Selective Isolation of Retroviruses from Extracellular Vesicles by Intact Virion Immunoprecipitation

Materials and Reagents

1. μ-Columns (Miltenyi Biotec, catalog number: 130-042-701 )
2. Microcentrifuge tubes (FroggaBio, catalog number: LMCT1.7B , or equivalent)
3. Pasteur pipettes (Fisher Scientific, catalog number: 13-678-20A , or equivalent)
4. PVDF membrane (Bio-Rad Laboratories, catalog number: 1620177 )
5. Serological pipettes, 10 ml (Corning, catalog number: 4488 , or equivalent)
6. Sterile 20 ml syringes with Luer-Lok (BD, catalog number: 302830 , or equivalent)
7. Sterile 450 nm Luer-Lok syringe filters (Pall, catalog number: 4614 , or equivalent)
8. Sterile 50 ml conical tubes (FroggaBio, catalog number: TB50-500 , or equivalent)
9. Sterile pipette tips (Diamed, DIATEC, catalog numbers: DIATEC520-5376 , DIATEC520-5876 , DIATEC520-6501 , or equivalent)
10. HEK 293T cells (ATCC, catalog number: CRL-3216 )
11. R187 Hybridoma (ATCC, catalog number: CRL-1912 )
12. μMACS GFP Isolation Kit (Miltenyi Biotec, catalog number: 130-091-125 )
13. 10 cm culture dishes (Corning, catalog number: 430167 , or equivalent)
14. 220 nm Steritop filters (Merck, catalog number: SCGPT10RE , or equivalent)
15. Anti-eGFP (Takara Bio, Clontech, catalog number: 632381 )
16. Anti-Flag, HRP conjugated (Sigma-Aldrich, catalog number: A8592-1MG )
17. Anti-Mouse IgG, HRP conjugated (Cell Signaling Technology, catalog number: 7076S )
18. Anti-Rabbit IgG, HRP conjugated (Abcam, catalog number: ab6721 )
19. Anti-Rat IgG, HRP conjugated (Sigma-Aldrich, catalog number: AP183P )
20. Anti-V5 (Merck, catalog number: AB3792 )
21. Dulbecco's modified Eagle’s medium (DMEM) high glucose, with L-glutamine, sodium pyruvate and phenol red (WISENT, catalog number: 319-005-CL , or equivalent)
22. Dynabeads M270-epoxy (Thermo Fisher Scientific, catalog number: 14321D )
23. ECL Substrates, i.e.:
    Clarity Western ECL Substrate (Bio-Rad Laboratories, catalog number: 1705060S , or equivalent)
    ClarityMax Western ECL Substrate (Bio-Rad Laboratories, catalog number: 1705062S , or equivalent)
24. Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 12483020 , or equivalent)
25. Glycine (Fisher Scientific, catalog number: BP381-5 )
26. HCl (36.5-38%) (Fisher Scientific, catalog number: A144S-500 )
27. Hybridoma-SFM (Thermo Fisher Scientific, Gibco^TM, catalog number: 12045076 )
28. KCl (Fisher Scientific, catalog number: BP366-500 ) 
29. KH₂PO₄ (Fisher Scientific, catalog number: P285-500 )
30. Methanol (VWR, catalog number: 56902-543 )
31. Milli-Q Water
32. Na₂HPO₄ (Fisher Scientific, catalog number: S393-3 )
33. NaCl (Fisher Scientific, catalog number: BP358-10 )
34. NaOH, 10N certified (Fisher Scientific, catalog number: SS255-1 )
35. NuPAGE^TM 4-12% Bis-Tris Gel (Thermo Fisher Scientific, Invitrogen^TM, catalog number: NP0335BOX )
36. NuPAGE^TM MOPS SDS Running Buffer (Thermo Fisher Scientific, Invitrogen^TM, catalog number: NP0001 )
37. Penicillin-Streptomycin (GE Healthcare, catalog number: SV30010 , or equivalent)
38. Polyethylenimine (PEI) (Polysciences, catalog number: 23966-1 , or equivalent)
39. Sucrose (WISENT, catalog number: 800-081-LG , or equivalent)
40. Tris Base (Fisher Scientific, catalog number: BP152-5 )
41. Tween^TM 20 (Fisher Scientific, catalog number: BP337-100 )
42. PBS (10x) (see Recipes)
43. 20% sucrose in PBS (see Recipes)
44. Complete DMEM (see Recipes)
45. Tris-Glycine Transfer Buffer (25x) (see Recipes)
    

Note: We used VWR as a distributor for Pall and Corning products.

Equipment

1. μMACS Separator (Miltenyi Biotec, catalog number: 130-042-602 )
2. 4 °C refrigerator
3. Balance (Fisher Scientific, catalog number: 01-919-358, or equivalent)
   Manufacturer: OHAUS, catalog number: 30100606/RM .
4. Biosafety cabinet (Thermo Fisher Scientific, catalog number: 1323TS , or equivalent)
5. Digital Imager (GE Healthcare, model: ImageQuant LAS 4000, catalog number: 28955810 , or equivalent)
6. Haemocytometer (Hausser Scientific, catalog number: 3100 , or equivalent)
7. MACS MultiStand (Miltenyi Biotec, catalog number: 130-042-303 )
8. Magnetic stand (Thermo Fisher Scientific, catalog number: 12321D )
9. Microscope (Fisher Scientific, catalog number: LMI6PH2, or equivalent)
   Manufacturer: Laxco, catalog number: LMI6PH2 . 
10. Pipettes (Gilson, catalog number: F167700 , or equivalent)
11. Refrigerated table-top centrifuge (Thermo Fisher Scientific, model: Sorvall^TM ST 40 , catalog number: 75004524, or equivalent)
12. Rocking platform (Maxi Rotator) (Labline Instruments, model: Model 4631 , or equivalent)
13. Tissue culture incubator, humidity, temperature and CO₂ regulated (Thermo Fisher Scientific, catalog number: 3110 , or equivalent)
14. Tube Revolver (Thermo Fisher Scientific, catalog number: 88881002 or equivalent)
15. Type 70Ti Rotor (Beckman Coulter, catalog number: 337922 , or equivalent)
16. Type 70Ti Tubes (Polycarbonate tubes and lids) (Beckman Coulter, catalog number: 355618 , or equivalent)
17. Ultracentrifuge (Beckman Coulter, catalog number: 969347 , or equivalent)
    

Procedure

1. Seed 10 cm dishes with 3 x 10⁶ 293T cells in 8 ml of complete DMEM. For this assay, we typically use at least 2 dishes per condition.
2. Allow the cells to grow at 37 °C in 5% CO₂ until they reach 70-80% confluence, this should take approximately 24 h.
3. Transfect viral plasmid(s) of interest. For M-MLV, we typically use 10 μg of plasmid DNA, though this may be optimized based on the virus (Note 1).
4. Return the cells to the incubator for virus production for a period of 72 h (Note 2).
5. Before collecting the viral supernatant, pre-cool the ultracentrifuge to 4 °C.
6. Collect the viral supernatant (roughly 15 ml) using a serological pipette, and transfer it into a 50 ml conical tube.
7. Centrifuge this supernatant for 5 min at 500 x g to clear cellular debris.
8. During this centrifugation step, prepare the appropriate number of syringes and 450 nm filters.
9. Transfer the cleared supernatant into a syringe and filter it directly into a Type 70Ti tube.
10. Top up each tube with media or PBS such that they contain approximately 5.5 ml below the maximum threshold.
11. Place a sterile Pasteur pipette into each 70Ti tube, with the thin side immersed in viral media. Refer to Figure 2.
12. Slowly add the sterile 20% sucrose solution through the Pasteur pipette so it may form a cushioning layer below the viral supernatant. Five milliliters of this solution is sufficient (Note 3). Refer to Figure 2.
13. Balance each tube appropriately for ultracentrifugation. Sterile media or PBS can be used to adjust the mass of viral samples.
14. Cap each tube, ensuring the O-rings and aluminum caps are sealed properly. Insert these tubes appropriately into the Type 70Ti rotor and insert the rotor into the ultracentrifuge (Note 3).
15. Ultracentrifuge these samples with an acceleration and deceleration set to 50%, for 3 h at 4 °C at 100,000 x g (Note 4).
16. Remove the tubes from the rotor, visualize the pellets and circle with a marker. As time passes after the centrifugation, these become more difficult to see so you need to proceed as fast as possible. Refer to Figure 2.
    
    
    Figure 2. Virus concentration by ultracentrifugation. A Pasteur pipette (A) is used to add a distinct underlayer of sucrose (B). After ultracentrifugation, the pellet is visualized and identified with a marker (C).
    
    
17. Gently remove supernatants using a serological pipette and resuspend the pellet in 1 ml of PBS (Note 4).
18. Incubate this concentrated viral sample with an excess of antibody-conjugated beads (we used 30 μl) targeting a surface antigen (i.e., anti-GFP) for 3 h at 4 °C with constant gentle rotation.
19. Using the μMACs system (Note 5), ready the μ-columns on the MultiStand.
20. Add PBS to prime the column prior to sample addition (Note 6).
21. Load the viral sample into a μ-column and allow PBS to flow through the column by gravity. Flow through from this stage can be collected for analysis with the enriched portion. Refer to Figure 3.
22. Wash the column with 5x volume of sterile PBS (5 ml for each 1 ml of virus).
23. Elute from the column as desired. Miltenyi describes multiple conditions for elution, both denaturing and non-denaturing. In our case, we desired to reduce non-specific elution and maintain viral integrity. To achieve this, remove the column from the magnetic stand, insert it into a microcentrifuge tube and add 250 μl of sterile PBS to the column, which is collected in the tube. The eluate will also contain the magnetic beads, so it will be a translucent brown colour as shown in Figure 3.
24. Samples can be stored short-term (< 1 day) at 4 °C, and long-term (weeks) at -80 °C.
    
    
    Figure 3. μMACS isolation of intact viruses and gentle elution. A μ-column is securely fit into the separator on the magnetic stand and a microcentrifuge tube can be placed below to collect flow through (A). Removal of the μ-column from the magnetic field (B) will enable mobility of the magnetic beads in any desired elution buffer. The beads will also elute from the column, giving the eluate a translucent brown colour (C).

Data analysis

1. SDS-PAGE analysis: Sample purity can be determined by quantifying integral viral components. Proteomic techniques, such as SDS-PAGE or ELISA, are recommended as this is a protein interaction-based isolation. However, viral genome quantifications are a suitable alternative to assess total virus isolation efficiency. In our study, M-MLV was probed for the p30 capsid protein (R187, rat monoclonal, 1μg/ml), the viral envelope glycoprotein Env-eGFP (anti-eGFP, JL-8, mouse monoclonal, 0.2μg/ml) and recombinant gPr80 (gPr93FV contains an N-proximal Flag-tag and C-terminal V5-tag, shifting its observed size from 80 kDa to 93 kDa; anti-V5, rabbit polyclonal, 0.2μg/ml). For the p30 capsid antibody, R187 cells were grown according to the conditions outlined by the ATCC using Hybridoma-SFM. The supernatant can either be used directly or purified using a Protein A or G resin. All other antibodies were purchased from the suppliers indicated in the Materials and Reagents section.
     For best resolution, we used NuPAGE 4% to 12% gradient gels in MOPS running buffer at 200 V for approximately 45-55 min. Transfer was done to a PVDF membrane using a Tris-Glycine Transfer buffer at 100 V for approximately 80 min. Blocking was done using 5% milk in PBS-T for 1 h at room temperature. Blocking, washing and antibody staining steps are best performed on a rocking platform. We achieved optimal results by performing primary antibody stainings overnight at 4 °C in blocking buffer, while secondary antibodies (conjugated to HRP) can be incubated at room temperature for 1 h at the proper dilutions in blocking buffer (anti-mouse IgG secondary 1:5,000; anti-rabbit IgG secondary at 0.1 μg/ml; anti-rat IgG secondary at 0.05 μg/ml). Detection was done using an ECL of appropriate intensity (i.e., Bio-Rad, Clarity, 1705060S or ClarityMax, 1705062S, or equivalents) and analyzed on a Digital Imager (GE LifeSciences, ImageQuant LAS 4000, or equivalent). It is good practice to analyze the unprocessed sample (input), flow through (unbound) and eluate (IP) of the immunoprecipitation using multiple antibodies targeting viral components as a way to monitor the performance of the IVIP assay.
2. Interpretation of results: The IVIP protocol described here was developed to characterize the differential association of the retroviral integral membrane protein glycogag (gPr80) with intact EVs and viruses, along with determining its membrane topology at the surface of M-MLV virions. To help characterize the association of gPr80 with virions and EVs, we developed gPr93FV, a recombinant gPr80 construct harboring N-proximal Flag and C-terminal V5 epitope tags. These features enabled us to determine the orientation of gPr93FV in the outer envelope of EVs and M-MLV by the way it interacts with antibodies directed against each epitope tag. Given that we previously demonstrated that the viral envelope glycoprotein, Env (or more specifically, Env-eGFP), is a selection marker abundantly found on the surface of M-MLV and rarely detected on EVs (Tang et al., 2017), it was therefore possible to distinguish EVs (unbound fraction) and viruses (bound fraction) following the eGFP-targeting IVIP protocol described above.
     The Input, Unbound and IP fractions were probed using three antibodies targeting viral proteins. Figure 4A shows that capsid (p30) was present in the Unbound, but enriched in the IP fraction as expected. Env-eGFP, the capture antigen, was present in the IP fraction but not in the unbound fraction, indicating a successful and pure intact virus preparation (Figure 4B). Using a V5 antibody, gPr93FV was detected in both the Unbound and IP fractions (Figure 4C). These data along with other experiments from our study enabled us to conclude that gPr80 can be inserted in the M-MLV envelope as a Type-I integral membrane protein in an N_exoC_cyto orientation, as its N terminus is accessible to the V5 antibody on the surface of intact virus particles. Development of this protocol has also lead to the identification of subpopulations of secreted particles (viruses, EVs, VLPs) that independently and differentially incorporate viral proteins, and would otherwise be indistinguishable by conventional methods that denature the structural integrity of such particles prior to SDS-PAGE analysis (Renner et al., 2018).
   
   
   Figure 4. Assessment of the purity and viral constituents of IVIP samples by SDS-PAGE. This figure has been adapted from Renner et al. (2018). M-MLV was co-produced with a Glycosylated Gag expression plasmid (gPr93FV) and isolated according to the IVIP protocol described above. A) Viral capsid (p30), B) viral envelope glycoprotein (eGFP) and C) gPr93FV (V5) were all visualized by SDS-PAGE using NuPAGE gels. The band pattern of gPr93FV is indicative of its multiple glycosylation states. The V5 antibody used visualized a non-specific band at approximately 65 kDa, however it is not a viral component as it is washed away during the IP procedure.

Notes

1. Transfection reagents or methods to produce virus from an expression plasmid can vary. We used polyethylenimine (PEI) and the protocol is extensively described in Longo et al. (2013).
2. Optimal production time may vary depending on the virus or cell type.
3. Be very careful when maneuvering with these tubes, as the sucrose layer can mix with the viral supernatant. It is important that these layers remain distinct and separated.
4. Steps 9-17 were carried out using standard methods for M-MLV concentration by ultracentrifugation. Spinning the virus through a sucrose cushion removes small cells debris, cytosolic material and impurities, and typically yields > 90% recovery on input virus infectivity. The pellet generated by ultracentrifugation is expected to be a small, viscous, slightly reddish accumulation at the bottom of the tube (if produced in DMEM with phenol red). Other concentration methods may be more suitable, and gentler, for other types of viruses (Rayaprolu et al., 2018). 
5. The μMACS system is one option of many which also allows protein G conjugation of any antibody. We have also used Dynabeads M270-epoxy (Thermo Fisher Scientific) in conjunction with the associated magnetic stand (Thermo Fisher Scientific), which allows for the use of any antibody with minimal antibody shedding from the beads. However, magnetic beads are highly recommended, as we find that these often produce reduced levels of non-specific binding when compared to sepharose or agarose beads.
6. If gravity flow does not initiate when preparing the column with PBS, a detergent-containing buffer (i.e., PBS + 0.1% Tween^TM 20) may be used to prime the column. Be sure to remove all traces of this buffer with a larger volume of detergent-free buffer (i.e., PBS), as the detergent will likely impact the integrity of enveloped viruses/vesicles.
   

Recipes

1. PBS (10x)
   NaCl 80 g/L
   KCl 2 g/L
   Na₂HPO₄ 7.63 g/L
   KH₂PO₄ 2.4 g/L
   Sterilize by passing through a Steritop 220 nm filter if to be stored for an extended period. Storage at room temperature
   Dilute as necessary to a 1x PBS solution
   Adjust pH to 7.4 using NaOH or HCl and filter sterilize
   Store anywhere between 4 °C and 25 °C
   Note: We typically make a 10x stock solution of PBS to reduce significance of the error associated with weighing these powders. This should be stored at room temperature; refrigeration may cause precipitation of salts.
2. 20% sucrose in PBS (m/v)
   200 g sucrose
   100 ml 10x PBS solution
   Volume brought to 1 L with sterile H₂O
   Adjust pH to 7.4 using NaOH or HCl
   Filter sterilize
   Store in fridge (4 °C)
3. Complete DMEM
   DMEM high glucose
   50 ml Fetal Bovine Serum
   5 ml penicillin/streptomycin solution
   Store in fridge (4 °C) 
4. Tris-Glycine Transfer buffer (25x)
   72.8 g Tris-Base
   360 g Glycine
   Volume brought to 2 L with sterile H₂O
   Store this at room temperature
   Just before use, dilute to 1x using 20% total volume methanol and the remainder sterile H₂O (i.e., 1 L total volume = 200 ml methanol, 40 ml 25x Transfer Buffer and 760 ml sterile H₂O).
   

Acknowledgments

M.-A.L. holds a Canada Research Chair in Molecular Virology and Intrinsic Immunity. This research was supported by a grant from the Canadian Institutes of Health Research (grant 89774) and an Early Researcher Award from the Ontario Ministry of Research and Innovation to M.-A.L. T.M.R. holds a QEII Graduate Scholarship of Ontario. This protocol was developed and briefly described in the Journal of Virology (Renner et al., 2018). Our gratitude goes to Vera A. Tang for helpful discussions and technical support, especially with regards to nanoscale flow cytometry.

Competing interests

The authors declare no conflicts of interest or competing interests.

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