title: Charge cluster-to-alanine scanning of UL128 for fine tuning of the endothelial cell tropism of human cytomegalovirus.
abstract: The viral genes UL128, UL130, and UL131A have been identified as major determinants of endothelial cell (EC) tropism of human cytomegalovirus (HCMV), with deletion of either gene causing a null phenotype. We hypothesized that a functional scanning of these genes by minor genetic modifications would allow for the generation of mutants with an intermediate phenotype. By combining charge cluster-to-alanine (CCTA) mutagenesis with markerless mutagenesis of a bacterial artificial chromosome-cloned endotheliotropic HCMV strain, we analyzed UL128 in order to identify functional sites and hence enable targeted modulation of the EC tropism of HCMV. A total of nine mutations in eight charge clusters were tested. Three of the CCTA mutations severely reduced EC tropism, three were irrelevant, two had a weak effect on cell tropism, and one mutation in the most C-terminal cluster caused an intermediate phenotype. All of the highly effective mutations were located in a core region (amino acids 72 to 106) which appears to be particularly crucial for EC tropism. The intermediate effect of mutations in the C-terminal cluster could be modulated by varying the number of amino acids replaced with alanine. This study provides a rational approach for targeted modulation of HCMV cell tropism, which may aid in the development of HCMV strains with a desired degree of attenuation.
authors.affiliation: Institut für Medizinische Virologie und Epidemiologie der Viruskrankheiten, Universität Tübingen, Elfriede-Aulhorn-Strasse 6, D-72076 Tübingen, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Schuessler
authors.forename: Kerstin Laib
authors.initials: KL
authors.lastname: Sampaio
authors.forename: Christian
authors.initials: C
authors.lastname: Sinzger