An Assay to Evaluate the Effect of Test Compounds on Viral Entry and Fusion in Host Cells

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 glycosaminoglycans during the early viral entry steps. Heparin, which is known to interfere with the entry of many viruses, is used as a positive control t....

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
MV.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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