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. Author manuscript; available in PMC: 2021 Jan 6.
Published in final edited form as: Methods Mol Biol. 2017;1628:53–63. doi: 10.1007/978-1-4939-7116-9_4

Production of Filovirus Glycoprotein-Pseudotyped Vesicular Stomatitis Virus for Study of Filovirus Entry Mechanisms

Rachel B Brouillette 1, Wendy Maury 1
PMCID: PMC7787361  NIHMSID: NIHMS1657320  PMID: 28573610

Abstract

Members of the family Filoviridae are filamentous, enveloped, and nonsegmented negative-stranded RNA viruses that can cause severe hemorrhagic disease in humans and nonhuman primates with high mortality rates. Current efforts to analyze the structure and biology of these viruses as well as the development of antivirals have been hindered by the necessity of biosafety level 4 containment (BSL4). Here, we outline how to produce and work with Ebola virus glycoprotein bearing vesicular stomatitis virus (VSV) pseudovirions. These pseudovirions can be safely used to evaluate early steps of the filovirus life cycle without need for BSL4 containment. Virus gene expression in the transduced cells is easy to assess since the pseudovirions encode a reporter gene in place of the VSV G glycoprotein gene. Adoption of VSV for use as a pseudovirion system for filovirus GP has significantly expanded access for researchers to study specific aspects of the viral life cycle outside of BSL4 containment and has allowed substantial growth of filovirus research.

Keywords: Filovirus, Pseudovirus, Pseudotyped-vesicular stomatitis virus, Entry

1. Introduction

Members of the family Filoviridae are filamentous, enveloped, and nonsegmented negative-stranded RNA viruses [1, 2] of which there are three genera: Ebolavirus, Cuevavirus, and Marburgvirus. Filoviruses can cause severe hemorrhagic disease in humans and nonhuman primates with high mortality rates [1, 2]. As a consequence of the absence of countermeasures against these viruses, their lethality, and their transmissibility between individuals, filoviruses are listed as a Category A pathogen by the NIH. Current efforts to analyze the structure and biology of these viruses as well as the development of antivirals have been hindered by the necessity of biosafety level 4 (BSL4) containment. After it was observed that VSV could efficiently incorporate foreign viral glycoproteins on its particles [3], VSV pseudovirions bearing filovirus glycoprotein (GP) were developed as a surrogate to investigate filoviral biology under more accessible BSL2 conditions [4–6].

VSV is a member of the family Rhabdoviridae, and like filoviruses, is within the taxonomic order of Mononegavirales and therefore has a similar genome structure. Although the concept of viral glycoprotein pseudotyping was discovered to occur during coinfections between VSV and other viruses in the 1980s [7], these initial studies included within the VSV genome an intact G gene encoding the native VSV glycoprotein. Thus the use of VSV pseudovirions to investigate the biology of the pseudotyped glycoprotein did not occur for more than another decade [3]. Subsequent studies used VSV genomes that deleted the G gene, thus allowing the study of the biology of the pseudotyped GP [4]. Early studies showed that Ebola virus GP conferred cellular tropism to VSV corresponding to the host range tropism of Ebola virus [4]. VSV pseudovirions have proven useful for the study of a variety of aspects of the biology of virion-associated glycoproteins from a number of high containment, bio-defense viral pathogens, including arenaviruses, paramyxoviruses, filoviruses, and bunyaviruses [8–15]. Additionally, VSV preparations are convenient for production of pseudoparticles; VSV grows in many animal and some insect cells and can be propagated in large quantities [16]. Adoption of VSV for use as a pseudovirion system for filovirus GP has significantly expanded access for researchers to study the virus outside of BSL4 containment and has allowed studies to emerge that demonstrate a range of findings from the basic structure of the Ebola glycoprotein, to entry, to its post internalization complex processing, to mechanisms of antibody-mediated neutralization and more.

The following protocol, as summarized in Fig. 1, describes the production of recombinant VSV containing a reporter gene, green fluorescent protein (GFP), instead of the VSV G protein gene, which thus are not infectious unless a gene expressing a viral glycoprotein responsible for receptor binding and membrane fusion is provided in trans (VSVΔG-GFP) [4].

Fig. 1.

Fig. 1

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Outline of VSV pseudovirion production. (a) Schematic of wild-type VSV genome, the VSV genome deleted for the G gene and insertion of the reporter gene, GFP, in place of G. (b) Production of EBOV GP pseudotyped VSV

2. Materials

2.1. Cell Culture Reagents

  1. Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin—streptomycin (P/S) (see Notes 1–3).

  2. DMEM supplemented with 1.5% FBS and 1% P/S.

  3. 1× phosphate buffered saline (PBS) (see Note 4).

  4. An inverted light microscope.

2.2. Cells

  1. HEK293T cells (Homo sapiens embryonic kidney epithelial cells containing the SV40 T antigen; ATCC CRL-3216).

  2. Vero cells (Cercopithecus aethiops kidney epithelial cells; ATCC CCL-81).

2.3. Transfection Reagents

  1. Polyethylenimine (PEI) solution (1 mg/mL) (see Note 5).

  2. 150 mM NaCl solution.

  3. DNA plasmid containing a filovirus glycoprotein gene driven by a mammalian enhancer/promoter, such as the CMV promoter. Using this protocol, we have generated VSV pseudovirions containing Ebola virus, Sudan virus, Bundibugyo, or Marburg virus GP (see Note 6).

2.4. Transduction Reagent

  1. VSVΔG-GFP pseudotyped with a viral glycoprotein that readily mediates entry into HEK293T cells (see Note 7). VSVΔG-stocks encoding a reporter gene (GFP, dsRed or luciferase) are available from Kerafast (http://www.kerafast.com/c-310-delta-g-vsv-pseudotyping-system.aspx).

2.5. Concentration/ Purification of Viral Particles (Optional)

  1. A high-speed centrifuge that can achieve ≥5400 × g.

  2. 250 or 500 mL sterile polypropylene bottles (depending on centrifuge rotor size).

  3. An ultracentrifuge that can achieve ≥80,000 × g.

  4. 3 mL sterile polycarbonate ultracentrifuge tubes for Beckman SW SW60Ti rotor or 30 mL sterile ultracentrifuge tube for Beckman SW32Ti rotor (or similar equipment if using an alternative ultracentrifuge).

  5. PBS.

  6. 20% sucrose in PBS (sterilized through a ≤0.45 μm filter).

2.6. Quantification of Viral Particles

  1. Flow cytometer.

  2. Flow cytometer analysis software.

  3. Accutase.

2.7. Normalization of Pseudovirus Stocks

  1. 5× lysis buffer (0.125% NP40 in PBS).

  2. 96-Well dot blot apparatus.

  3. Primary/secondary antibodies and blocking buffer for immunostaining (see Note 8).

3. Methods

3.1. Preparation of 1 mg/mL PEI Solution

  1. Dissolve 100 mg of PEI powder in 80–90 mL water on a stir plate with a magnetic bar.

  2. Adjust the pH to 7.0 with HCl (the solution clarifies as the correct pH is approached; full dissolution may take several hours).

  3. Add water until a final volume of 100 mL is reached.

  4. Sterilize the solution through a 0.22 μm filter and dispense into aliquots.

  5. Store aliquots at −20 °C or −80 °C for long term storage; an in-use stock can be kept at 4 °C for up to several months (see Note 5).

3.2. Transfection of HEK293T Cells

  1. In the afternoon, seed tissue culture plates to achieve 70–80% confluency 24 h after seeding in DMEM supplemented with 10% FBS and 1% P/S. See Table 1 for cell numbers.

  2. Twenty four hours following seeding, transfect cells with filovirus glycoprotein plasmid DNA (see Notes 9–11).

  3. Prepare two tubes for transfection (see Table 1): Tube 1—Mix 25 μL of NaCl with every 1 μg of DNA. Tube 2—Mix 22 μL of NaCl with 3 μL of PEI for every 1 μg of DNA in Tube 1.

  4. Combine the two tubes, vortex well (10–15 s), and incubate at room temperature for 7–20 min.

  5. Add the total volume of DNA/PEI/NaCl solution dropwise to cells and place tissue culture plate(s) back in 37 °C incubator with 5% CO2 until transduction.

Table 1.

Quantity of reagents needed for transfection performed with different sized plates

Tissue culture plate Transfection tube 1
 Transfection tube 2
 Total volume (μL)
# Cells to seed μg DNA μL NaCl μL PEI μL NaCl
6-Well 5.00E+05 2 per well 50 per well 6 per well 44 per well 100 per well
10 cm 3.50E+06 16 400 48 352 800
15 cm 1.00E+07 32 800 96 704 1600
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3.3. Transduction with VSVG-GFP

  1. Add a stock of GP pseudotyped VSVΔG at an MOI of ∼1–3 to cells 16–24 h post transfection (there is no need to change the medium). The MOI of the seeding virus added will need to be empirically determined for your conditions and seeding stock. The stock we typically use is a Lassa virus GPC-pseudotyped VSVΔG containing GFP (see Note 7).

  2. Maintain the seeding stock on cells at 37 °C for 2–4 h. As filovirus GP-pseudotyped VSV particles are produced as early as 6 h following transduction, be sure to continue with step 3 before then.

  3. Remove media and the wash cells with PBS twice to remove unbound input virus before replacing with DMEM supplemented with 1.5% FBS and 1% P/S.

  4. Return plate(s) to the 37 °C incubator 5% CO2.

3.4. Collection of Filovirus GP-Pseudotyped VSV G-GFP

  1. Collect medium in sterile syringes at 24 h following transduction and filter them through 0.45 μm filters into sterile containers.

  2. If making small stocks of pseudovirus, a single collection at 24 h may be sufficient. Store in ∼0.5 mL aliquots and discard used culture plates. However, if making large stocks of pseudovirus, add fresh DMEM to cells and repeat collection at 48 h following transduction.

  3. Store collected stocks at −80 °C or continue directly to ultracentrifugation, if desired (see Notes 12 and 13).

3.5. Concentration and Purification via Ultracentrifugation Through a Sucrose Cushion (Optional) (See Note 13)

  1. If collecting ≥30 mL of supernatant pseudovirus, first combine all collections into a single sterile bottle and spin for at least 16 h at 5400 × g in a high speed centrifuge at 4 °C to pellet the viral particles. Resuspend the pellet in ∼1 mL sterile PBS. If concentrating <30 mL of supernatant pseudovirus, begin at step 2, using the instructions for the SW32Ti rotor and the larger centrifuge tubes.

  2. Add 0.5 mL of sterile 20% sucrose PBS to the bottom of a 3 mL polycarbonate ultracentrifuge tube if using the SW60Ti rotor; or add 3 mL of sterile 20% sucrose PBS to the bottom of a 30 mL ultracentrifuge tube if using the SW32Ti rotor (see Note 14).

  3. Without penetrating the sucrose cushion, carefully layer either the supernatant collection or the resuspended virus pellet into the tube. Completely fill the tube, using PBS as necessary, to prevent collapse of the tube during centrifugation.

  4. Balance the rotor according to the ultracentrifuge manufacturer’s instructions and spin at 80,000 × g for 2 h at 4 °C.

  5. Discard liquid from the tubes and wipe out any remaining liquid with a sterile swab. Place the pellets on ice, add sterile PBS (to a desired volume) and resuspend the pellet. It is best to allow the pellet to loosen by incubating on ice for at least 30 min prior to attempting to resuspend. With highly concentrated stocks, resuspension of virion clumps can take several hours and may require scraping with a pipette tip and/ or extensive vortexing. Highly concentrated stocks will have a milky appearance.

  6. Distribute resuspension into aliquots and store at −80 °C for future use.

3.6. Quantification of Pseudovirus Stocks

  1. To determine the pseudovirion titer (transducing units/mL) in Vero cells, seed 50,000 Vero cells per well in a 48-well format in DMEM supplemented with 10% FBS and 1% P/S in the afternoon.

  2. On the following day, make several dilutions of your pseudovirus stock in DMEM supplemented with 1.5% FBS and 1% P/S. Remove medium from Vero cells and add 250 μL of each concentration of pseudovirus to duplicate or triplicate wells. In our lab, to titer a typically concentrated and purified virus stock, a series of several dilutions (two-, five-, or ten-fold) is assessed. See Fig. 2 for a typical detailed dilution curve.

  3. Incubate plates in a 5% CO2 incubator at 37 °C for 18–24 h.

  4. Remove media and detach cells with 100 μL 37 °C Accutase per well. Transfer cells into 4 mL polystyrene round-bottom tubes appropriate for use on a flow cytometer.

  5. Quantify the number of GFP-expressing cells using a flow cytometer. Using values in the linear range of your dilution curve, calculate transducing units/mL after analysis with the flow cytometer software: [proportion of GFP-positive cells] × [75,000 cells]/[volume of pseudovirus in mL] as shown in Fig. 2 (see Note 15).

Fig. 2.

Fig. 2

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Typical dilution curve of transducing VSV/GFP and calculations to determine titers of stocks

3.7. Normalization of Pseudovirus Stocks

For many experiments, equivalent numbers of virions from different stocks need to be compared, and, to do this, you need to normalize the number of virions added. For these types of studies, we normalize by comparing the amount of VSV matrix protein in each stock.

  1. Mix an aliquot containing an equivalent volume of each pseudovirus stock with PBS and a sufficient quantity of 5× lysis buffer to achieve a 1× lysis buffer in the final concentration. For stocks concentrated by ultracentrifugation, as little as 1–6 μL of stock can be used for normalization studies. The total sample volume then should be brought to 160 μL with PBS and 40 μL of 5× lysis buffer added to achieve a final sample size of 200 μL. For unconcentrated supernatant virus stocks, 160 μL of stock can be mixed directly with 5× lysis buffer. Incubate virions in the lysis buffer for at least 2 min.

  2. Make several two-fold dilutions of your lysed virions in PBS.

  3. Apply 100 μL of each dilution of each pseudovirus directly to a nitrocellulose membrane pre-wetted with PBS through a 96-well dot blot apparatus attached to a vacuum pump. Wash each well with 1× buffer several times.

  4. Proceed with immunoblotting according to a standard protocol. Primary antibodies targeting either the filovirus GP or the VSV matrix protein are appropriate. For detection of VSV matrix protein, monoclonal antibody 23H12 (1 μg/mL) works well and is available from Kerafast.com. Antisera and monoclonal antibodies (0.1–0.5 μg/mL) for the detection of Ebola virus glycoprotein by immunoblotting are available from IBT Bioservices (see Notes 8 and 16).

  5. Quantify densities of dots and calculate the amount of particles relative to a standard stock as shown in Fig. 3. Immune detection of VSV matrix protein in our lab is performed using secondary antibodies conjugated to far red fluorophores and imaged using a LiCor Odyssey imaging system. However, other quantitative imaging approaches also work.

  6. For further experiments, the amount of a new pseudovirion preparation used should then be adjusted based on its relative reactivity in dot blot compared to the standard stock. A sample calculation is presented in Fig. 3.

Fig. 3.

Fig. 3

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Analysis and normalization of concentrated virus stocks in dot blots. Shown are eight different virus stocks (including a standard stock) that are to be normalized for the quantity of VSV capsid. A solution of 6 μL of each stock was assessed as well as two 2-fold dilutions. In the example provided, 1.82 μL of Virus 1 should be added for every 1.00 μL of Standard Stock virus in order to be considered roughly equivalent in particle numbers

Acknowledgments

This work was supported by the National Institutes of Health R21 AI123616 (W.M.).

Footnotes

1.

It is important to use premium FBS, as HEK293T cells may not transfect or transduce well under suboptimal growth conditions.

2.

Stock penicillin—streptomycin (P/S) can be purchased commercially as a 100× solution, which contains 10,000 units/mL penicillin and 10,000 μg/mL streptomycin.

3.

All reagents used in transfections, transductions, and general cell culture must be sterile.

4.

To prevent potential bacterial contamination, you may supplement PBS with 1% P/S for washing cells. For 1 L 20× PBS: 160 g NaCl, 4 g KCl, 28.8 g Na2PO4, 4.8 g KH2PO4; adjust to pH 7.4 after dilution to 1× concentration.

5.

Sterile PEI solutions may be frozen at −20 °C for long term storage. Upon thawing, 1 mg/mL concentrations of PEI tend to precipitate out of solution. To resuspend precipitant, heat PEI solution in a 37 °C water bath for 30 min to an hour and vortex well. The PEI transfection method provided here was optimized for our lab’s conditions. Protocols for PEI transfection vary widely from lab to lab, so we encourage you to troubleshoot accordingly if transfection efficiency is low (less than 60–70% of an HEK293T culture).

6.

VSV recruits its viral glycoprotein at the plasma membrane. Those viral glycoproteins that do not traffic to the plasma membrane, such as flaviviruses, pseudotype VSV poorly. For those viral glycoproteins, other systems such as transcription-and replication-competent virus-like particle (trVLP) approaches should be used to study entry.

7.

The VSVΔG-GFP stock used to generate your new stocks of VSVΔG-GFP bearing filovirus GP needs to robustly transduce HEK293T cells. Viral glycoproteins that mediate strong transduction of HEK293T cells include Lassa virus GPC, VSV G, or Venezuelan Equine Encephalitis Virus GP. If you wish to use your previously generated filovirus GP-VSVΔG-eGFP stock as your seed stock, see Note 11.

8.

5× lysis buffer contains low concentrations of a mild detergent (NP40) that does not affect the detection of the native conformation of viral glycoproteins; therefore conformationally dependent antibodies will work in the dot blot immunostaining protocol.

9.

To achieve the goal of a high titer stock with this transfection/ transduction protocol, optimal levels of transfection are needed. Ideally, close to 100% of the HEK 293 T cells should be expressing the transfected viral glycoprotein. Introduction of pseudotyped VSVΔG stock into an untransfected cell will generate defective VSVΔG that do not bear a viral glycoprotein and, consequently, produce only defective viral particles.

10.

It is not required to refresh the media before PEI transfection of HEK293T cells, but if you choose to do so, make sure the new media is warmed in 37 °C at 5% CO2 for at least 30 min before transfection.

11.

Filovirus glycoproteins on the surface of the VSV pseudovirions do not mediate robust entry into HEK293T cells. This allows only one efficient cycle of pseudovirus production. To amplify production of filovirus GP pseudotyped VSV, you may co-transfect the HEK293T cells with a plasmid expressing TIM-1cDNA (9:1 ratio of GP expressing to TIM-1 expressing plasmid). TIM-1 is a cell surface, phosphatidylserine receptor for filoviruses. Including a TIM-1 expressing plasmid in the transfection protocol enhances filovirus pseudovirion production by allowing multi-cycle amplification.

12.

After collection of supernatants, cells can be incubated with a sterile 5 mM EDTA/PBS solution for 5 min to release any cell-associated particles. You may pool this collection with the supernatant collection; however, it is only advised to do so if you plan to concentrate and purify by ultracentrifugation, as EDTA can affect downstream applications. EDTA also has the potential to lift cells from the plate, so in the event that too many cells accompanied your collection of supernatant pseudovirus spin the collection at 170 × g for 3–5 min to pellet the cells and debris before filtering.

13.

Pseudovirus can be used directly as supernatant collections. Preparations can also be concentrated by overnight centrifugation in a high speed centrifuge (at least 16 h at 5400 × g at 4 °C) and/or concentrated and purified via ultracentrifugation (80,000 × g for 2 h at 4 °C). The method will depend on your application of interest.

14.

The ultracentrifugation protocol is based on using the Beckman Coulter Optima™ L-90K (Class S) model using rotors SW60Ti or SW32Ti. If using alternative equipment, modifications should be made accordingly.

15.

While we find that titering the viral stock based on flow cytometric analysis of transduced cells provides the most accurate titer determination, an alternative titering approach is to serially dilute your stock in an end point dilution assay in a 96-well format. For these assays, stocks are usually diluted in three- or fivefold serial dilutions and as many as eight replicates are assessed at each dilution. End point dilution titers are evaluated at day 5 after infection by assessing GFP expression in each well using an inverted fluorescent microscope.

16.

Primary antibodies or antisera used for the dot blot normalization assays must not react with nonspecific background proteins. In a similar manner, use of affinity purified secondary antibodies will prevent detection of irrelevant background proteins.

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