title: Construction and characterization of an infectious murine gammaherpesivrus-68 bacterial artificial chromosome.
abstract: 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.
authors.affiliation: Department of Molecular and Medical Pharmacology, School of Medicine, University of California at Los Angeles, 650 Charles E Young Drive, CHS 23-120, Los Angeles, CA 90095, USA.
authors.forename: Ting-Ting
authors.initials: TT
authors.lastname: Wu
authors.forename: Hsiang-I
authors.initials: HI
authors.lastname: Liao
authors.forename: Leming
authors.initials: L
authors.lastname: Tong
authors.forename: Ronika Sitapara
authors.initials: RS
authors.lastname: Leang
authors.forename: Greg
authors.initials: G
authors.lastname: Smith
authors.forename: Ren
authors.initials: R
authors.lastname: Sun
mesh_terms: D000818:Animals; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D004587:Electrophoresis, Agar Gel; D005260:Female; D018142:Gammaherpesvirinae / Q000235:genetics* / Q000472:pathogenicity; D016679:Genome, Viral; D008168:Lung / Q000821:virology; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames / Q000235:genetics; D013154:Spleen / Q000821:virology