Procedures for the Identification of SARS-CoV-2 Entry Inhibitors as Potential Antivirals using MLV-Based Pseudoviruses

Our research focuses on identifying inhibitors that block SARS-CoV-2 entry using pseudovirus systems. Studying SARS-CoV-2 entry often requires high biosafety containment laboratories, typically BSL-3. This protocol enables safe antiviral screening used in pseudoviruses in a standard BSL-2 laboratories.

To begin, place a six well tissue culture plate in the biosafety cabinet and seed 7.5 times 10 to the power of five HEK 293 derived cells per well in two milliliters of complete medium. Incubate the plate at 37 degrees Celsius with 5%carbon dioxide overnight. The following day, confirm that the cells have reached approximately 50 to 60%confluency.

On the next day, prepare the polyethylenimine or PEI solution by adding five microliters of one milligram per milliliter PEI stock to 50 microliters of reduced serum medium in a 1.5 milliliter micro centrifuge tube. In a separate 1.5 milliliter micro centrifuge tube, prepare the DNA solution by adding 2.5 micrograms of GFP expressing plasmid to 50 microliters of the same medium. Mix each tube briefly by vortexing, then spin down for five seconds and incubate at room temperature.

During the 10 minute incubation period, carefully remove the medium from each well of the six well plate without disrupting the cell monolayer and add one milliliter of complete transfection medium devoid of antibiotics to each well. After the incubation, add 50 microliters of the DNA solution to the tube containing the PEI solution. Mix quickly by pipetting six times and incubate at room temperature for exactly three minutes.

Then quickly add the PEI DNA complex dropwise to the corresponding well. Now, seal the borders of the plate with parafilm and centrifuge at 1000 G for 30 minutes at room temperature without break using a swing rotor and a plate bucket. After centrifugation, gently remove the parafilm and incubate the plate at 37 degrees Celsius with 5%carbon dioxide for three hours.

Then gently replace the transfection medium in each well with two milliliters of prewarm complete DMEM medium. Incubate the transfected cells at 37 degrees Celsius with 5%carbon dioxide for 48 hours, and assess the transfection efficiency by evaluating GFP expression using fluorescence microscopy. Seed HEK 293T derived cells into two 6 well plates as demonstrated earlier.

Designate one plate for SARS-CoV-2 spike pseudo virus generation and the other for VSVG pseudo viruses. Prepare the transfection mix and PEI DNA complexes on the day of the experiment and incubate for 10 minutes at room temperature. During the incubation, carefully remove the medium from each well of the six well plates without disrupting the cell monolayer.

Slowly add one milliliter of prewarmed complete transfection medium without antibiotics to each well, allowing the medium to run down the wall of the well. Add the prepared transfection complexes to the cells to transfect them as demonstrated previously. After 48 hours of incubation post transfection, collect the supernatants from each well into a 50 milliliter sterile conical centrifuge tube and centrifuge at 290 G for seven minutes at four degrees Celsius.

Collect the cell-free supernatant and filter it through a sterile 0.45 micrometer polyvinylidene fluoride top bottle filter unit. Prepare one milliliter aliquots of the filtered supernatant and store them at minus 80 degrees Celsius Seed 2.5 times 10 to the power of four HSI cells, a highly SARS-CoV-2 susceptible cell line in 50 microliters of complete DMEM per well in a 96 well tissue culture plate. Prepare and labeled triplicate wells for each condition to be tested, including sparkless pseudo virus infection and non-infected controls.

Incubate the plate at 37 degrees Celsius with 5%carbon dioxide overnight. On the day of transduction, thaw one aliquot of each pseudo virus batch on ice and gently mix by inversion, including one aliquot of the spikeless control. Add the required volumes of pseudo virus preparation directly to the designated wells.

Adjust the volume of each well to 150 microliters with complete DMEM. Now, seal the plate with parafilm and centrifuge at 2, 500 G for 45 minutes at room temperature without break. Remove the parafilm seal and incubate the plate for 72 hours at 37 degrees Celsius with 5%carbon dioxide.

After incubation, prepare the luciferase assay reagents according to the manufacturer's instructions. Thaw the luciferase substrate and the 5X luciferase assay lysis buffer at room temperature for 10 to 15 minutes. Dilute the 5X luciferase assay lysis buffer to 1X with sterile water.

Carefully aspirate and discard the supernatant. Next, add 30 microliters of 1X lysis buffer to each well. Place the plate on a plate shaker and shake for 10 minutes at 150 revolutions per minute at room temperature.

Verify complete cell lysis under an inverted microscope to confirm that cells are fully lysed and not distinguishable. Then transfer the lysates to an opaque white 96 well plate. Add 50 microliters of luciferase substrate to each well.

Mix briefly by gently moving the plate in a circular motion and place the plate into the luminometer and measure luminescence. Seed 2.5 times 10 to the power of four HSI cells in 50 microliters of complete DMEM per well in a 96 well plate as previously described. Incubate overnight at 37 degrees Celsius with 5%carbon dioxide.

The next day prepare one to three serial dilution of the compounds to be tested such as arbidol in complete DMEM containing 0.5%dimethyl sulfoxide. Using a multi-channel pipette, gently aspirate the medium from each well. Add 50 microliters of the corresponding compound dilution to each well while adding complete DMEM alone to the column 12 control wells.

Incubate for one hour at 37 degrees Celsius with 5%carbon dioxide. Without removing the compounds, directly add 50 microliters of SARS-CoV-2 spike pseudo virus to each well diluted in complete medium containing the same compound concentrations. Seal the plate with parfilm.

Centrifuge at 2, 500 G for 45 minutes at room temperature without break. After removing the parfilm, incubate the plate at 37 degrees Celsius with 5%carbon dioxide for 72 hours. Finally, quantify infectivity using a luciferase assay as demonstrated previously.

Transfection of HEK 293T derived cells with branched PEI resulted in high transfection efficiency resulting in roughly 70%GFP positive cells. 48 hours after transfection. Infection of susceptible HSI cells with decreasing amounts of SARS-CoV-2 spike pseudo viruses or VSVG pseudo viruses showed a dose dependent increase in luminescence with increasing pseudo virus input.

Spike less pseudo viruses and uninfected cells showed very low levels of luminescence. The compound arbidol was tested for its ability to inhibit pseudo viral entry and it produced clear dose response inhibition of luciferase activity generated by spike pseudo viruses with less inhibition observed for VSVG pseudo viruses. The assay showed a Z prime factor of 0.54, indicating suitability for screening purposes.

Treatment with arbidol at concentrations starting at 30 micromolar did not significantly affect cell viability compared to the cells treated with 0.5%dimethyl sulfoxide vehicle alone. This protocol enabled the production of high titer pseudo viruses through optimized conditions. This allows researchers to measure pseudo viral entry and identify compounds that inhibit SARS-CoV-2 spike mediated infection.

High transfection efficiency is critical to generate high titer pseudo viruses and obtain reliable infection signals in downstream assays. This system supports high throughput antiviral screening and devaluation of entry inhibitors against emerging SARS-CoV-2 variants and can be readily adapted to generate pseudo viruses displaying envelope proteins from other viruses.