human cytomegaloviruses expressing yellow fluorescent fusion proteins - characterization and use in antiviral screening人类巨细胞病毒表达黄色荧光融合蛋白-表征和使用抗病毒药物筛选.pdfVIP

Human Cytomegaloviruses Expressing Yellow Fluorescent Fusion Proteins - Characterization and Use in Antiviral Screening 1 3 1 3 1 Sarah Straschewski , Martin Warmer , Giada Frascaroli , Heinrich Hohenberg , Thomas Mertens , Michael Winkler1,2* ¨ 1 Institute of Virology, Ulm University Hospital, Ulm, Germany, 2 Institute for Infection Medicine, Universitatsklinikum Schleswig-Holstein Campus Kiel, Kiel, Germany, 3 Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg, Hamburg, Germany Abstract Recombinant viruses labelled with fluorescent proteins are useful tools in molecular virology with multiple applications (e.g., studies on intracellular trafficking, protein localization, or gene activity). We generated by homologous recombination three recombinant cytomegaloviruses carrying the enhanced yellow fluorescent protein (EYFP) fused with the viral proteins IE-2, ppUL32 (pp150), and ppUL83 (pp65). In growth kinetics, the three viruses behaved all like wild type, even at low multiplicity of infection (MOI). The expression of all three fusion proteins was detected, and their respective localizations were the same as for the unmodified proteins in wild-type virus–infected cells. We established the in vivo measurement of fluorescence intensity and used the recombinant viruses to measure inhibition of viral replication by neutralizing antibodies or antiviral substances. The use of these viruses in a pilot screen based on fluorescence intensity and high-content analysis identified cellular kinase inhibitors that block viral replication. In summary, these viruses w