Cloning and sequencing of a highly productive, endotheliotropic virus strain derived from human cytomegalovirus TB40/E.
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.
Sinzger
Christian
C
Institut für Medizinische Virologie, Eberhard-Karls-Universität, Tübingen, Germany.
Hahn
Gabriele
G
Laboratoriumsmedizin, Klinikum Ingolstadt, Germany.
Digel
Margarete
M
Institut für Medizinische Virologie, Eberhard-Karls-Universität, Tübingen, Germany.
Katona
Ruth
R
Institut für Medizinische Virologie, Eberhard-Karls-Universität, Tübingen, Germany.
Sampaio
Kerstin Laib
KL
Institut für Medizinische Virologie, Eberhard-Karls-Universität, Tübingen, Germany.
Messerle
Martin
M
Abteilung für Virologie, Medizinische Hochschule Hannover, Germany.
Hengel
Hartmut
H
Institut für Virologie, Heinrich-Heine-Universität, Düsseldorf, Germany.
Koszinowski
Ulrich
U
Max von Pettenkofer-Institut für Virologie, Ludwig-Maximilians-Universität München, Germany.
Brune
Wolfram
W
Fachgebiet Virale Infektionen, Robert Koch-Institut, Berlin, Germany.
Adler
Barbara
B
Max von Pettenkofer-Institut für Virologie, Ludwig-Maximilians-Universität München, Germany.
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