Protocol for characterizing the inhibition of SARS-CoV-2 infection by a protein of interest in cultured cells

Summary Here, we present a protocol to characterize the antiviral ability of a protein of interest to SARS-CoV-2 infection in cultured cells, using MUC1 as an example. We use SARS-CoV-2 ΔN trVLP system, which utilizes transcription and replication-competent SARS-CoV-2 virus-like particles lacking nucleocapsid gene. We describe the optimized procedure to analyze protein interference of viral attachment and entry into cells, and qRT-PCR-based quantification of viral infection. The protocol can be applied to characterize more antiviral candidates and clarify their functioning stage. For complete details on the use and execution of this protocol, please refer to Lai et al. (2022).


SUMMARY
Here, we present a protocol to characterize the antiviral ability of a protein of interest to SARS-CoV-2 infection in cultured cells, using MUC1 as an example. We use SARS-CoV-2 DN trVLP system, which utilizes transcription and replication-competent SARS-CoV-2 virus-like particles lacking nucleocapsid gene. We describe the optimized procedure to analyze protein interference of viral attachment and entry into cells, and qRT-PCR-based quantification of viral infection. The protocol can be applied to characterize more antiviral candidates and clarify their functioning stage. For complete details on the use and execution of this protocol, please refer to Lai et al. (2022).

BEFORE YOU BEGIN
The protocol below describes detailed procedures for researchers to investigate the ability of a protein of interest to inhibit SARS-CoV-2 infection in a transcription and replication-competent SARS-CoV-2 virus-like particles lacking nucleocapsid (N) gene (SARS-CoV-2 DN trVLP) infection system.
Here we evaluate the antiviral effect of a protein of interest using human breast milk component of mucin1 (MUC1) as an example in a cell infection system of SARS-CoV-2 DN trVLP infecting Caco-2-N cells to mimic authentic SARS-CoV-2 infection (Lai et al., 2022;Ju et al., 2021).
Note: Caco-2 are epithelial cells isolated from colon tissue derived from a 72-year-old, white, male patient with colorectal adenocarcinoma (ATCC HTB-37ä, https://www.atcc.org/ products/htb-37). Caco-2-N cells are established to stably express SARS-CoV-2 N protein by lentiviral transduction. The reason why we choose Caco-2 as infection cell model is that Caco-2 cells express high level of SARS-CoV-2 receptors of ACE2 and TMPRSS2, which are permissive to SARS-CoV-2. Caco-2 cells were widely used to study infection with SARS-CoV and are now again being used to study SARS-CoV2 infections (Bojkova et al., 2020).
Note: SARS-CoV-2 DN trVLP expresses a reporter gene (green fluorescent protein, GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly. The lack of the viral N protein could be genetically complemented in trans by ectopic expression in Caco-2-N cells. SARS-CoV-2 DN trVLP could be propagated and passaged in Caco-2-N cells. If you would like to know more details about the Caco-2-N production, trVLP production and viral titration, you are highly recommended to refer to another published detailed protocol well-written by Prof. Qiang Ding, Tsinghua University .
The protocol here might also provide clue for other kinds of viruses, such as hepatitis B virus (HBV), hepatitis C virus (HCV), HIV, CMV, dengue virus, Zika virus, etc., to identify the stages of action of their own antiviral candidates by employing different temperature settings.
Before beginning the experiments, we need prepare the protein of interest (commercial MUC1) stock media. According to the product data sheet, the MUC1 commercial product is dissolved in PBS containing 5% Trehalose, pH7.2 to make a 0.1 mg/mL stock.
Note: PBS containing 5% Trehalose is more suitable for dissolving because it can avoid the loss of MUC1 caused by attaching on the tube inner wall.
CRITICAL: Centrifuge MUC1 lyophilized protein vial at 10,000 3 g for 1 min prior to opening. Do not mix by vortex or pipetting. Store at 4 C for 1 week, or store aliquots at À80 C for 3 months. Avoid repeated freezing-thawing.

Experimental design considerations
The viral infection process can be separated roughly into 3 stages, attachment, entry and post-entry stage, based on the relative position between the virus and cell (Fan et al., 2020;Malone et al., 2022). The attachment stage means that the virus binds to its receptor, which can still happen even at a low temperature of 4 C. The entry stage is a subsequent conformational transition that needs transmembrane serine protease 2 to cleave the spike protein. At 4 C, the enzyme activity is limited so that the conformational transition stops at entry stage. Meanwhile the cell itself uses ATP to achieve cytoskeleton shape change and provides sufficient ATP to overcome the natural barrier between the virus and the cellular membranes to achieve membrane fusion and internalization of viral particles. At 4 C, the ATP enzyme activity is also restricted so that the conformational transition stops. The entry stage happens quickly usually within 1 h at 37 C and costs much energy. The postentry stage means the virus has been internalized and starts to replicate, which still costs much energy and needs 37 C temperature set (Shang et al., 2020;Fan et al., 2020).
Based on the characteristics of three stages, it works that we use different temperature sets to restrict the viral infection into certain stages. Details of temperature sets are listed below at corresponding section.
A list of all reagents, buffer and equipment for this protocol are described in the key resources table. Make sure all materials required for the experiments are available and/or prepared in advance.

Institutional permissions
The institution's Research Ethics Board approval is required when working with human biological material. The human derived samples used in this study was collected with informed consent. The study was approved by the ethics committees of the Medical Center. SARS-CoV-2 is a biosafety level 3 (BSL-3) pathogen. Even though the artificial replicon of trVLP could be used in the BSL-2 laboratory, an appropriate facility and biosafety training for researchers before starting the experiment is needed.

Cell culture
Timing: 5-7 days ll OPEN ACCESS 1. Thaw a vial of Caco-2-N cells in a 37 C water bath and immediately plate the cells in 10 cm cell culture dishes.
2. Caco-2-N cells (recommend passages: 3-10) are cultured in Basic Dulbecco's modified Eagle's media (DMEM) with high glucose and pyruvate and supplemented with 1% Streptomycin/ Penicillin and 10% fetal bovine serum (FBS), at 37 C, in 5% CO 2 . a. Cells are cultured in 10 cm dishes and split in cell culture media twice per week at a 1:6 ratio. If you need more cells, you can transfer cells to a 175 cm 2 rectangular angled neck TC-treated culture flask with vent cap (Corning, cat# 431080). b. Culture cells for at least 5-7 days after thawing before using them for viral infection to ensure that they are healthy.
CRITICAL: All the cells and reagents must be sterile. A strict aseptic environment should be acquired for all the procedures.

MATERIALS AND EQUIPMENT
Note: Media should be stored at 4 C for no more than 2 months.

Timing: 30 min
To obtain Caco-2-N cells for SARS-CoV-2 DN trVLP infection, cells are firstly collected from the cultured dish.
1. Remove the cell culture media and add 5 mL of sterile PBS to wash the Caco-2-N cells, when Caco-2-N cells grows in a monolayer configuration and at 90% of confluence. 2. Aspirate the PBS and add 2 mL trypsin 0.25% EDTA solution. 3. Incubate at 37 C in the cell culture incubator for 2-5 min. 4. Add 2 mL of complete DMEM media and resuspend the cells.
Note: Pipette the media with cells up-and-down using a 10 mL pipette to better detach and dissociate the cells.
5. Collect the cell suspension in a 15 mL centrifuge tubes. 6. Take an aliquot of 10 mL of cell suspension in a 1.7 mL microcentrifuge tube to count the cell numbers. a. Mix the 10 mL cell suspension aliquot with 10 mL trypan blue solution and transfer the 20 mL mixture into a cell counting slide. b. Count live cells manually with a hemocytometer or with an automated cell counter to determine the cell density. 7. Prepare the proper volume of cells at the concentration of 2 3 10 5 cells/mL with the cell culture media.
Note: For example, in this experiment, we have 5 experiment groups and each group has three replicates so that we have to prepare 15 wells of cells. Considering the possible media loss, we are recommended to prepare total volume of 4.5 mL cells for 18 wells (each well needs 50,000 cells in the 250 mL of media). Therefore, we need 18 3 50,000=9 3 10 5 cells in total. Then, we need prepare 4.5 mL of cell media with cell concentration of 2 3 10 5 cells/mL. If the cell density of the stock is 2 3 10 6 cells/mL, we need prepare and mix 450 mL of the stock and 4,050 mL of culture media.
8. Seed 250 mL of cell suspension solution per well in a new 48-well plate.
Note: The data points should be performed at least in triplicates and it should always include a positive control. As for our lab, we choose 2 mg/mL human breast milk sample (A17) which has been tested to inhibit SARS-CoV-2 infection effectively (Fan et al., 2020). Remdesivir is also recommended. As for this test, we test four different concentrations of MUC1 (0.002, 0.0004, 0.00008 and 0 mg/mL).
9. Incubate cells for 18-24 h at 37 C in a CO 2 (5%) incubator to allow cells to recover and attach to the plate.

Viral infection-Day 2
Timing: 3 h At this section, we use SARS-CoV-2 DN trVLP to infect Caco-2-N cell. We expect to find different infection ratios by adding different concentrations of a protein of interest (e.g., MUC1).
10. Prepare the SARS-CoV-2 DN trVLP infection media by adding viral stocks into complete DMEM cell culture media.
Note: In our lab, the viral stock's TCID 50 exceeds 10 5 . We use SARS-CoV-2 DN trVLP to infect Caco-2-N at MOI=1, which can establish stable viral infection in 2 days. The trVLP titration method is in another published protocol .
11. Prepare different concentrations of a protein of interest (e.g., MUC1) for treating trVLP as follows.
Note: We still need a positive control and a negative control as mentioned above and we recommend you add different volume of stock solution to replace a series of dilution from previous concentration in this experiment actually.  e. Incubate for 5 min at 25 C for random hexamer primed synthesis, followed by 60 min at 42 C and terminate the reaction by heating at 70 C for 5 min. 19. Set up and load a 96-well plate using the 23 PowerUp TM SYBR Green Master Mix (Applied Biosystems) according to the manufacturer's instruction (https://www.thermofisher.cn/cn/zh/ home/life-science/pcr/real-time-pcr/real-time-pcr-reagents/sybr-green-real-time-master-mixes/ powerup-sybr-green-master-mix.html). The qPCR primers for viral RNA are as follows: SARS-CoV-2-RNA-F: 5 0 -CGAAAGG TAAGATGGAGAGCC-3 0 . SARS-CoV-2-RNA-R: 5 0 -TGTTGACGTGCCTCTGATAAG-3 0 . RPS11-F: 5 0 -GCCGAGACTATCTGCACTAC-3 0 . RPS11-R: 5 0 -ATGTCCAGCCTCAGAACTTC-3 0 . RPS11 is used to normalize all the data.
a. Prepare PCR mixture for each well of the 96-well plate: Note: You can make a premix of 23 PowerUp TM SYBR Green Master Mix, Primer-F and Primer-R. If you do so, you need to prepare the 1.2 times of premix volume needed because of the liquid loss during pipetting.
b. Mix the components thoroughly, then centrifuge briefly to spin down the liquid and eliminate any air bubbles. c. Transfer the appropriate volume of each reaction to each well of an optical plate. d. Seal the plate with an MicroAmpä Optical Adhesive Film, then centrifuge briefly to spin down the contents and eliminate any air bubbles. 20. Set up and run the qRT-PCR instrument.
a. Place the reaction plate in the qRT-PCR instrument. b. Set the thermal cycling conditions using the default PCR thermal cycling conditions specified in the following tables according to the instrument cycling parameters and melting temperatures of the specific primers.
21. Analyze data and determine the infection ratio and inhibition effects. MUC1 showed high inhibitory activity to SARS-CoV-2 infection (Figure 1).
Note: In our lab, we choose 2 mg/mL of human breast milk sample (A17) which has been tested to inhibit SARS-CoV-2 infection effectively (Fan et al., 2020). The inhibition rate test of MUC1 was performed to calculate relative expression compared to the negative control.

Timing: 4-5 days
The viral infection process can be separated roughly into 3 stages of attachment, entry and post-entry stage. Thus, we use different temperature sets to restrict the viral infection into certain stages. Details of temperature set are listed below at corresponding section.
22. Identification of the antiviral ability of a protein of interest (e.g., MUC1) at attachment stage.
Note: The experiment is roughly similar, only the infection process changes. In order to emphasize the differences, the experiments below are focused on the different procedures and simplify the same procedures. The 48 well plate for infection is recommended. The data points should be performed in triplicates.
a. Prepare three groups of culture media designed as follows: i. trVLP infection stock (dilute 10 times).
Note: Mix these 3 groups respectively and put at 4 C for 1 h.
b. Add the MUC1-trVLP mixtures mentioned above into the wells for infection and incubate at 4 C for 2 h to allow full viral attachment to cells. c. Wash the cells with 300 mL PBS per well for 3 times to remove free MUC1, viral particles and reload 250 mL/well of culture media and culture for 96 h. d. Harvest the cells, extract the RNA and carry out qRT-PCR experiment as mentioned above.
Note: To better wash away all the free trVLP in the culture media, shake the plates gently after adding the PBS into the wells.
23. Identification of the antiviral ability of a protein of interest (e.g., MUC1) at entry stage. At this section, we seed Caco-2-N cells one day prior to viral infection as mentioned above. Caco-2-N cells are kept with trVLP at 4 C for viral attachment, added with MUC1 and carried out to permit viral entry at 37 C. Detailed procedures are listed below: a. Dilute the trVLP with complete DMEM cell culture media to MOI=1, then add the viral media into the wells.

OPEN ACCESS
b. Put the plates into a refrigerator at 4 C for 2 h to allow viral attachment enough to cells. c. Wash the cells with PBS for 3 times to exclude free virus as mentioned above. d. Prepare three groups of media designed as follows: i. Fresh media only.
ii. Fresh media with 0.002 mg/mL of MUC1.
iii. Fresh media with 2 mg/mL of A17. e. Add these 3 groups of media respectively into the wells and transfer to an incubator at 37 C for 1 h to allow viral internalization into cells. f. Wash the cells with PBS for 3 times to exclude free viral particles, MUC1 or A17 as mentioned above and reload fresh media and culture for 96 h. g. Harvest the cells and analyze the antiviral ability by detecting viral mRNA with qRT-PCR as mentioned above. 24. Identification of the antiviral ability of protein of interest (e.g., MUC1) at post-entry stage. At this section, we seed Caco-2-N cells one day prior to viral infection as mentioned above. a. Dilute the trVLP with complete culture media to MOI=1, then add it into the wells and transfer into an incubator at 37 C for 2 h to allow trVLPs fully enter cells. b. Wash the cells with PBS for 3 times to exclude free virus. c. Prepare fresh media designed as follows: i. Fresh media only, ii. Fresh media with 0.002 mg/mL of MUC1.
iii. Fresh media with 2 mg/mL of A17. d. Add the prepared media above respectively into the wells and incubate at 37 C, in 5% CO 2 for 96 h. e. Harvest the cells, extracted viral RNA and measure by qRT-PCR to calculate the antiviral ability.
Possibility of a protein of interest (e.g., MUC1) to inhibit SARS-CoV-2 attaching to heparan sulfate proteoglycans at the attachment stage

Timing: 4-5 days
It is reported that SARS-CoV infection can be inhibited by targeting heparan sulfate proteoglycans (HSPG) (Lang et al., 2011) and then SARS-CoV-2 infection depends on both cellular heparan sulfate and ACE2 (Clausen et al., 2020). To determine its potential involvement of a protein of interest (e.g., MUC1) in the inhibition of SARS-CoV-2 binding to HSPG, we treated the cells with MUC1 combined with different concentrations of heparin (Lai et al., 2022).
Note: Heparin is an analog of HSPG and can also inhibit viral infection by competitively binding to SARS-CoV-2 thereby preventing viral attachment to HSPG.
In this section, we test and identify whether MUC1 inhibits SARS-CoV-2 attaching to HSPG by using the commercial HSPG analog of heparin. We seed Caco-2-N cells one day prior to viral infection as mentioned above. 26. Add the MUC1-heparin-trVLP mixtures respectively into the wells and incubate at 4 C for 2 h to allow fully viral attachment to cells. 27. Discard the media, wash the cells with 300 mL of PBS per well for 3 times, replenish with fresh media and incubate at 37 C in 5% CO 2 for 96 h. 28. Collect the supernatant and harvest the cells to extract RNA for further viral RNA quantification by qRT-PCR as mentioned above.

EXPECTED OUTCOMES
The protein of interest (MUC1) inhibits SARS-CoV-2 infection and replication in a dose dependent manner (Figure 1). To further determine how these proteins impact viral infection, the exact viral attachment, entry and post-entry experiments were designed to analyze it. It was confirmed that the protein of interest (MUC1) plays a critical role in blocking the steps of viral attachment, entry and post-entry replication (Figure 2).
For the HSPG experiment, we expect that the protein of interest (MUC1) to interfere with the binding of Heparin to SARS-CoV-2. At low concentrations of Heparin, MUC1 is free to bind to HSPG and thereby can block viral attachment. An increase in concentration of Heparin to 10 U/mL increases Heparin-MUC1 binding thereby leaving HSPG free to bind to SARS-CoV-2 making viral attachment possible. Since Heparin can also act as an inhibitor of SARS-CoV-2 binding to HSPG, further increase in Heparin concentration competitively inhibits binding of SARS-CoV-2 to HSPG thereby blocking viral attachment (Figure 3).

LIMITATIONS
The experiment design is based on the SARS-CoV-2 DN trVLP and Caco-2-N cells infection system which can mimic the authentic infection in BSL-2 laboratory. But some limitations do exist. First of all, the trVLP system is an artificial replicon which can't mimic the whole life cycle of SARS-CoV-2. To overcome this limitation, the experiments should be repeated in the live SARS-CoV-2 infection system to prove the proteins interference of viral infection. Moreover, we do not test whether the Caco-2 cells can be replaced by other cell lines (Vero E6 and Huh7.5 expressing SARS-CoV-2 N). We also do find during the passage, SARS-CoV-2 N will decrease its expression probably due to some immunological reasons (The better passages are 5-10 for the experiments). In addition, the construction of trVLP is laborious but the SARS-CoV-2 variants are emerging quickly, various kinds of trVLPs should be developed especially SARS-CoV-2 spike protein replaced by reporter genes (Ricardo-Lax et al., 2021).
When it comes to the protocol details, there are still some limitations. Firstly, we use qRT-PCR to test the MUC1 inhibition ratio, which are likely to be false-positive due to its high sensitivity. Moreover, different kinds of cell lines tolerate temperature shift differently, which will surely influence the MUC1 inhibition efficiency.

Potential solution
Check the trVLP titration. In addition, Caco-2-N cells may express low N protein. Add 5 mg/mL of Blasticidin to screen N protein expressing cells.

Problem 2
Minimal or unexpected changes in MUC1 treatment (step 11).

Potential solution
The major cause of variation might be improper in MUC1 stock preparation. Uneven cell numbers may also result in unexpected changes. Therefore, it is important to make sure the proper MUC1 stock preparation according to the reconstitution sheet is made. And the same number of cells is seeded in each well of replicates.

Problem 3
Step 23: very little detected signal exists during the experiment.

Potential solution
The reason might be the incubation time is not enough for viral attaching to the cells. To solve this, different time points should be tested to help us select the best time point for the experiment.

Problem 4
Step 24: unexpected result exists in the repeated experiments.

Potential solution
The virus attaching ability to the cells in the 4 C treatment step is not very tight, which is easily washed away during the wash step. To solve this, it is better to shake the cells gently by the shaker with same time during the wash step.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Kuanhui Xiang (kxiang@bjmu.edu.cn).

Materials availability
This study did not generate new unique reagents.

Data and code availability
This study did not generate or analyze datasets or code.