title: Reconstruction of the complete human cytomegalovirus genome in a BAC reveals RL13 to be a potent inhibitor of replication.
abstract: Human cytomegalovirus (HCMV) in clinical material cannot replicate efficiently in vitro until it has adapted by mutation. Consequently, wild-type HCMV differ fundamentally from the passaged strains used for research. To generate a genetically intact source of HCMV, we cloned strain Merlin into a self-excising BAC. The Merlin BAC clone had mutations in the RL13 gene and UL128 locus that were acquired during limited replication in vitro prior to cloning. The complete wild-type HCMV gene complement was reconstructed by reference to the original clinical sample. Characterization of viruses generated from repaired BACs revealed that RL13 efficiently repressed HCMV replication in multiple cell types; moreover, RL13 mutants rapidly and reproducibly emerged in transfectants. Virus also acquired mutations in genes UL128, UL130, or UL131A, which inhibited virus growth specifically in fibroblast cells in wild-type form. We further report that RL13 encodes a highly glycosylated virion envelope protein and thus has the potential to modulate tropism. To overcome rapid emergence of mutations in genetically intact HCMV, we developed a system in which RL13 and UL131A were conditionally repressed during virus propagation. This technological advance now permits studies to be undertaken with a clonal, characterized HCMV strain containing the complete wild-type gene complement and promises to enhance the clinical relevance of fundamental research on HCMV.
authors.affiliation: Section of Medical Microbiology, Department of Infection, Immunity and Biochemistry, School of Medicine, Cardiff University, Cardiff, UK. StantonRJ@cf.ac.uk
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Stanton
authors.forename: Katarina
authors.initials: K
authors.lastname: Baluchova
authors.forename: Derrick J
authors.initials: DJ
authors.lastname: Dargan
authors.forename: Charles
authors.initials: C
authors.lastname: Cunningham
authors.forename: Orla
authors.initials: O
authors.lastname: Sheehy
authors.forename: Sepehr
authors.initials: S
authors.lastname: Seirafian
authors.forename: Brian P
authors.initials: BP
authors.lastname: McSharry
authors.forename: M Lynne
authors.initials: ML
authors.lastname: Neale
authors.forename: James A
authors.initials: JA
authors.lastname: Davies
authors.forename: Peter
authors.initials: P
authors.lastname: Tomasec
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Davison
authors.forename: Gavin W G
authors.initials: GW
authors.lastname: Wilkinson
mesh_terms: D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial; D003587:Cytomegalovirus / Q000235:genetics* / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism / Q000821:virology; D005814:Genes, Viral; D016679:Genome, Viral; D009154:Mutation; D018523:Tropism / Q000235:genetics; D014771:Virion / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication