Pseudovirus (PSV) Assay

Pseudotyped lentivirus protocol for coronaviruses

1) Generation of host cells

• For human coronavirus infection, 3 stable expression cell lines were used: HeLa-ACE2 (for SARS-CoV-1, SARS-CoV-2, NL63), HeLa-DPP4 (for MERS) and HeLa-ANPEP (for 229E). The cell lines were generated from WT HeLa cells (CCL-2, ATCC) by VSV-G lentivirus infection (see part 2 for viral packaging details), and FACS sorted with the corresponding spike probe conjugated with FITC for high expression (top 1%) populations of the respective viral receptors. The cell lines were cultured in DMEM complete medium (10% FBS, 1%Pen/Strep and 1% Glutamax).

2) SARS-CoV-1, SARS-CoV-2, MERS, NL63, 229E pseudotyped viruses (PSVs) production

• Seed HEK293T cells to 6-well plate (1 million/well) or 10cm dish (5 million cells/dish) one day before transfection.
• Check for cell viability and confluency before transfection. The cells should be 70-80% confluent when you start to do viral packaging. 
• Gently aspirate media, add 2 ml (6-well plate) or 10 mL (10cm dish) fresh DMEM (Gibco, 10569010) complete medium (with 10% Heat Inactivated FBS and 1% P/S) to 6-well plate or 10cm dish before transfection.
• Tube A: Prepare a mixture of the 3 transfection plasmids in 200uL (or 1mL, if using 10cm dish) Opti-MEM medium:

          (The viral packaging plasmids should be prepped by transfection grade kit to remove any possible contamination of endotoxin.)

          a) Envelope: 

          SARS2-D18 (Addgene # 170442): 0.5ug for 6-well plate; 2.5ug for 10cm dish

          SARS1-D28 (Addgene # 170447): 1ug for 6-well plate; 5ug for 10cm dish

          VSV-G (Addgene # 8454): 0.5ug for 6-well plate; 2.5ug for 10cm dish

          MERS-D12 (Addgene # 170448):  1ug for 6-well plate; 5ug for 10cm dish

          NL63-D14 (Addgene # 172666): 1ug for 6-well plate; 5ug for 10cm dish

          229E-D15 (addgene # 188908): 1ug for 6-well plate; 5ug for 10cm dish

          b) Gag/Pol: 

          pCMV-delta-R8.2 (Addgene # 12263)

          2.5ug for 6-well plate, 12.5ug for 10cm dish.

          c) Reporter: 

          pBOBI-FLuc (Addgene # 170674) for lentivirus 

          2ug for 6-well plate; 10ug for 10cm dish 

• Use Lipofectamine2000 from Invitrogen to do the transfection. Add 2.5uL Lipo2000/ 1ug plasmid into a new tube (tube B) of Opti-MEM (same volume as tube A). Incubate at room temperature for 3-5 min, and mix tube B with tube A. Incubate at RT for 15-20min, then add the mixture gently into cell culture. Be careful not to disperse the cells.

        (Other transfection reagents may be used according to manufacturer’s instructions. However, as we have tested, Lipo2000 / Lipo3000 work better than PEI)

• 12-16 hours post transfection, gently aspirate the medium and add 5mL (or 25mL) fresh medium to each well of 6-well plate or 10cm dish respectively.
• 48 hours post transfection, collect the supernatant and spin down at 1500g for 10min to get rid of any cell pellets (Alternative: pass through 0.45um filter). Collect the supernatant and aliquot and freeze at -80C for long-term storage. The pseudotyped virus produced can be stored at -80C for up to 6 months.

         Optional: You can add another 5 / 25mL fresh medium, incubate for another 24 hours and collect PSV again (72 hours post transfection). 

3) PSV infection assay

Serially dilute antibody or serum with a suitable starting concentration from Row A to H in 96-well plate. Transfer 25ul of diluted antibody or sera to each well of 96-well half area well plate (Corning® 96 Half Area, # 3688) Add 25ul virus to columns 1-11, and 25ul fresh medium to column 12 (Cells only control). You can do a serial dilution of virus to infect host cells before doing the neutralization assay to get a rough idea of your viral titer.  Spin down and incubate at incubator for 1h. Prepare HeLa-ACE2 (or DPP4) cells in the meanwhile. Count and dilute cells to 200,000/ml. Add DEAE-dextran (Stock 10mg/ml, 500 X, Sigma, # 93556-1G) to the HeLa-ACE2 cells at a final concentration of 20ug/ml. Directly add 50ul of cells to the Ab/Virus or Serum/Virus mixture into 96-well plate. Incubate 48 hours in the incubator. Read the plate Prepare 1 X lysis buffer (25mM Gly-Gly pH 7.8, 15mM MgSO4, 4mM EGTA, 1% Triton X-100, you can make 10X stock and store at 4 degree) Prepare luciferase substrate: Dilute Bright-glo substrate (Promega, #E2650) 10 fold in 1 x lysis buffer.  Aspirate medium from the wells Add 50ul/well diluted luciferase substrate and incubate for >1min. (Do not incubate for more than 10min)  Luciferase intensity is read on a luminometer.   Layout template.     Note: If you still have questions, please refer to the Protocol for Neutralizing Antibody Assay for HIV-1 in TZM-bl Cells. You can find the equation to calculate % neutralization of serum or antibody. https://www.hiv.lanl.gov/content/nab-reference-strains/html/Protocol-for-Neutralizing-Antibody-Assay-for-HIV-1-in-TZMbl-cells_Nov2018.pdf https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4040342/

Figures

Viral titer measure: (a) Serial dilution (x2 fold) for different PSVs involved. RLU: relative luminescence unit.  (b) Different c-terminal deletions affect viral titer. FL: full length. Δn: n amino acid deletion from the c-terminal. Each version with the highest viral titer is marked with an asterisk. All the PSVs were tested on HeLa-ACE2 cells.  (c) MERS viral titer in different cell lines.

Protocol credit to Deli Huang, Linghang Peng and David Nemazee

Contact nemazee@scripps.edu for more information