title: Cloning and sequencing of a highly productive, endotheliotropic virus strain derived from human cytomegalovirus TB40/E.
abstract: Human cytomegalovirus (HCMV) strain TB40/E, replicates efficiently, exhibits a broad cell tropism and is widely used for infection of endothelial cells and monocyte-derived cells yet has not been available in a phenotypically homogeneous form compatible with genetic analysis. To overcome this problem, we cloned the TB40/E strain into a bacterial artificial chromosome (BAC) vector. Both highly endotheliotropic and poorly endotheliotropic virus clones, representing three distinct restriction fragment patterns, were reconstituted after transfection of BAC clones derived from previously plaque-purified strain TB40/E. For one of the highly endotheliotropic clones, TB40-BAC4, we provide the genome sequence. Two BACs with identical restriction fragment patterns but different cell tropism were further analysed in the UL128-UL131A gene region. Sequence analysis revealed one coding-relevant adenine insertion at position 332 of UL128 in the BAC of the poorly endotheliotropic virus, which caused a frameshift in the C-terminal part of the coding sequence. Removal of this insertion by markerless mutagenesis restored the highly endotheliotropic phenotype, indicating that the loss of endothelial cell tropism was caused by this insertion. In conclusion, HCMV strain TB40/E, which combines the high endothelial cell tropism of a clinical isolate with the high titre growth of a cell culture adapted strain, is now available as a BAC clone suitable for genetic engineering. The results also suggest BAC cloning as a suitable method for selection of genetically defined virus clones.
authors.affiliation: Institut für Medizinische Virologie, Eberhard-Karls-Universität, Tübingen, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Sinzger
authors.affiliation: Laboratoriumsmedizin, Klinikum Ingolstadt, Germany.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Hahn
authors.affiliation: Institut für Medizinische Virologie, Eberhard-Karls-Universität, Tübingen, Germany.
authors.forename: Margarete
authors.initials: M
authors.lastname: Digel
authors.affiliation: Institut für Medizinische Virologie, Eberhard-Karls-Universität, Tübingen, Germany.
authors.forename: Ruth
authors.initials: R
authors.lastname: Katona
authors.affiliation: Institut für Medizinische Virologie, Eberhard-Karls-Universität, Tübingen, Germany.
authors.forename: Kerstin Laib
authors.initials: KL
authors.lastname: Sampaio
authors.affiliation: Abteilung für Virologie, Medizinische Hochschule Hannover, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Messerle
authors.affiliation: Institut für Virologie, Heinrich-Heine-Universität, Düsseldorf, Germany.
authors.forename: Hartmut
authors.initials: H
authors.lastname: Hengel
authors.affiliation: Max von Pettenkofer-Institut für Virologie, Ludwig-Maximilians-Universität München, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Koszinowski
authors.affiliation: Fachgebiet Virale Infektionen, Robert Koch-Institut, Berlin, Germany.
authors.forename: Wolfram
authors.initials: W
authors.lastname: Brune
authors.affiliation: Max von Pettenkofer-Institut für Virologie, Ludwig-Maximilians-Universität München, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Adler
mesh_terms: D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000145:classification / Q000235:genetics*; D042783:Endothelial Cells / Q000821:virology*; D005347:Fibroblasts / Q000821:virology; D016679:Genome, Viral; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017421:Sequence Analysis; D018523:Tropism