An Assay to Evaluate the Effect of Test Compounds on Viral Entry and Fusion in Host Cells
Published: February 29, 2024
Abstract
Source: Tai, C., et al. Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds. J. Vis. Exp. (2015).
This video demonstrates an assay to evaluate antiviral agents that target specific steps of the viral entry into host cells and fusion with the endosome. The cells are incubated at a low temperature and then infected with HCV. The low temperature permits viral binding to host cells but inhibits internalization via endocytosis. The test antiviral compound is added, and the cells are incubated at physiological temperature. This shift to a higher temperature facilitates viral entry and allows assessment of the impact of the test compound on viral envelope fusion with endosomes.
Protocol
Note: Ensure that all procedures involving cell culture and virus infection are conducted in certified biosafety hoods that are appropriate for the biosafety level of the samples being handled. For the purpose of describing the protocols, Gaussia luciferase reporter-tagged HCV is used as a model virus. In the context of the representative results, the compounds chebulagic acid (CHLA) and punicalagin (PUG) are used as candidate antivirals that target viral glycoprotein interactions with cell surface glyco…
Representative Results
Table 1: Host cell type for viral infection. The cell type used for each viral infection system described in the representative results is indicated. Virus Cell Type HCMV HEL HCV Huh-7.5 DENV-2 Vero <…
Disclosures
The authors have nothing to disclose.
Materials
| DMEM | GIBCO | 11995-040 | |
| FBS | GIBCO | 26140-079 | |
| Penicillin-Streptomycin | GIBCO | 15070-063 | |
| Amphotericin B | GIBCO | 15290-018 | |
| DMSO | Sigma | D5879 | |
| In vitro toxicology assay kit, XTT-based | Sigma | TOX2 | |
| PBS pH 7.4 | GIBCO | 10010023 | |
| Microplate reader | Bio-Tek Instrument, Inc. | ELx800 | |
| Microcentrifuge | Thermo Scientific | 75002420 | |
| BioLux Gaussia luciferase assay kit | New England Biolabs | E3300L | |
| Luminometer | Promega | GloMax-20/20 | |
| Sodium citrate, dihydrate | Sigma | 71402 | |
| Potassium chloride | Sigma | P5405 |
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Cite This Article
An Assay to Evaluate the Effect of Test Compounds on Viral Entry and Fusion in Host Cells. J. Vis. Exp. (Pending Publication), e22000, doi: (2024).