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J. Virol. doi:10.1128/JVI.01069-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Charge-cluster-to-alanine scanning of UL128 for fine tuning of the endothelial cell tropism of human cytomegalovirus

Institute of Medical Virology and Epidemiology of Virus Diseases, University of Tuebingen, D-72076 Tuebingen, Germany

^* To whom correspondence should be addressed. Email: christian.sinzger{at}med.uni-tuebingen.de.

	Abstract

The viral genes UL128, UL130 and UL131A have been identified as major determinants of endothelial cell (EC) tropism of human cytomegalovirus (HCMV), 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 BAC-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 9 mutations in 8 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.