cell culturesPublished by Elsevier ,2004
- Efficient Infection by a Recombinant Kaposi's Sarcoma-Associated Herpesvirus Cloned in a Bacterial Artificial Chromosome: Application for Genetic AnalysisJournal of Virology, 2002
- Human Cytomegalovirus with IE-2 (UL122) Deleted Fails To Express Early Lytic GenesJournal of Virology, 2001
- Molecular Cloning of the Guinea Pig Cytomegalovirus (GPCMV) Genome as an Infectious Bacterial Artificial Chromosome (BAC) in Escherichia coliMolecular Genetics and Metabolism, 2001
- Allele replacement: an application that permits rapid manipulation of herpes simplex virus type 1 genomesGene Therapy, 1999
- The herpes simplex virus amplicon: A new eucaryotic defective-virus cloning-amplifying vectorCell, 1982
- Bacteriophage P1 site-specific recombinationJournal of Molecular Biology, 1981
- Regulation of α genes of herpes simplex virus: Expression of chimeric genes produced by fusion of thymidine kinase with α gene promotersCell, 1981
- The pathogenicity of thymidine kinase-deficient mutants of herpes simplex virus in miceEpidemiology and Infection, 1978
- Characterization of Herpes Simplex Virus Strains Differing in their Effects on Social Behaviour of Infected CellsJournal of General Virology, 1968