High-content screening to distinguish between attachment and post-attachment steps of human cytomegalovirus entry into fibroblasts and epithelial cells
| Autor: | David Woodhall, Adam L. Feire, Marjo Götte, Yvonne Ibig-Rehm, Teresa Compton, Nathan Brown, Daniela Gabriel, Ashley Shea |
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| Rok vydání: | 2010 |
| Předmět: |
Human cytomegalovirus
Cell type viruses Green Fluorescent Proteins Drug Evaluation Preclinical Cytomegalovirus Biology medicine.disease_cause Antiviral Agents Virus Herpesviridae Green fluorescent protein Viral entry Genes Reporter Virology medicine Humans Pharmacology Staining and Labeling Epithelial Cells Fibroblasts Virus Internalization medicine.disease Entry inhibitor High-content screening medicine.drug |
| Zdroj: | Antiviral research. 89(3) |
| ISSN: | 1872-9096 |
| Popis: | Human cytomegalovirus (HCMV) enters cells through a complex pathway involving the interaction of multiple viral glycoproteins and cellular receptors. While HCMV clinical isolates enter a wide range of cell types, entry has historically been studied using a laboratory strain of virus that can only infect fibroblasts. Herein, we have constructed a HCMV reporter strain that contains GFP fused to the abundant tegument protein pp65 to allow for the direct visualization of virus attachment and entry. Furthermore, the UL131 gene of this strain was restored to clinical isolate sequence to expand our studies of entry into physiologically relevant epithelial cell types. Using the HCMV-GFP reporter virus, we developed an image-based assay and screened a library containing 65,000 compounds for the inhibition of virus entry into fibroblasts. In addition to assessing the effect on virus entry, automated image analysis provided information on compound toxicity and whether the compounds acted as attachment or post-attachment inhibitors. To identify therapeutically viable inhibitors capable of blocking entry in multiple cell types, the inhibitors were screened further for their ability to inhibit virus entry into epithelial cells. Compounds were identified that were able to inhibit virus entry into both cell types at either attachment or post-attachment steps. |
| Databáze: | OpenAIRE |
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