title: Development of a high-content screen for the identification of inhibitors directed against the early steps of the cytomegalovirus infectious cycle.
abstract: Human cytomegalovirus (CMV) is a latent and persistent virus whose proliferation increases morbidity and mortality of immune-compromised individuals. The current anti-CMV therapeutics targeting the viral DNA polymerase or the major immediate-early (MIE) gene locus are somewhat effective at limiting CMV-associated disease. However, due to low bioavailability, severe toxicity, and the development of drug resistant CMV strains following prolonged treatment, current anti-CMV therapeutics are insufficient. To help address this shortfall, we established a high-content assay to identify inhibitors targeting CMV entry and the early steps of infection. The infection of primary human fibroblasts with a variant of the CMV laboratory strain AD169 expressing a chimeric IE2-yellow fluorescence protein (YFP) (AD169IE2-YFP) provided the basis for the high-content assay. The localization of IE2-YFP to the nucleus shortly following an AD169IE2-YFP infection induced a robust fluorescent signal that was quantified using confocal microscopy. The assay was optimized to achieve outstanding assay fitness and high Z' scores. We then screened a bioactive chemical library consisting of 2080 compounds and identified hit compounds based on the decrease of fluorescence signal from IE2-YFP nuclear expression. The hit compounds likely target various cellular processes involved in the early steps of infection including capsid transport, chromatin remodeling, and viral gene expression. Extensive secondary assays confirmed the ability of a hit compound, convallatoxin, to inhibit infection of both laboratory and clinical CMV strains and limit virus proliferation. Collectively, the data demonstrate that we have established a robust high-content screen to identify compounds that limit the early steps of the CMV life cycle, and that novel inhibitors of early infection events may serve as viable CMV therapeutics.
authors.affiliation: Icahn School of Medicine at Mount Sinai, Department of Microbiology, New York, NY 10029, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Gardner
authors.affiliation: Icahn School of Medicine at Mount Sinai, Department of Microbiology, New York, NY 10029, USA.
authors.forename: Tobias
authors.initials: T
authors.lastname: Cohen
authors.affiliation: Icahn School of Medicine at Mount Sinai, Department of Microbiology, New York, NY 10029, USA.
authors.forename: Veronika
authors.initials: V
authors.lastname: Redmann
authors.affiliation: Icahn School of Medicine at Mount Sinai, Integrated Screening Core, Experimental Therapeutics Institute, One Gustave L. Levy Place, New York, NY 10029, USA.
authors.forename: Zerlina
authors.initials: Z
authors.lastname: Lau
authors.affiliation: Icahn School of Medicine at Mount Sinai, Integrated Screening Core, Experimental Therapeutics Institute, One Gustave L. Levy Place, New York, NY 10029, USA.
authors.forename: Dan
authors.initials: D
authors.lastname: Felsenfeld
authors.affiliation: Icahn School of Medicine at Mount Sinai, Department of Microbiology, New York, NY 10029, USA.
authors.forename: Domenico
authors.initials: D
authors.lastname: Tortorella
mesh_terms: D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003587:Cytomegalovirus / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D004279:DNA, Viral / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism; D008826:Microbial Sensitivity Tests / Q000379:methods*; D054852:Small Molecule Libraries / Q000737:chemistry*; D013328:Strophanthins / Q000494:pharmacology*; D015534:Trans-Activators / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D004279:DNA, Viral; C069186:IE2 protein, Cytomegalovirus; D017874:Immediate-Early Proteins; D054852:Small Molecule Libraries; D013328:Strophanthins; D015534:Trans-Activators; C000505:convallatoxin
keywords: Antivirals; Cardiac glycoside; High throughput; Human cytomegalovirus; Small-molecule screen; Therapeutics