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Mar 2018

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Selective Isolation of Retroviruses from Extracellular Vesicles by Intact Virion Immunoprecipitation
通过完整病毒免疫沉淀技术从细胞外囊泡选择性分离逆转录病毒   

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Abstract

There exists a wide variety of techniques to isolate and purify viral particles from cell culture supernatants. However, these techniques vary greatly in ease of use, purity, yield and impact on viral structural integrity. Most importantly, it is becoming evident that secreted extracellular vesicles (EVs) co-purify with retroviruses using nearly all purification methods due to nearly indistinguishable biophysical characteristics such as size, buoyant density and nucleic acid content. Recently, our group has illustrated a means of isolating intact and highly enriched retroviral virions from EV-containing cell supernatants using an immunoprecipitation approach targeting the viral envelope glycoprotein of the Moloney Murine Leukemia Virus (Renner et al., 2018). This technique, that we call intact virion immunoprecipitation (IVIP), enabled us to characterize the accessibility of epitopes on the surface of these retroviruses and assess the orientation of the virus-encoded integral membrane protein Glycogag (gPr80) in the viral envelope. Proper implementation of this protocol enables fast, simple and reproducible preparations of intact and highly purified retroviral particles devoid of detectable EV contaminants.

Keywords: Retrovirus purification (逆转录病毒纯化), Extracellular vesicles (细胞外囊泡), Exosomes (外泌体), MLV (MLV), HIV (HIV), Immunoprecipitation (免疫共沉淀), Glycogag (糖胺多糖), gPr80 (gPr80), Nanoscale flow cytometry (纳米流式细胞术), Flow virometry (病毒流式仪), Intact virion immunoprecipitation (完整病毒免疫沉淀技术), IVIP (IVIP)

Background

Widely used approaches for isolating retroviruses, such as the Human Immunodeficiency Virus (HIV) and Murine Leukemia Virus (MLV), include precipitation, chromatography, ultrafiltration, ultracentrifugation, as well as various other means of particle separation (Reviewed in Nestola et al., 2015). While each technique has its specific advantages, drawbacks and limitations, a common concern for all methods is the co-purification of cell secreted extracellular vesicles (EVs).

EVs constitute a heterogeneous population of membrane-derived vesicles secreted by all cell types (Yanez-Mo et al., 2015). There are strikingly similar biophysical and biochemical characteristics between retroviruses and EVs (Table 1), especially with the small 50-150 nm vesicles secreted through the endosomal pathway, better known as exosomes (Reviewed by Nolte-'t Hoen et al., 2016). Some retroviruses, such as HIV and MLVs, can also share with exosomes the pathways of biogenesis and egress through the endocytic system (Orenstein et al., 1988; Raposo et al., 2002; Houzet et al., 2006; Sandrin and Cosset, 2006; Akers et al., 2013; Madison and Okeoma, 2015; Martin et al., 2016; Nolte-'t Hoen et al., 2016). This imparts inherent biochemical composition similarities between the two types of particles, which extend to their cargo (e.g., proteins, mRNAs, miRNAs) and host-derived surface membrane proteins and antigens (e.g., CD9, CD63, CD81), which inevitably increases difficulties in telling them apart (Eckwahl et al., 2016; Nolte-'t Hoen et al., 2016; Telesnitsky and Wolin, 2016). Given such similarities, selective isolation of retroviruses requires a unique identifying marker to confidently discriminate them from exosomes and EVs in general.

Table 1. Retroviruses and EVs are nearly indistinguishable by their biochemical and biophysical characteristics


Nanoscale flow cytometry (NFC), also called flow virometry or NanoFACS, is an optimization of flow cytometry techniques, sample preparations and hardware for the analysis of particles smaller than 200 nm, which is the average detection limit of most commercial flow cytometers (Tang et al., 2016 and 2017; Lippe, 2018). This technology is especially useful for the immune phenotypic profiling of markers on the surface of viruses and EVs. By using this approach, we previously determined that a fluorescently tagged viral envelope glycoprotein (Env-eGFP) of the Moloney MLV (M-MLV) was almost exclusively expressed on the surface of these virus particles, and thereby constituted a very reliable selection marker (Figure 1) (Tang et al., 2017).


Figure 1. Env-eGFP represents a selection marker for identifying retroviruses by nanoscale flow cytometry. This figure has been adapted from Tang et al. (2017). 293T cells were mock transfected with an empty plasmid (A), with Env-eGFP (B) or eGFP (D) expression plasmids, or with the Env-eGFP expressing M-MLV viral plasmid (C). The M-MLV used in this study contained an eGFP reporter inserted into the proline-rich region of the extracellular domain of the envelope glycoprotein (Sliva et al., 2004). Supernatants were 450 nm-syringe filtered prior to NFC analysis. Transfection efficiency was monitored by eGFP expression in the transfected cells and was similar in each relevant condition (data not shown). For NFC analysis, particles were detected by triggering off of side-scattered light (SSC). A square gate was set above background in the Mock sample where eGFP+ events are expected (A). Side and top boundaries of this gate were determined by the limits of the eGFP+ events in the MLVeGFP sample (C). Numbers in green represent eGFP+ particles detected and enumerated in the gate during a fixed acquisition time window, which was the same for all samples analyzed. For NFC analysis, SSC is more sensitive than forward scattered (FSC) light to detect particles smaller than 200nm on our instrument (Tang et al., 2016). The results show that, in our system, the membrane-expressed Env-eGFP does not substantially associate with EVs (B). However, cytosolic eGFP is incorporated as cargo inside EVs (D). Env-eGFP is highly enriched only on the surface of viruses (C).

The protocol described here was specifically developed to study an enigmatic virus-encoded integral membrane protein called Glycogag (or gPr80) inserted in the envelope of M-MLV (Pillemer et al., 1986; Fujisawa et al., 1997 and 2001; Rosales Gerpe et al., 2015; Renner et al., 2018). Our goal was to assess the incorporation and orientation of full-length gPr80 in the envelope of M-MLV. A major caveat to this particular study was the release of EVs by the infected cells that contaminated our virus samples. This proved to be especially problematic, as we found that the gPr80 protein associated with both EVs and virions (Renner et al., 2018). But given that Env-eGFP was highly enriched on the surface of M-MLV virions and poorly incorporated on EVs (Figure 1) (Tang et al., 2017), we thus developed an intact virion immunoprecipitation (IVIP) assay designed to specifically isolate structurally intact viral particles expressing Env-eGFP on their surface. Using this approach, we successfully identified the orientation of gPr80 as a Type-I integral membrane protein on virions but as a Type-II integral membrane protein on EVs that are devoid of Env-eGFP (Renner et al., 2018). In conclusion, IVIP has the ability to selectively isolate and discriminate retroviruses from EVs with minimal physical manipulation and without compromising the structural integrity of either particle type.

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, GibcoTM, 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, GibcoTM, 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. NuPAGETM 4-12% Bis-Tris Gel (Thermo Fisher Scientific, InvitrogenTM, catalog number: NP0335BOX )
  36. NuPAGETM MOPS SDS Running Buffer (Thermo Fisher Scientific, InvitrogenTM, 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. TweenTM 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: SorvallTM 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 NexoCcyto 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% TweenTM 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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  25. Pillemer, E. A., Kooistra, D. A., Witte, O. N. and Weissman, I. L. (1986). Monoclonal antibody to the amino-terminal L sequence of murine leukemia virus glycosylated gag polyproteins demonstrates their unusual orientation in the cell membrane. J Virol 57(2): 413-421.
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简介

存在多种从细胞培养上清液中分离和纯化病毒颗粒的技术。然而,这些技术在易用性,纯度,产量和对病毒结构完整性的影响方面差异很大。最重要的是,由于几乎无法区分的生物物理特征,例如大小,浮力密度和核酸含量,使用几乎所有纯化方法分泌的细胞外囊泡(EV)与逆转录病毒共同纯化变得明显。最近,我们小组已经阐明了一种利用针对Moloney鼠白血病病毒的病毒包膜糖蛋白的免疫沉淀方法从含有EV的细胞上清液中分离完整和高度富集的逆转录病毒病毒颗粒的方法(Renner et al。, 2018)。这种技术,我们称之为完整的病毒粒子免疫沉淀(IVIP),使我们能够表征这些逆转录病毒表面上表位的可及性,并评估病毒包膜中病毒编码的整合膜蛋白Glycogag(gPr80)的方向。该方案的正确实施使得能够快速,简单且可重复地制备完整且高度纯化的逆转录病毒颗粒,其没有可检测的EV污染物。

【背景】广泛使用的分离逆转录病毒的方法,如人类免疫缺陷病毒(HIV)和小鼠白血病病毒(MLV),包括沉淀,色谱,超滤,超速离心,以及各种其他粒子分离方法(评论在Nestola et al。,2015)。虽然每种技术都有其特定的优点,缺点和局限性,但所有方法的共同关注点是细胞分泌的细胞外囊泡(EV)的共同纯化。

EV构成由所有细胞类型分泌的膜衍生囊泡的异质群体(Yanez-Mo 等人,,2015)。在逆转录病毒和EV之间存在惊人相似的生物物理和生物化学特征(表1),特别是通过内体途径分泌的小的50-150nm囊泡,更好地称为外来体(由Nolte-'t Hoen 等人评论。 ,2016)。一些逆转录病毒,如HIV和MLV,也可以通过内吞系统与外泌体共享生物发生和流出的途径(Orenstein et al。,1988; Raposo et al。 ,2002; Houzet et al。,2006; Sandrin和Cosset,2006; Akers et al。,2013; Madison and Okeoma,2015; Martin et al。 ,2016; Nolte-'t Hoen et al。,2016)。这赋予了两种类型颗粒之间固有的生化组成相似性,这些颗粒延伸到它们的货物(例如,,蛋白质,mRNA,miRNA)和宿主衍生的表面膜蛋白和抗原(例如,CD9,CD63,CD81),这不可避免地增加了将它们分开的困难(Eckwahl et al。,2016; Nolte-'t Hoen et al。,2016 ; Telesnitsky和Wolin,2016)。鉴于这种相似性,逆转录病毒的选择性分离需要独特的鉴定标记,以便将它们与外来体和EV一般地区别开来。

表1.逆转录病毒和EV几乎无法通过其生化和生物物理特征进行区分


纳米级流式细胞仪(NFC),也称为流式病毒测定法或NanoFACS,是流式细胞仪技术,样品制备和硬件的优化,用于分析小于200 nm的颗粒,这是大多数商业流式细胞仪的平均检测限(Tang et al。,2016和2017; Lippe,2018)。该技术尤其适用于病毒和EV表面上标记物的免疫表型分析。通过使用这种方法,我们以前确定Moloney MLV(M-MLV)的荧光标记的病毒包膜糖蛋白(Env-eGFP)几乎仅在这些病毒颗粒的表面上表达,从而构成非常可靠的选择标记(图1)(Tang et al。,2017)。


图1. Env-eGFP代表通过纳米级流式细胞仪鉴定逆转录病毒的选择标记。该图已经改编自Tang 等人(2017)。用空质粒(A),Env-eGFP(B)或eGFP(D)表达质粒或用表达Env-eGFP的M-MLV病毒质粒(C)模拟转染293T细胞。本研究中使用的M-MLV含有插入包膜糖蛋白细胞外结构域的富含脯氨酸区域的eGFP报告基因(Sliva 等人,2004)。在NFC分析之前,将上清液进行450nm注射过滤。通过转染细胞中的eGFP表达监测转染效率,并且在每种相关条件下相似(数据未显示)。对于NFC分析,通过触发侧向散射光(SSC)来检测粒子。在模拟样本中将方形门设置在背景之上,其中预期eGFP +事件(A)。该门的侧边界和顶边界由MLVeGFP样品(C)中eGFP +事件的极限确定。绿色数字表示在固定采集时间窗口期间在门中检测和计数的eGFP +颗粒,对于所有分析的样品是相同的。对于NFC分析,SSC比前向散射(FSC)光更灵敏,可以检测仪器上小于200nm的粒子(Tang et al。,2016)。结果表明,在我们的系统中,膜表达的Env-eGFP基本上不与EV结合(B)。然而,细胞溶质eGFP作为货物并入EV(D)中。 Env-eGFP仅在病毒表面高度富集(C)。

这里描述的方案专门开发用于研究插入M-MLV包膜中的神秘病毒编码的整合膜蛋白Glycogag(或gPr80)(Pillemer et al。,1986; Fujisawa 等人,,1997和2001; Rosales Gerpe 等人,2015; Renner 等人,2018)。我们的目标是评估全长gPr80在M-MLV包膜中的掺入和定向。这项特殊研究的一个主要警告是被我们的病毒样本污染的受感染细胞释放EV。这被证明是特别成问题的,因为我们发现gPr80蛋白与EV和病毒粒子相关(Renner et al。,2018)。但鉴于Env-eGFP在M-MLV病毒体表面高度富集并且很少掺入EV(图1)(Tang et al。,2017),因此我们开发了完整的病毒粒子免疫沉淀( IVIP)测定法设计用于在其表面上特异性分离表达Env-eGFP的结构完整的病毒颗粒。使用这种方法,我们成功地确定了gPr80作为病毒体上I型整合膜蛋白的定向,但作为不含Env-eGFP的EV上的II型整合膜蛋白(Renner et al。,2018)。总之,IVIP能够以最小的物理操作选择性地从EV中分离和区分逆转录病毒,并且不损害任一种颗粒类型的结构完整性。

关键字:逆转录病毒纯化, 细胞外囊泡, 外泌体, MLV, HIV, 免疫共沉淀, 糖胺多糖, gPr80, 纳米流式细胞术, 病毒流式仪, 完整病毒免疫沉淀技术, IVIP

材料和试剂

  1. μ-Columns(Miltenyi Biotec,目录号:130-042-701)
  2. 微量离心管(FroggaBio,目录号:LMCT1.7B,或同等产品)
  3. 巴斯德吸管(Fisher Scientific,目录号:13-678-20A,或同等产品)
  4. PVDF膜(Bio-Rad Laboratories,目录号:1620177)
  5. 血清移液管,10 ml(Corning,目录号:4488,或同等学历)
  6. Luer-Lok无菌20 ml注射器(BD,目录号:302830,或同等产品)
  7. 无菌450 nm Luer-Lok注射器过滤器(颇尔,目录号:4614,或等效物)
  8. 无菌50毫升锥形管(FroggaBio,目录号:TB50-500,或等效物)
  9. 无菌移液器吸头(Diamed,DIATEC,目录号:DIATEC520-5376,DIATEC520-5876,DIATEC520-6501或同等产品)
  10. HEK 293T电池(ATCC,目录号:CRL-3216)
  11. R187杂交瘤(ATCC,目录号:CRL-1912)
  12. μMACSGFP分离试剂盒(Miltenyi Biotec,目录号:130-091-125)
  13. 10厘米培养皿(康宁,目录号:430167,或等效物)
  14. 220 nm Steritop过滤器(Merck,目录号:SCGPT10RE,或同等产品)
  15. 抗eGFP(Takara Bio,Clontech,目录号:632381)
  16. 抗Flag,HRP共轭(Sigma-Aldrich,目录号:A8592-1MG)
  17. 抗小鼠IgG,HRP共轭(Cell Signaling Technology,目录号:7076S)
  18. 抗兔IgG,HRP结合(Abcam,目录号:ab6721)
  19. 抗大鼠IgG,HRP结合(Sigma-Aldrich,目录号:AP183P)
  20. Anti-V5(默克,产品目录号:AB3792)
  21. Dulbecco改良Eagle's培养基(DMEM)高葡萄糖,含L-谷氨酰胺,丙酮酸钠和酚红(WISENT,目录号:319-005-CL,或等效物)
  22. Dynabeads M270-epoxy(赛默飞世尔科技,目录号:14321D)
  23. ECL Substrates,即:
    Clarity Western ECL Substrate(Bio-Rad Laboratories,目录号:1705060S,或同等产品)
    ClarityMax Western ECL Substrate(Bio-Rad Laboratories,目录号:1705062S,或同等产品)
  24. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM ,目录号:12483020,或等同物)
  25. 甘氨酸(Fisher Scientific,目录号:BP381-5)
  26. HCl(36.5-38%)(Fisher Scientific,目录号:A144S-500)
  27. Hybridoma-SFM(Thermo Fisher Scientific,Gibco TM ,目录号:12045076)
  28. KCl(Fisher Scientific,目录号:BP366-500)&nbsp;
  29. KH 2 PO 4 (Fisher Scientific,目录号:P285-500)
  30. 甲醇(VWR,目录号:56902-543)
  31. Milli-Q水
  32. Na 2 HPO 4 (Fisher Scientific,目录号:S393-3)
  33. NaCl(Fisher Scientific,目录号:BP358-10)
  34. NaOH,10N认证(Fisher Scientific,目录号:SS255-1)
  35. NuPAGE TM 4-12%Bis-Tris凝胶(Thermo Fisher Scientific,Invitrogen TM ,目录号:NP0335BOX)
  36. NuPAGE TM MOPS SDS运行缓冲液(Thermo Fisher Scientific,Invitrogen TM ,目录号:NP0001)
  37. 青霉素 - 链霉素(GE Healthcare,目录号:SV30010,或同等学历)
  38. 聚乙烯亚胺(PEI)(Polysciences,目录号:23966-1,或同等学历)
  39. 蔗糖(WISENT,目录号:800-081-LG,或同等学历)
  40. Tris Base(Fisher Scientific,目录号:BP152-5)
  41. Tween TM 20(Fisher Scientific,目录号:BP337-100)
  42. PBS(10x)(见食谱)
  43. PBS中20%蔗糖(参见食谱)
  44. 完整的DMEM(见食谱)
  45. Tris-Glycine转移缓冲液(25x)(见食谱)

注意:我们使用VWR作为颇尔和康宁产品的分销商。

设备

  1. μMACS分离器(Miltenyi Biotec,目录号:130-042-602)
  2. 4°C冰箱
  3. 平衡(Fisher Scientific,目录号:01-919-358,或等效物)
    制造商:OHAUS,目录号:30100606 / RM。
  4. 生物安全柜(Thermo Fisher Scientific,目录号:1323TS,或同等产品)
  5. Digital Imager(GE Healthcare,型号:ImageQuant LAS 4000,目录号:28955810,或同等产品)
  6. 血细胞计数器(Hausser Scientific,目录号:3100,或同等学历)
  7. MACS MultiStand(Miltenyi Biotec,目录号:130-042-303)
  8. 磁力架(Thermo Fisher Scientific,目录号:12321D)
  9. 显微镜(Fisher Scientific,目录号:LMI6PH2,或等效物)
    制造商:Laxco,目录号:LMI6PH2。&nbsp;
  10. 移液器(Gilson,目录号:F167700,或等效物)
  11. 冷藏台式离心机(Thermo Fisher Scientific,型号:Sorvall TM ST 40,目录号:75004524,或同等产品)
  12. 摇摆平台(Maxi Rotator)(Labline仪器,型号:4631型或同等产品)
  13. 组织培养箱,湿度,温度和CO 2 调节(Thermo Fisher Scientific,目录号:3110,或等效物)
  14. Tube Revolver(Thermo Fisher Scientific,目录号:88881002或同等产品)
  15. 70Ti型转子(Beckman Coulter,目录号:337922,或同等产品)
  16. 70Ti型管(聚碳酸酯管和盖子)(Beckman Coulter,目录号:355618,或同等产品)
  17. 超速离心机(Beckman Coulter,目录号:969347,或同等产品)

程序

  1. 在8ml完全DMEM中用3×10 6个 293T细胞接种10cm培养皿。对于该测定,我们通常每种条件使用至少2个培养皿。
  2. 让细胞在37℃,5%CO 2 下生长,直至达到70-80%汇合,这应该需要大约24小时。
  3. 转染感兴趣的病毒质粒。对于M-MLV,我们通常使用10μg质粒DNA,但可以根据病毒进行优化(注1)。
  4. 将细胞放回培养箱中进行病毒生产72小时(注2)。
  5. 在收集病毒上清液之前,将超速离心机预冷至4°C。
  6. 使用血清移液管收集病毒上清液(约15ml),并将其转移到50ml锥形管中。
  7. 将该上清液在500 x g 下离心5分钟以清除细胞碎片。
  8. 在该离心步骤期间,准备适当数量的注射器和450nm过滤器。
  9. 将澄清的上清液转移到注射器中,并将其直接过滤到70Ti型管中。
  10. 用培养基或PBS填充每个管,使其含有低于最大阈值约5.5ml。
  11. 将无菌巴斯德吸管放入每个70Ti管中,将薄侧浸入病毒培养基中。参见图2。
  12. 通过巴斯德吸管缓慢加入无菌20%蔗糖溶液,使其在病毒上清液下方形成缓冲层。 5毫升这种溶液就足够了(注3)。参见图2。
  13. 适当平衡每个管以进行超速离心。无菌培养基或PBS可用于调节病毒样品的质量。
  14. 盖上每根管子,确保O形圈和铝盖正确密封。将这些管子适当地插入70Ti型转子中,然后将转子插入超速离心机(注3)。
  15. 超速离心这些样品,加速和减速设定为50%,在4°C,100,000 x g 3小时(注4)。
  16. 从转子上取下管子,观察颗粒并用标记圈住。随着离心后的时间过去,这些变得更难以看到,因此您需要尽快进行。参见图2.


    图2.通过超速离心的病毒浓度。巴斯德吸管(A)用于添加不同的蔗糖底层(B)。超速离心后,将沉淀物显现并用标记物(C)鉴定。

  17. 用血清移液管轻轻取出上清液,将沉淀重悬于1 ml PBS中(注4)。
  18. 将该浓缩的病毒样品与过量的抗体偶联的珠子(我们使用30μl)一起孵育,靶向表面抗原(即,抗-GFP),在4℃下持续温和旋转3小时。
  19. 使用μMAC系统(注5),准备好MultiStand上的μ列。
  20. 在添加样品之前添加PBS以填充色谱柱(注释6)。
  21. 将病毒样品加载到μ柱中,让PBS通过重力流过柱子。可以收集来自该阶段的流量以用富集部分进行分析。参见图3。
  22. 用5倍体积的无菌PBS(每1ml病毒5ml)洗涤柱子。
  23. 根据需要从色谱柱中洗脱。 Miltenyi描述了洗脱的多种条件,包括变性和非变性。在我们的案例中,我们希望减少非特异性洗脱并保持病毒的完整性。为此,从磁力架上取下色谱柱,将其插入微量离心管中,向色谱柱中加入250μl无菌PBS,将其收集在试管中。洗脱液也含有磁珠,因此它将是半透明的棕色,如图3所示。
  24. 样品可以在4°C下短期(<1天)储存,并在-80°C下长期储存(数周)。


    图3.完整病毒的μMACS分离和温和洗脱。将μ-柱牢固地装入磁力架上的分离器中,并且可以在下方放置微量离心管以收集流过(A)的流量。从磁场(B)中除去μ-柱将使磁珠在任何所需的洗脱缓冲液中移动。珠子也将从柱子中洗脱,使洗脱液呈半透明的棕色(C)。

数据分析

  1. SDS-PAGE分析:样品纯度可通过定量整合病毒组分来确定。建议使用蛋白质组学技术,如SDS-PAGE或ELISA,因为这是基于蛋白质相互作用的分离。然而,病毒基因组定量是评估总病毒分离效率的合适替代方案。在我们的研究中,M-MLV被探测p30衣壳蛋白(R187,大鼠单克隆,1μg/ ml),病毒包膜糖蛋白Env-eGFP(抗eGFP,JL-8,小鼠单克隆,0.2μg/ ml)和重组gPr80(gPr93FV含有N-近端Flag-标签和C-末端V5-标签,将其观察到的大小从80kDa转移至93kDa;抗-V5,兔多克隆,0.2μg/ ml)。对于p30衣壳抗体,根据ATCC使用Hybridoma-SFM概述的条件培养R187细胞。上清液可以直接使用或使用蛋白A或G树脂纯化。所有其他抗体均购自材料和试剂部分中指出的供应商。
    &NBSP;为了获得最佳分辨率,我们在MOPS运行缓冲液中使用NuPAGE 4%至12%梯度凝胶在200 V下保持约45-55分钟。使用Tris-甘氨酸转移缓冲液在100V下对PVDF膜进行转移约80分钟。使用PBS-T中的5%牛奶在室温下封闭1小时。阻塞,洗涤和抗体染色步骤最好在摇摆平台上进行。我们通过在封闭缓冲液中在4°C下进行一级抗体染色来获得最佳结果,而二级抗体(与HRP缀合)可以在封闭缓冲液中以适当的稀释度在室温下孵育1小时(抗小鼠IgG二级1: 5,000;抗兔IgG继发0.1μg/ ml;抗大鼠IgG二次,0.05μg/ ml)。使用适当强度的ECL(即,Bio-Rad,Clarity,1705060S或ClarityMax,1705062S或等同物)进行检测,并在数字成像仪(GE LifeSciences,ImageQuant LAS 4000或等效物)上进行分析。 )。优良作法是使用靶向病毒组分的多种抗体分析未处理的样品(输入),流过(未结合)和洗脱(IP)免疫沉淀,作为监测IVIP测定性能的方式。
  2. 结果解释:这里描述的IVIP方案被开发用于表征逆转录病毒整合膜蛋白质糖蛋白(gPr80)与完整EV和病毒的差异关联,以及确定其在M表面的膜结构。 -MLV病毒粒子。为了帮助表征gPr80与病毒粒子和EV的关联,我们开发了gPr93FV,一种含有N-近端Flag和C-末端V5表位标签的重组gPr80构建体。这些特征使我们能够通过与针对每个表位标签的抗体相互作用的方式确定EV和M-MLV的外包膜中gPr93FV的方向。鉴于我们之前已经证明病毒包膜糖蛋白,Env(或更具体地,Env-eGFP)是在M-MLV表面上大量发现的选择标记,并且很少在EV上检测到(Tang et al。 &NBSP;使用靶向病毒蛋白的三种抗体探测输入,未结合和IP级分。图4A显示衣壳(p30)存在于未结合中,但如预期的那样富集IP部分。 Env-eGFP(捕获抗原)存在于IP部分中但不存在于未结合部分中,表明成功且纯的完整病毒制剂(图4B)。使用V5抗体,在未结合和IP部分中检测到gPr93FV(图4C)。这些数据以及我们研究中的其他实验使我们得出结论,gPr80可作为N exo C cyto中的I型整合膜蛋白插入M-MLV包膜中

    图4.通过SDS-PAGE评估IVIP样品的纯度和病毒成分。该图已经改编自Renner 等人(2018)。 M-MLV与糖基化的Gag表达质粒(gPr93FV)共同产生,并根据上述IVIP方案分离。 A)病毒衣壳(p30),B)病毒包膜糖蛋白(eGFP)和C)gPr93FV(V5)均使用NuPAGE凝胶通过SDS-PAGE显现。 gPr93FV的条带模式指示其多种糖基化状态。使用的V5抗体显示约65kDa的非特异性条带,但它不是病毒组分,因为它在IP过程中被洗掉。

笔记

  1. 从表达质粒产生病毒的转染试剂或方法可以变化。我们使用聚乙烯亚胺(PEI),该方案在Longo 等人(2013)中有详细描述。
  2. 最佳生产时间可能因病毒或细胞类型而异。
  3. 当用这些管操纵时要非常小心,因为蔗糖层可以与病毒上清液混合。重要的是这些层保持清晰和分离。
  4. 使用超速离心M-MLV浓度的标准方法进行步骤9-17。通过蔗糖垫旋转病毒除去小细胞碎片,细胞溶质材料和杂质,并且通常产生> 1。输入病毒感染率恢复90%。通过超速离心产生的颗粒预计在管底部是小的,粘稠的,略带红色的积聚物(如果在具有酚红的DMEM中产生)。对于其他类型的病毒,其他浓度方法可能更合适,更温和(Rayaprolu et al。,2018)。&nbsp;
  5. μMACS系统是许多中的一种选择,其还允许任何抗体的蛋白G缀合。我们还将Dynabeads M270-epoxy(Thermo Fisher Scientific)与相关的磁力架(Thermo Fisher Scientific)结合使用,该磁力架允许使用任何抗体,其中珠子的抗体脱落最少。然而,强烈推荐磁珠,因为我们发现与琼脂糖凝胶或琼脂糖珠相比,这些磁珠通常产生降低水平的非特异性结合。
  6. 如果在用PBS制备色谱柱时没有开始重力流动,可以使用含有洗涤剂的缓冲液(即,PBS + 0.1%吐温 TM 20)来填充色谱柱。一定要用较大量的无去污剂缓冲液(即,PBS)去除所有缓冲液的痕迹,因为去污剂可能会影响包膜病毒/囊泡的完整性。

食谱

  1. PBS(10x)
    NaCl 80 g / L
    KCl 2 g / L
    Na 2 HPO 4 7.63 g / L
    KH 2 PO 4 2.4 g / L
    如果要长时间储存,请通过Steritop 220 nm过滤器进行灭菌。在室温下储存
    根据需要稀释1x PBS溶液
    使用NaOH或HCl将pH调节至7.4并过滤灭菌
    储存在4°C至25°C之间
    注意:我们通常制作10倍的PBS原液,以降低与称量这些粉末相关的误差的重要性。这应该在室温下储存;冷藏可能导致盐沉淀。
  2. PBS中的20%蔗糖(m / v)
    200克蔗糖
    100毫升10倍PBS溶液
    用无菌H 2 O将体积调至1L 使用NaOH或HCl将pH调节至7.4
    过滤消毒
    存放在冰箱(4°C)
  3. 完成DMEM
    DMEM高糖葡萄糖
    50毫升胎牛血清
    5毫升青霉素/链霉素溶液
    存放在冰箱(4°C)&nbsp;
  4. Tris-甘氨酸转移缓冲液(25x)
    72.8克Tris-Base
    360克甘氨酸
    用无菌H 2 O将体积调至2L 在室温下储存
    在使用前,用20%总体积的甲醇稀释至1倍,剩余的无菌H 2 O(即,1 L总体积= 200 ml甲醇,40 ml 25x转移缓冲液和760ml无菌H 2 O)。

致谢

M.-A.L.拥有加拿大分子病毒学和内在免疫研究主席。这项研究得到了加拿大卫生研究院的资助(拨款89774)和安大略省研究与创新部颁发给M.-A.L的早期研究员奖。 T.M.R.持有安大略省QEII研究生奖学金。该方案在Journal of Virology(Renner et al。,2018)中开发并简要描述。我们感谢Vera A. Tang提供的有益讨论和技术支持,尤其是纳米级流式细胞仪。

利益争夺

作者声明没有利益冲突或竞争利益。

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引用:Renner, T. M., Bélanger, K. and Langlois, M. (2018). Selective Isolation of Retroviruses from Extracellular Vesicles by Intact Virion Immunoprecipitation. Bio-protocol 8(17): e3005. DOI: 10.21769/BioProtoc.3005.
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