Name: an assay to evaluate the effect of test compounds on viral entry and fusion in host cells
Uploaded: 2024-02-29T13:13:41.000+00:00
Duration: 1 min 26 s
Description: Note: Ensure that all procedures involving cell culture and virus infection are conducted in certified biosafety hoods that are appropriate for the ...

an assay to evaluate the effect of test compounds on viral entry and fusion in host cells

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

For assaying viral fusion and entry, pre-chill the cell culture plate at 4 degrees Celsius for one hour after the overnight incubation for cell attachment. Remove the existing medium from the wells, and infect the cells with 10 to the second FFU HCV on ice.
Incubate the plate at 4 degrees Celsius for three hours. Gently wash the cells twice with 200 microliters of ice-cold PBS to remove the non-adhered virus. Then, on ice, treat the cells with a test compound dissolved in basal medium or basal medium with 1% DMSO as a control, and incubate the plate at 37 degrees Celsius for three hours. This allows assessment of the test compounds on viral entry, which occurs at 37 degrees Celsius.
Aspirate the medium and wash the non-internalized viruses twice with 200 microliters of citrate buffer or PBS. Add 100 microliters of basal medium per well, and incubate at 37 degrees Celsius for another 72 hours. Collect the supernatant and perform the Gaussia luciferase assay as demonstrated before for detecting the released luciferase reporter.

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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 treatment in such a context.
1. Cell Culture, Compound Preparation, and Compound Cytotoxicity
<ol>
	<li>Grow the respective cell line for the virus infection system to be analyzed (Table 1). For HCV, grow the Huh-7.5 cells in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 200 U/ml penicillin G, 200 &#181;g/ml streptomycin, and 0.5 &#181;g/ml amphotericin B.</li>
	<li>Prepare the test compounds and controls using their respective solvents: for example, dissolve CHLA and PUG in dimethyl sulfoxide (DMSO); prepare heparin in sterile double-distilled water. For all subsequent dilutions, use culture media. 
	Note: The final concentration of DMSO in the test compound treatments is less than 1% in the experiments; 1% DMSO is included as a negative control treatment in the assays for comparison.</li>
	<li>Determine the cytotoxicity of the test compounds (e.g., CHLA and PUG) on the cells for the viral infection by using cell viability determining reagent such as XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-phenylamino)-carbonyl]-2H-tetrazolium hydroxide):
	<ol>
		<li>For HCV, seed Huh-7.5 cells in a 96-well plate (1 &#215; 104&#160;cells per well) and incubate at 37 &#176;C in a 5% CO2&#160;incubator O/N to obtain a monolayer.</li>
		<li>Apply DMSO control (1%) or increasing concentrations of the test compounds CHLA and PUG (ex. 0, 10, 50, 100, and 500 &#956;M) to the culture wells in triplicate.</li>
		<li>Incubate at 37 &#176;C for 72 hr, then discard the medium in the plate and wash the cells with 200 &#956;l of phosphate-buffered saline (PBS) twice.</li>
		<li>Add 100 &#181;l of assaying solution from the XTT-based&#160;in vitro&#160;toxicology assay kit to each well and incubate the plates at 37 &#176;C for another 3 hr to allow XTT formazan production.</li>
		<li>Determine the absorbance with a microplate reader at a test wavelength of 492 nm and a reference wavelength of 690 nm.</li>
		<li>Calculate the percentage of surviving cells using the following formula: cell viability (%) = At / As &#215; 100%, where &#39;At&#39; and &#39;As&#39; refer to the absorbance of the test compounds and the solvent control (ex. 1% DMSO) treatments, respectively. Determine the concentration of 50% cellular cytotoxicity (CC50) of the test compounds from an analytical software such as GraphPad Prism according to the manufacturer&#39;s protocol.</li>
	</ol>
	</li>
</ol>
2. Readout of Viral Infection
Note: The readout of viral infection depends on the virus system used and can involve methods such as plaque assays or measuring reporter signals from reporter-tagged viruses. The method for detecting reporter-HCV infection based on the luciferase reporter activity is described below.
<ol>
	<li>Collect the supernatants from the infected wells and clarify at 17,000 x g in a microcentrifuge for 5 min at 4 &#176;C.</li>
	<li>Mix 20 &#956;l of test supernatant to 50 &#956;l of luciferase substrate from the Gaussia luciferase assay kit and directly measure with a luminometer according to the manufacturer&#39;s instructions.</li>
	<li>Express HCV infectivity as log10 of relative light units (RLU) for determining viral inhibition (%) and calculate the 50% effective concentration (EC50) of the test compounds against HCV infection using algorithms from GraphPad Prism software according to the manufacturer&#39;s protocol.</li>
</ol>
3. Viral Entry/Fusion Assay
Note: Examples of incubation periods and viral doses for various viruses are listed in Figure 1A &#39;Entry/Fusion&#39;. Higher concentrations of the virus can also be tested by increasing the MOI/PFU.
<ol>
	<li>Seed Huh-7.5 cells in a 96-well plate (1 &#215; 104 cells per well), and incubate at 37 &#176;C in a 5% CO2 incubator O/N to obtain a monolayer.</li>
	<li>Pre-chill the cell monolayers in plates at 4 &#176;C for 1 hr.</li>
	<li>Infect the cells with HCV (multiplicity of infection, MOI = 0.01) at 4 &#176;C for 3 hr. For example, use a 100 &#956;l virus inoculum containing 1 x 102 focus forming units (FFU). 
	Note: Perform the addition of the viral inoculum on ice and the ensuing incubation in a 4 &#176;C refrigerator to maintain the temperature at 4 &#176;C, which permits viral binding but not entry.</li>
	<li>Remove the supernatant and gently wash the cell monolayers with 200 &#956;l of ice-cold PBS twice. 
	Note: Perform the washes gently to avoid lifting the cells.</li>
	<li>Treat the wells with the test compounds or controls (final concentrations are: CHLA = 50 &#956;M; PUG = 50 &#956;M; heparin = 1,000 &#956;g/ml; DMSO = 1%) and incubate at 37 &#176;C for 3 hr. For example, add 10 &#956;l of a 500 &#956;M CHLA working dilution to 90 &#956;l of media, mix, and treat the wells; this yields CHLA treatment at a final concentration of 50 &#956;M. 
	Note: The shift from 4 &#176;C to 37 &#176;C now facilitates the viral entry/fusion event and therefore allows assessment of test compounds&#39; effect on this particular step.</li>
	<li>Aspirate the drug-containing supernatant and remove non-internalized extracellular viruses by either washing with 200 &#956;l of citrate buffer (50 mM sodium citrate, 4 mM potassium chloride, pH 3.0) or PBS. Apply 100 &#956;l of the basal medium before incubating at 37 &#176;C for 72 hr.</li>
	<li>Analyze the resulting infection by assaying the supernatant for luciferase activity as described in &#39;2. Readout of Viral Infection&#39;.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>GIBCO</td>
			<td>11995-040</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>GIBCO</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>GIBCO</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>GIBCO</td>
			<td>15290-018</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D5879</td>
			<td></td>
		</tr>
		<tr>
			<td>In vitro toxicology assay kit, XTT-based</td>
			<td>Sigma</td>
			<td>TOX2</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS pH 7.4</td>
			<td>GIBCO</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Bio-Tek Instrument, Inc.</td>
			<td>ELx800</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Scientific</td>
			<td>75002420</td>
			<td></td>
		</tr>
		<tr>
			<td>BioLux Gaussia luciferase assay kit</td>
			<td>New England Biolabs</td>
			<td>E3300L</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Promega</td>
			<td>GloMax-20/20</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate, dihydrate</td>
			<td>Sigma</td>
			<td>71402</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Tai, C., et al. <a href="https://app.jove.com/v/53124">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> 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.

Table 1: Host cell type for viral infection.&#160;The cell type used for each viral infection system described in the representative results is indicated.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Virus</td>
			<td>Cell Type</td>
		</tr>
		<tr>
			<td>HCMV</td>
			<td>HEL</td>
		</tr>
		<tr>
			<td>HCV</td>
			<td>Huh-7.5</td>
		</tr>
		<tr>
			<td>DENV-2</td>
			<td>Vero</td>
		</tr>
		<tr>
			<...

Note: Ensure that all procedures involving cell culture and virus infection are conducted in certified biosafety hoods that are appropriate for the ...

Take liver cancer cells, and incubate at a low temperature to impair endocytosis.
Introduce a recombinant Hepatitis C virus or HCV, comprising an engineered RNA genome encoding a secreted luciferase reporter.
The viral lipoproteins interact with the proteoglycans on the host cells, while the viral envelope glycoproteins bind to specific host receptors.
In selected wells, add the test compound that binds to viral glycoproteins.
Upon incubation at physiological temperature, the bound viruses are endocytosed.
Discard the non-internalized viruses, add a growth media, and incubate.
In the untreated wells, the glycoproteins aid viral envelope-endosome fusion, releasing the nucleocapsid, while compound masking impairs fusion in the treated wells.
Upon release, the RNA expresses viral proteins, including luciferase which is secreted extracellularly.
Harvest the luciferase-containing supernatant and add a bioluminescent substrate, which is oxidized by luciferase to emit light.
A decreased luminescence in the treated wells compared to the untreated indicates inhibition of viral envelope-endosome fusion.

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This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.

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

Transcription

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