Open AccessNovel Yeast Cell-Based Assay To Screen for Inhibitors of Human Cytomegalovirus Protease in a High-Throughput Format
Author(s) -
Valérie Cottier,
Alcide Barberis,
Urs Lüthi
Publication year - 2006
Publication title -
antimicrobial agents and chemotherapy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.07
H-Index - 259
eISSN - 1070-6283
pISSN - 0066-4804
DOI - 10.1128/aac.50.2.565-571.2006
Subject(s) - protease , saccharomyces cerevisiae , biology , human cytomegalovirus , yeast , cleavage (geology) , high throughput screening , enzyme , cell growth , viral replication , biochemistry , microbiology and biotechnology , virology , virus , gene , paleontology , fracture (geology)
The protease encoded by the human cytomegalovirus (HCMV) is an attractive target for antiviral drug development because of its essential function in viral replication. We describe here a cellular assay in the yeastSaccharomyces cerevisiae for the identification of small molecule inhibitors of HCMV protease by conditional growth in selective medium. In this system, the protease cleavage sequence is inserted into theN -(5′-phosphoribosyl)anthranilate isomerase (Trp1p), a yeast protein essential for cell proliferation in the absence of tryptophan. Coexpression of HCMV protease with the engineered Trp1p substrate in yeast cells results in site-specific cleavage and functional inactivation of the Trp1p enzyme, thereby leading to an arrest of cell proliferation. This growth arrest can be suppressed by the addition of validated HCMV protease inhibitors. The growth selection system presented here provides the basis for a high-throughput screen to identify HCMV protease inhibitors that are active in eukaryotic cells.