MuLV Psueduvirus

SARS pseudoviruses that can readily infect Vero E6 cells

G: pUMVC, pCMV-Gag-Pol  plasmid ID 388

V: pCMV-VSVG: plasmid ID 389

nonreplicating Moloney murine leukemia virus (MuLV)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7127165/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5181643/

MuLV packaging cell line TELCeB6 (Schnierle et al., 1997) was transfected with pcDNA-S, pHCMV-S, pHCMV-G (Burns et al., 1993), or pLTR-gp140 (HIV-1DH12; Kim et al., 2001) using  Lipofectin.

pHCMV-G vector expresses high level of vesicular stomatitis virus (VSV) G pcDNA-S: spike in pCDNA pHCMV-S: Spike in pHCMV-G (Spike + VSVG) both pcDNA-S and pHCMV-S constructs were able to pseudotype MuLV particles to produce SARS pseudoviruses that could readily infect Vero E6 cells (Fig. 3A) . None of the other cell lines we tested, including HeLa, A549, 293T, and BS-C-1, were susceptible. This is in contrast to VSV-G pseudotyped viruses, which could infect all cell lines

Protocol 

https://www.jove.com/t/59010/production-pseudotyped-particles-to-study-highly-pathogenic

Plasmid/reagent	Quantity
pCMV-MLVgagpol MLV gag and pol encoding plasmid	300 ng
pTG-Luc transfer vector with luciferase reporter	400 ng
pcDNA-SARS-S, pcDNA-MERS-S or empty vector	300 ng
Reduced serum cell culture medium	To 50 µL

Reagent	Quantity
Transfection reagent	3 µL
Reduced serum cell culture medium	47 µL

Materials

Name	Company	Catalog Number	Comments
Human embryonic kidney (HEK) HEK-293T/17 cells	ATCC	CRL-11268	Clone 17 cells are highly competent for transfection.
African green monkey kidney epithelial Vero-E6 cells	ATCC	CRL-1586
Human hepatic Huh-7 cells	Japan National Institutes of Biomedical Innovation, Health and Nutrition	JCRB0403
Inverted light microscope with 10 × objective	Nikon	TS100
Dulbecco’s modification of Eagle's medium (DMEM) with 4.5 g/L glucose, L-glutamine without sodium pyruvate	Corning Mediatech	10-017-CV
Heat-inactivated fetal bovine serum (FBS)	Thermo Fisher Scientific, Gibco	1614071
1 M N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES)	Corning Mediatech	25-060-Cl
100 × penicillin-streptomycin (PS) solution	Corning Mediatech	30-002-Cl
Dulbecco’s phosphate buffered saline (DPBS) with Ca2+ and Mg2+	Corning Mediatech	21-030-CV
0.25% trypsin, 2.21 mM ethylenediaminetetraacetic acid (EDTA) 1 × solution	Corning Mediatech	25-053-Cl
Cell counting slides with grids	Kova	87144
Opti-minimal essential medium (Opti-MEM)	Thermo Fisher Scientific, Gibco	31985-070
Lipofectamine 2000 transfection reagent	Thermo Fisher Scientific, Invitrogen	11668-027
0.45 µm pore-size sterile filter	Pall	4184
10 mL syringes	BD	309604
5 × luciferase assay lysis buffer	Promega	E1531
Luciferin, substrate for luciferase assay	Promega	E1501
Sterile water	VWR	E476-1L
GloMax 20/20 luminometer	Promega	2030-100

Cell Seeding for Pseudotyped Particle Production

NOTE: Perform this step in the biosafety cabinet.

1.By standard cell culture techniques, obtain an 80–90% confluent 75 cm² flask of HEK-293T/17 cells passaged in complete Dulbecco's Modified Eagle's Medium (DMEM-C) containing 10% (vol/vol) fetal bovine serum (FBS), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 100 IU/mL penicillin, and 100 µg/mL streptomycin. Prepare DMEM-T medium for transfections (its composition is the same as DMEM-C but without antibiotics).

2. Wash cells with 10 mL of pre-warmed (37 °C) Dulbecco's Phosphate Buffered Saline (DPBS) twice.

NOTE: Handle HEK293T/17 cells with care as they easily detach.

Aspirate the supernatant and detach cells with 1 mL of 0.25% trypsin solution pre-warmed at 37 °C. Place the flask of cells at 37 °C, 5% CO₂ incubator for 3−5 min or until cells start detaching.

NOTE: Avoid incubating cells with trypsin for more than 5 min as this typically leads to cell clumping.

3. Deactivate trypsin by adding 4 mL of DMEM-C medium and count cells using a cell counting slide and light microscope.

NOTE: To avoid having to count too many cells, an additional dilution step may be required beforehand. Remember to factor in this dilution when calculating the actual cell density of trypsinized cells.

4. Dilute cells to 5 x 10⁵ cells/mL with DMEM-C.

5. Seed 6-well tissue culture plate with 2 mL of cell solution per well and gently move the plate back and forth and side to side to evenly distribute cells, avoiding circular motion.

NOTE: This is a key step. Evenly distributed cells will ensure that cells do not clump at the center of wells. In turn, this will ensure good transfection efficiencies and pseudotyped particle production.

6. Incubate the plate overnight (16–18 h) in a 37 °C, 5% CO₂ cell culture incubator.

7. Three-plasmid Co-transfection

NOTE: Perform this step in the biosafety cabinet.

Observe cells under an inverted light microscope to check for the cell morphology and density.

NOTE: Ideally, cell density should be in the 40–60% confluency range. It is critical that cells are neither too confluent (80−90% confluent) nor too sparsely distributed (20−30% confluent) in each well. A cell density of 40−60% confluency will ensure good pseudotyped particles production.

Plasmids mix

1. Calculate the plasmid mix for each envelope glycoprotein following the quantities for one well of a 6-well plate shown in Table 1. Multiply quantities if transfecting several wells and include an extra well to avoid running out of mix.

NOTE: Along with the SARS-CoV S and MERS-CoV S encoding plasmids, include empty vector control for the generation of negative control particles which lack viral envelope glycoproteins (Δenv particles) along with a positive control glycoprotein such as vesicular stomatitis virus (VSV) G glycoprotein that is known to robustly infect a very wide range of cells (VSV-Gpp).  

2. Mix calculated volumes of plasmids and reduced serum cell culture medium (see Table of Materials) in a microcentrifuge tube.

Lipid-based transfection reagent mix (see Table of Materials)

1. Calculate the volumes for the transfection reagent mix from the quantities shown in Table 2 for one well (1:3 transfection ratio, multiply quantities as needed). Include extra wells to avoid running out of transfection reagent mix.
2. Mix calculated volumes of lipid-based transfection reagent (3 µL per well) and reduced serum cell culture medium (47 µL per well) in a microcentrifuge tube, making sure to add the transfection reagent into the reduced serum cell culture medium and not the other way around.
3. Incubate both mixes (for one well: 50 µL of plasmids mix and 50 µL of lipid-based transfection reagent mix) separately for 5 min at room temperature.
4. Add the contents of the transfection reagent mix to the plasmids mix at a 1:1 ratio (for 1 well: 50 µL of each mix)
5. Perform thorough up-down pipetting of the resulting mix.
6. Incubate the mix for at least 20 min at room temperature.
7. Aspirate the spent medium of cells.
8. Add gently 1 mL of pre-warmed (37 °C) reduced serum cell culture medium per well.
9. Add dropwise 100 µL of transfection mix to each well. 

 NOTE: Exercise care when adding the transfection mix to wells of HEK-293T as they detach easily.

10. Incubate cells in a 37 °C, 5% CO₂ cell culture incubator for 4–6 h.

11. Add 1 mL per well of pre-warmed (37 °C) DMEM-T medium, which does not contain antibiotics

NOTE: This is a key step. Transfection reagents increase cell permeability and increase sensitivity to antibiotics. To ensure good transfection efficiency and pseudotyped particle production, it is important to avoid using cell culture medium containing antibiotics

12. Incubate cells in a 37 °C, 5% CO₂ cell culture incubator for 48 h.

3. Pseudotyped Particles Collection

NOTE: Perform this step in the biosafety cabinet.

1. Observe cells under inverted light microscope to check for cell morphology and general condition. Also check the color of the medium which should be light pink/slightly orange.

NOTE: This is an important step. If there is too much cell death associated with the transfection or the medium color turned orange/yellow (acidic pH), this will typically be associated with lower yields in infectious pseudotyped particles

2. Transfer supernatants of transfected cells to 50 mL conical centrifuge tubes.
3. Centrifuge tubes at 290 x g for 7 min to remove cell debris.
4. Filter clarified supernatants through a sterile 0.45 µm pore-sized filter.
5. Make small volume aliquots (e.g., 500 µL or 1 mL) of pseudotyped virus solution in cryovials.
6. Store at -80 °C.

NOTE: The protocol can be paused here. Pseudotyped particles are stable at -80 °C for many months but once thawed, avoid re-freezing them as they will lose infectivity

4. Pseudotyped Particle Infection of Susceptible Cells

NOTE: Perform this step in the biosafety cabinet.

1. Cell seeding of susceptible cells in 24-well plate
   1. Obtain by standard cell culture techniques 80−90% confluent 75 cm² flask of susceptible cells: Vero-E6 cells for SARS-CoV pseudotyped particles (SARS-Spp) and Huh-7 cells for MERS-CoV S pseudotyped particles (MERS-Spp).

NOTE: To confirm whether the pseudotyped particles that have been produced are infectious, it is important to carefully choose an appropriate susceptible cell line for pseudovirion infectivity assays. Using poorly permissive cells will lead to low infectivity.

2. Wash cells twice with 10 mL of pre-warmed (37 °C) DPBS.
3. Aspirate the supernatant and detach cells with 1 mL of 0.25% trypsin solution pre-warmed at 37 °C. Place the flask of cells at 37 °C, 5% CO₂ incubator for 3−5 min or until cells start detaching.

NOTE: Avoid incubating cells with trypsin for more than 5 minutes as this typically leads to cell clumping. Huh-7 cells are especially sensitive to this effect.

4. Deactivate trypsin by adding DMEM-C medium and count cells using a cell counting slide and light microscope.
5. Dilute cells to 5 x 10⁵ cells/mL with DMEM-C.
6. Seed wells of a 24-well plate with 0.5 mL of cell solution per well and gently move the plate back and forth and side to side to evenly distribute cells, avoiding circular motion

NOTE: This is a key step. Evenly distributed cells will ensure that cells do not clump at the center of wells. In turn, this will ensure good infectivity assays. For each pseudotyped particle (SARS-Spp, MERS-Spp) and condition, prepare three wells for three experimental replicates. Include wells for the non-infected (N.I.), empty vector Δenv particles and positive control particles such as VSV-Gpp

7. Incubate the plate overnight (16−18 h) in a 37 °C, 5% CO₂

Pseudotyped particle infection

1. Observe cells under light microscope and visually confirm that there is a confluent carpet of cells.
2. Bring cryovials of pseudotyped virus to thaw on ice.
3. Wash cells three times with 0.5 mL pre-warmed (37 °C) DPBS

NOTE: This is a key step. Cells that are not properly rinsed prior to infection typically lead to poor infectivity readouts

4. Aspirate the supernatants of cells.
5. Inoculate cells with 200 µL of thawed pseudotyped particle solution.
6. Incubate cells in a 37 °C, 5% CO₂ cell culture incubator for 1−2 h.
7. Add 300 µL of pre-warmed (37 °C) DMEM-C medium.
8. Incubate cells in a 37 °C, 5% CO₂ cell culture incubator for 72 h.

5. Infectivity Quantification by Luciferase Assay Readout

NOTE: Perform initial steps in the biosafety cabinet.

1. Thaw luciferin substrate (stored at -80 °C) and 5x luciferase assay lysis buffer (stored at -20 °C) until they reach room temperature.
2. Dilute luciferase assay lysis buffer to 1x with sterile water.
3. Aspirate supernatants of cells infected with pseudotyped particles.
4. Add 100 µL of 1x luciferase assay lysis buffer to each well.
5. Place the plate on a rocker and incubate for 15 min with rocking at room temperature (from this point onwards the plate can be handled outside of a biosafety cabinet).
6. Prepare microcentrifuge tubes for each well by adding 20 µL of luciferin substrate in each tube.
7. Turn on the luminometer.
8. Perform luciferase activity measurement one well at a time by transferring 10 µL of lysate to one tube containing 20 µL of luciferin substrate.
9. Flick the tube gently to mix contents, but avoid displacing the liquid on walls of tube.
10. Place the tube in device and close lid.
11. Measure the luminescence value of the tube by using the luminometer.
12. Record the relative light unit's measurement.
13. Repeat steps 5.8–5.12 until all wells are analyzed.

NOTE: With the appropriate equipment such as a plate reading luminometer, this process can be performed automatically. The assay will need to be scaled to the plate format (e.g., 96-well plate format).

6. Data Analysis

1. Calculation and plotting of relative luciferase units' averages and standard deviations
   1. Use a graph plotting software to calculate luciferase assay measurement averages and standard deviations of experimental and biological replicates.
   2. Plot data as bar chart with standard deviations.

NOTE: When performing statistical analyses on data, make sure to include at least three biological replicates in data sets.

VSV G pseudotyped positive control particles (VSV-Gpp) gave very high average infectivity in the 106 to 107 relative luciferase units (RLU) range respectively. For SARS-CoV S pseudotyped particles (Figure 1A) infection of susceptible Vero-E6 cells, a strong average infectivity was measured at around 9.8 x 104 RLU. This value is almost 3 orders of magnitude higher than the values measured for the non-infected control (1.1 x 102 RLU), or the Δenv particles (1.5 x 102 RLU), which do not harbor any viral envelope glycoproteins at their surface. Similarly, for MERS-CoV S pseudotyped particles (Figure 1B) infection of Huh-7 cells, a high average infectivity was measured at around 1.0 x 106 RLU. This is almost 4 orders of magnitude higher than the values measured for the non-infected control (0.8 x 102 RLU), or the Δenv particles (2.0 x 102 RLU).

one 10-cm-diameter dish of subconfluently grown 293T or Anjou65 cells was transfected with 7.5 μg of pMP71-eGFP-pre (37), 12.5 μg of pSV-Mo-MLVgagpol, and 1 to 4 μg of env expression plasmids by using a calcium phosphate transfection kit (Sigma, Taufkirchen, Germany). The medium was replaced 6 to 8 h after transfection. Pseudotype vector-containing supernatants were harvested after an additional 24, 39, and 48 h; filtered through a 0.45 μm-pore-size Millex-HV filter (Millipore, Schwalbach, Germany); and used for transduction.

Deletion mutant CΔ19 (with 19 amino acids deleted from the C terminus) yielded about 100-fold-higher pseudotype titers than the wt S protein. Independently prepared pseudotype particles showed variations in titers, ranging from 4 × 103 to 1 × 104 TU/ml. Further truncations involved highly conserved cysteine residues and resulted in decreased pseudotype titers (Fig. 3A). Particles without envelope protein failed to infect Vero cells (not shown), whereas VSV and LCMV glycoprotein pseudotypes yielded two- to sevenfold-higher titers than the SARS S CΔ19 construct.