key: cord-0773523-fpdx4klo
authors: Eichler, Madeleine; Aksi, Ebru; Pfeilschifter, Josef; Imre, Gergely
title: Application of pseudotyped virus particles to monitor Ebola virus and SARS-CoV-2 viral entry in human cell lines
date: 2021-08-27
journal: STAR Protoc
DOI: 10.1016/j.xpro.2021.100818
sha: 03c17376cbb4ace4952545bc4b512d05ec0623f6
doc_id: 773523
cord_uid: fpdx4klo

Experimental work on highly pathogenic viruses such as Ebola virus (EBOV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) requires high-level biosafety facilities. Here we provide a detailed step-by-step protocol which details the production and application of replication-incompetent murine leukemia virus (MLV)-based pseudotyped particles to monitor and quantify the viral entry efficiency in human cell lines under bio safety level-2 conditions. We describe the use of viral particles encoding luciferase gene and the quantification of transduction efficiency by measuring luciferase activity.

Experimental work on highly pathogenic viruses such as Ebola virus (EBOV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) requires high-level biosafety facilities. Here we provide a detailed step-by-step protocol which details the production and application of replicationincompetent murine leukemia virus (MLV)-based pseudotyped particles to monitor and quantify the viral entry efficiency in human cell lines under bio safety level-2 conditions. We describe the use of viral particles encoding luciferase gene and the quantification of transduction efficiency by measuring luciferase activity.

For complete details on the use and execution of this protocol, please refer to Imre et al., 2021.

Here, we describe a protocol for the production of murine leukemia virus (MLV) based pseudoviruses. To generate pseudotyped vectors of highly pathogenic viruses, lentiviral vectors such as human immunodeficiency virus (HIV) and simian immunodeficiency virus based vectors are commonly employed as a first choice. Apart from the safety concerns of HIV based particles, it has been reported that in several cases higher yield can be achieved by the application of vesicular stomatitis virus (VSV) and MLV derived systems when compared to lentiviral vectors (Temperton et al., 2007; Cosset et al., 2009) . In comparison to the VSV based viral vectors MLV-pseudoviruses are smaller. VSV viral particles are 200 nm in length and have a slightly elongated structure. In contrast, MLV viral vectors are 80-100 nm in size and have a spherical shape (Chen et al., 2019) . The smaller size of the viral particles can result in higher yields and improved filtration efficacy of the viral particles. Similarly to replication incompetent lentiviral and VSV pseudoviruses, MLV pseudoviruses accomplish a single round of infection, since instead of harboring viral RNA the viral particles only harbor the firefly luciferase RNA (Millet et al., 2019) . Upon successful transduction of the MLV particles, the Luciferase gene integrates in the host genome and the light generating enzyme Luciferase is produced. This enables quantitative analysis of pseudovirus infectivity by measuring luciferase activity proportional to the detected luminescence intensity.

Any work involving genetically modified virus particles should be approved by national and institutional biosafety committees. Before starting, please make sure that your lab complies all the necessary criteria and documentations required to accomplish biosafety Level-2 (BSL-2) work.

Timing: 5-7 days 1. Thaw HEK293T, EA.hy926 and HuH-7 cells ( Figure 1A b. EA.hy926 and HuH-7 cells: Remove medium and rinse the cells twice with 5-5 ml 1x DPBS.

Add 3.5 ml Trypsin/EDTA (0.05%), rinse the cells by gently swirling the cell culture flask and remove the rest of the Trypsin/EDTA solution, leaving just a thin layer covering the whole surface. Incubate it for 5 minutes at 37 o C. Resuspend the cells in fresh, prewarmed complete medium. The splitting ratios for the cells are the following: HEK293T: 1:5; EA.hy926: 1:10; HuH-7: 1:5.

Timing: 3-4 days 4. Four plasmids need to be prepared in advance ( Figure 2 ): a. Plasmid containing the Murine leukemia virus (MLV) gag and pol genes: pCMV-MLVgag-pol (Bartosch et al., 2003; Millet et al., 2016; Millet et al., 2019) .

b. Reporter vector encoding the firefly luciferase (Luc) gene with a viral packaging sequence: pCMV-Luc (Bartosch et al., 2003; Millet et al., 2016; Millet et al., 2019) .

c. Plasmids encoding viral envelope proteins of interests: pcDNA3.1-Ebola virus glycoprotein (EBOV-GP) or pcDNA3.1-SARS-CoV-2-Spike (SARS-CoV-2-S). d. Empty vector as control plasmid: pcDNA3-Flag-HA. 5. Transform bacterial competent cells XL-1 Blue (Agilent) by employing standard heat shock transformation procedure. 6. Grow the transformed bacteria on agar plates containing ampicillin (100 µg/ml) overnight (16 h) at 37 o C. 7. Pick a single colony and incubate it in 100 ml LB media supplemented with 100 μg/ml ampicillin overnight at 37 o C in a shaker incubator. 8. Lyse the bacteria and extract the produced plasmid DNAs by employing standard midi prep columns. We use Nucleo Bond DNA extraction kit (Macherey Nagel, Cat#740573.100). 9. Determine the concentration of the plasmid DNAs by nanodrop method and adjust it to 1 µg/ml. 

Add the following ingredients to RPMI1640 (containing 2 mM glutamine, 2 g/l glucose):

Final concentration FBS 10 % Streptomycin 100 µg/ml Penicillin 100 units/ml Storage at 4 o C (max. till date of expire)

Alternatives: We have employed Luciferase Lysis Buffer (Cat#E1531) provided by the company Promega, however lysis buffer can be also self-prepared by using the following recipe:

Final concentration Tris-phosphate (pH 7.8) 25 mM Dithiothreitol (DTT) 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (DCTA) 2 mM Glycerol 10 % (w/v) Triton X-100 1 % (w/v) Note: Store Luciferase Lysis Buffer at -20 °C (max. 1 month) or at -80 °C (max. 1 year).

Alternatives: We have used Luciferase Assay System, including Luciferase Assay Substrate (luciferin, lyophilized) and Luciferase Assay Buffer for Luciferase activity detection (Cat#E1500, Promega), however alternatively the following Luciferase Substrate Protocol adapted from Ratnapriya et al. 2020 can also be applied: 

Dissolve lyophilized D-Luciferin (#122799, Perkin Elmer) in ddH2O adjusted to pH 6.3. Prepare aliquots with 225 µg/ml concentration and store them at -20 °C (max. 1 month) or at -80 °C (max. 1 year).

Step-by-step method details

In these steps, the MLV-EBOV-GP or MLV-SARS-CoV-2-S pseudovirus particles will be produced by employing co-transfection of three plasmids in HEK293T cells.

1. Day 1. Keep HEK293T cells in cell culture for at least 2 passages (Ratnapriya et al., 2020) and maximum 20 passages before starting the experiment. The cells should grow exponentially and exhibit healthy morphology ( Figure 1A ). Wash HEK293T cells gently with 1x DPBS and resuspend them in fresh complete DMEM medium.

2. Detach the cells with repeated up and down pipetting.

3. Count the cells by using a hemocytometer and adjust the cell concentration to 5x10 5 cells/well in a 6-well plate. Ideally, the cells should reach 50-60% confluency at the day of transfection.

Critical: At this point, continue the work in a BSL-2 facility. Before starting, make sure that all the required equipment for BSL-2 work, waste management and decontamination are available and properly function. Prepare all the necessary solutions for decontamination in a sufficient amount.

a. Replace the medium with 1 ml fresh complete DMEM medium/well. b. Prepare the transfection complex solution in a 1.5 ml tube. Table 1 shows the amount of reagents needed for 1 well of a 6-well plate. After mixing the OPTI-MEM and Genejuice, briefly vortex and spin (200 x g, 30 sec.) the solution, and incubate it for 5 minutes at room temperature (RT; 20-23 ℃).

Final Amount OPTI-MEM 100 µl Genejuice 7.2 µl pCMV-MLV-gag-pol 0.6 µg pCMV-Luc 1.2 µg Viral envelope protein vector/control vector 0.6 µg Table 1 . Transfection complex solution Components of the transfection mixture. The final amounts for the transfection complex solution are sufficient for 1 well of a 6-well plate. The ratio of total plasmid to Genejuice is 1:3.

Alternative: Other commercially available or self-prepared transfection reagents can also be employed. We describe the protocol by employing Genejuice transfection reagent. 7. Thaw the pseudoviral supernatants and pool the two batches. Next, filter them by employing a 0.45 µm PVDF filter unit to remove cell debris. Store the filtered pseudoviral supernatants at -80 o C.

Optional: Centrifugation can be employed prior to filtration to remove cell debris (600-900 x g for 8 minutes at 4 o C). This might help to avoid clotting of the cells in the filter.

Note: Prepare aliquots of the pseudoviral supernatants (e.g. 1-1 ml), which can be completely used up for a single experiment in order to avoid freeze-thaw cycles.

Pause Point: At this point, viral supernatants can be kept at -80 o C until further usage.

8. In order to confirm efficient transfection, wash and lyse the remaining cells (1 well/6-well) in plate employing 55 µl luciferase lysis buffer. Transfer 50 µl cell lysate to a 96-well white microtiter plate and incubate it for 15 minutes at RT. Add 90 µl luciferin substrate to the lysates and measure the J o u r n a l P r e -p r o o f luciferase activity immediately by a luminometer with 500 ms integration. We typically measure ~8-10x10 7 Relative Luminescence Unit (RLU) in 1 well of a 6-well plate of HEK293T cells.

We employed EA.hy926 endothelial hybridoma cells because they are susceptible to Ebola virus infection (Imre et al., 2021) . In these steps, human EAhy.926 cells are transduced by MLV-EBOV-GP pseudoviral particles. The pseudovirus entry efficiency is quantified by measuring luciferase activity.

Timing: 3 days 9. Day 1. Keep EA.hy926 cells in cell culture. In our experiments, we leave cells in cell culture for at least 2 passages before starting the transduction. The cells should grow exponentially and exhibit healthy morphology ( Figure 1B ). Wash EA.hy926 cells with 1x DPBS and detach the cells by employing Trypsin/EDTA (0.05%) typically incubating less than 5 minutes at 37 o C. Resuspend the cells in 10 ml pre-warmed fresh complete RPMI1640 medium and count the cells. 10. Seed 2x10 5 cells in 1 ml/well in a 12-well plate to reach 70-80% confluency on the next day. 11. Day 2. Thaw MLV-control/MLV-EBOV-GP viral supernatants and prewarm them at 37 o C (not longer than 15 minutes prior to experiment). Completely remove medium on the cells and replace it with 1-1 ml pseudoviral supernatant. Incubate the cells at 37 o C in 5% CO2.

In our experiments, the optimal titer to infect the cells is the undiluted pseudoviral supernatant.

Optional: Prior to first measurement, it is recommended to titrate the pseudoviruses. Make 1:2 serial dilution of the supernatants as shown in Figure 3A . Measure luciferase activity as described at step 12 and plot a Titer/Relative Luminescence Unit (RLU) curve to determine the most efficient volume for transduction ( Figure 3B ). Note: Alternatively to the use of Luciferase Substrate from Promega, after cell lysis and transfer of the cell lysates into 96-Well microplate (see step 12i.), add 100 µl of Firefly Luciferase Assay Buffer (Solution A, see Materials and equipment) and 50 µl Luciferin Substrate Solution (Solution B, see Materials and equipment). Measure after 20 seconds incubation at RT with luminometer (see step 12k.).

Since the peak intensity of detected light starts to slowly decline typically in 3-4 minutes after adding the substrate, it is recommended to use multichannel pipette for simultaneous sample detection of multiple samples. Alternatively, plate readers equipped with injectors can be employed, which ensure unified time interval between substrate injection and detection.

In general, longer incubation time than 24 hours results in a minimal increase of the detected luminescence intensity. However, 48 hours incubation leads to higher variability of the results. Therefore, we typically used 24 hours for detection. Figure 4A and 4B show the comparison between 24 and 48 hours measurements. Others reported that the luciferase activity peaks at 24 h (Ebola-GP) and 48 h (SARS-CoV-2-S) by employing VSV pseudotyped viruses (Lay Mendoza et al., 2020) .

HuH-7 cells were selected for transduction experiments because they express angiotensin-converting enzyme 2 (ACE-2), the host binding site of the SARS-CoV-2-Spike protein and therefore these cells are susceptible to infection (Hoffmann et al., 2020) . In these steps, human HuH-7 cells are transduced by MLV-SARS-CoV-2-S pseudoviral particles. The pseudovirus entry efficiency is quantified by measuring luciferase activity at 24 hours.

13. Day 1. Keep HuH-7 cells in cell culture for at least 2 passages before starting the experiment. The cells should grow exponentially and exhibit healthy morphology ( Figure 1C ). Wash HuH-7 cells with 1x DPBS and detach the cells by trypsinization typically incubating less than 5 minutes at 37 o C. Resuspend the cells in 10 ml prewarmed fresh complete DMEM medium.

Here we describe the transduction of HuH-7 cells, which are permissive to corona viruses (Freymuth et al., 2005) , however other permissive cell lines, such as Caco2 cells can be also transduced by MLV-SARS-CoV-2-S pseudoviruses (Hoffmann et al., 2020) .

14. Seed 2x10 5 cells/well in a 12-well plate to reach 70-80% confluency on the next day. 15. Day 2. Thaw MLV-control/MLV-SARS-CoV-2-S pseudoviral supernatants and prewarm them at 37 o C. Completely remove medium on the cells and replace it with 1-1 ml pseudoviral supernatant. Incubate the cells at 37 o C in 5% CO2. 16. Day 3. Detection of the luciferase activity. The next steps are identical to step 12. Please follow the sub-steps described under step 12.

The detection of luminescence typically results in approximately 2 orders of magnitude increase in relative luminescence unit (RLU) by employing the MLV-EBOV-GP particles in EA.hy926 cells ( Figure  4A ) and 1-2 orders of magnitude increase by employing the MLV-SARS-CoV-2-S particles in HuH-7 cells ( Figure 4B ).

All experiments have been repeated at least three times. To test statistical significance of the results Student´s t test (unpaired, two tailed) was applied. The significance is indicated as ***p<0.001 or nsnon significant.

The produced MLV pseudoviruses are replication incompetent. Thus, this method is not suitable for studies aiming at investigating the processes of de novo virus production, viral replication and viral budding. In addition, in combination application of chemical compounds that result in additional light emission/light absorption might interfere with luciferase activity measurement leading to false positive/negative results. For instance, employing the fluorescent compound Doxorubicin increases the detected light intensity (Calvert et al., 2013) and the application of carbon nanotubes may lead to reduced measured light intensity in luminescence detection assays (Szymanski et al., 2020) .

Troubleshooting Problem 1. No virus production due to poor plasmid yield.

If the luciferase measurement of the transfected HEK293T cells exhibited no sufficient intensity increase (see step 8), it can indicate low yield of plasmids.

Potential solutions:

Make sure that the amount of the plasmids is sufficient by measuring the concentration of the DNA. If necessary, prepare new plasmid stocks ("before you begin" steps 5-9). Check, if the plasmids harbor the correct, not mutated, inserts by employing gene sequencing. b) Transfection reagent is expired or was stored at room temperature for longer period of time (>24h). Use/prepare fresh transfection reagent.

Problem 3. Pseudovirus loss during filtration.

The Luminescence intensity is low in pseudovirus transduced cells due to not sufficient pseudovirus concentration. If the luciferase measurement of the transfected HEK293T cells exhibited sufficient intensity increase (see step 8), it indicates efficient transfection of the plasmids. Therefore, the problem might have occurred in the subsequent (steps 9-12) steps, possibly at the pseudovirus filtration.

Potential solution:

Make sure that you use filter units with 0.45 µm pores. Based on our experiments, application of 0.22 µm filters might also work, however they result in significantly less yields. In addition, check the manufacturer´s site for the specification of the filters. The application of filters with reduced binding capacity is required to avoid the attachment of viral particle to the filters. Furthermore, increased mechanical stress by intense up and down pipetting may also lead to destruction of the pseudovirus particles.

Problem 4. Too many freeze-thaw cycles of the pseudoviral supernatants.

In addition, repeated freeze-thaw cycles can result in loss of transduction efficiency of the pseudoviruses.

Potential solution:

Please make sure to prepare aliquots as suggested at step 7. Try to avoid repeated freeze-thaw cycles. Repeated freeze-thaw cycles can strongly influence pseudovirus infectivity. Store the viral supernatants at -80 o C. The best infectivity is achieved, if the pseudoviruses used immediately after filtration without freezing.

The luminescence intensity is low in pseudovirus infected cell lysates, however western blot analysis of the transduced lysates detects high amount of luciferase. This indicates that the pseudovirus transduction of the cells worked efficiently and the cells produced Luciferase protein. 

J o u r n a l P r e -p r o o f A. Dilution scheme on a 12-well plate using three technical replicates. The increasing darkness of the color represents the increasing concentration of the pseudovirus particles in the actual well. MLVcontrol is depicted with grey and MLV-EBOV-GP is depicted with orange color. The red numbers represent the volume of the pseudovirus supernatants and the black numbers show the volume of complete medium in one well (total volume is 1 ml/well). B. The detected light intensity by employing a 1:2 serial dilution of the viral supernatants measured by luminometer. EA.hy926 cells were transduced with indicated volumes of MLV-control and MLV-EBOV-GP pseudoviral supernatants. The samples were harvested at 24 hours and luciferase activity was measured as described in the protocol (n=3). The data represent means +/-sd. A. EA.hy926 cells were transduced with 1-1 ml MLV-control and MLV-EBOV-GP pseudoviral supernatants. The samples were harvested after 24 and 48 hours (24 h and 48 h) and luciferase activity was measured as described in the protocol. The data represent means +/-sd. To test significance Student´s t-test was performed. ***p<0.001; ****p<0.0001; ns-non significant. n=4 (24 h) and n=3 (48 h). B. HuH-7 cells were transduced with 1-1 ml MLV-control and MLV-SARS-CoV-2-S pseudoviral supernatants. The samples were harvested and luciferase activity was measured at 24 and 48 hours as described in the protocol. The data represent means +/-sd. To test significance Student´s t-test was performed. ***p<0.001; ns-non significant. n=4 (24 h) and n=3 (48 h).

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We thank to Professor Dr. Stefan Pöhlmann (Infection Biology Unit, German Primate Center -Leibniz Institute for Primate Research, Faculty of Biology and Psychology, University Göttingen) for the plasmids pCMV-Luc and pCMV-MLV-gag-pol. The project is supported by the Goethe Corona Fonds Frankfurt to J.P. (80301283) 

The authors declare no competing interest.