Construction and Characterization of an Infectious Murine Gammaherpesivrus-68 Bacterial Artificial Chromosome

Here we describe the cloning of a sequenced WUMS isolate of murine gammaherpesvirus-68 (MHV-68, γHV-68, also known as MuHV-4) as a bacterial artificial chromosome (BAC). We engineered the insertion of the BAC sequence flanked by loxP sites into the left end of the viral genome before the M1 open rea...

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Bibliographic Details
Published in:BioMed research international 2011-01, Vol.2011 (2011), p.1-11
Main Authors: Liao, Hsiang-I, Wu, Ting-Ting, Tong, Leming, Leang, Ronika Sitapara, Smith, Greg, Sun, Ren
Format: Article
Language:English
Subjects:
Animals
Care and treatment
Cell Line
Chromosomes, Artificial, Bacterial - genetics
Cloning, Molecular - methods
Cricetinae
Electrophoresis, Agar Gel
Female
Gammaherpesvirinae - genetics
Gammaherpesvirinae - pathogenicity
Genes
Genome, Viral
Genomes
Infection
Lung - virology
Mice
Mice, Inbred BALB C
Microbiology
Mutagenesis, Site-Directed
Mutation
Open Reading Frames - genetics
Proteins
Risk factors
Spleen - virology
Viral infections
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Summary:Here we describe the cloning of a sequenced WUMS isolate of murine gammaherpesvirus-68 (MHV-68, γHV-68, also known as MuHV-4) as a bacterial artificial chromosome (BAC). We engineered the insertion of the BAC sequence flanked by loxP sites into the left end of the viral genome before the M1 open reading frame. The infectious viruses were reconstituted following transfection of the MHV-68 BAC DNA into cells. The MHV-68 BAC-derived virus replicated indistinguishably from the wild-type virus in cultured cells. Excision of the BAC insert was efficiently achieved by coexpressing the Cre recombinase. Although the BAC insertion did not significantly affect acute productive infection in the lung, it severely compromised the ability of MHV-68 to establish splenic latency. Removal of the BAC sequence restored the wild-type level of latency. Site-specific mutagenesis was carried out by RecA-mediated recombination to demonstrate that this infectious BAC clone can be used for genetic studies of MHV-68.
ISSN:1110-7243
2314-6133
1110-7251
2314-6141
DOI:10.1155/2011/926258