Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

Summary

This protocol describes an experimental process to produce high-titer infectious viral pseudotyped particles (pp) with envelope glycoproteins from two influenza A strains and how to determine their infectivity. This protocol is highly adaptable to develop pps of any other type of enveloped viruses with different envelope glycoproteins.

Abstract

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious public health issues and suggest the possibility of an epidemic. However, our molecular understanding of the virus is rudimentary, and it is necessary to study the biological properties of its envelope proteins as therapeutic targets and to develop strategies to control infection. We developed a solid viral pseudotyped particle (pp) platform to study avian influenza virus, including the functional analysis of its hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins, the reassortment characteristics of the HAs and NAs, receptors, tropisms, neutralizing antibodies, diagnosis, infectivity, for the purposes of drug development and vaccine design. Here, we describe an experimental procedure to establish pps with the envelope glycoproteins (HA, NA) from two influenza A strains (HAPI H5N1 and 2013 avian H7N9). Their generation is based on the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins into a pp. In addition, we also detail how these pps are quantified with RT-qPCR, and the infectivity detection of native and mismatched virus pps depending on the origin of the HAs and NAs. This system is highly flexible and adaptable and can be used to establish viral pps with envelope glycoproteins that can be incorporated in any other type of enveloped virus. Thus, this viral particle platform can be used to study wild viruses in many research investigations.

Introduction

The mission of a viral particle is to transport its genome from an infected host cell to a non-infected host cell and to deliver it into the cytoplasm or the nucleus in a replication-competent form¹. This process is initially triggered by binding to host cell receptors, followed by fusion of virion and cellular membranes. For enveloped viruses, like influenza viruses, the spike glycoproteins are responsible for receptor binding and fusion^1,2. Viral envelope glycoproteins (e.g., pyrogens, antigens), are involved in many important properties and events, such as virus lifecycle initiation (binding and fusion), viral pathogenesis, immunogenicity, host cell apoptosis and cellular tropism, the cellular endocytic pathway, as well as interspecies transmission and reassortment^1,3,4,5,6,7. Research on viral envelope glycoproteins will help us understand many aspects of the viral infection process. Pseudotyped viral particles (pp), also termed pseudovirions or pseudoparticles, can be generated through a pseudotyping technique^8,9,10. This technology has been used to develop pseudotyped particles of many viruses, including hepatitis C^11,12, hepatitis B¹³, vesicular stomatitis virus (VSV)^14,15, and influenza virus^16,17,18,19. This technology is based on the Gag-Pol protein of lentiviruses or other retroviruses.

Pseudotyped viral particles can be obtained using a three-plasmid system by cotransfecting a viral envelope glycoprotein expression plasmid, a retroviral packaging plasmid missing the envelope env gene, and a separate reporter plasmid into pp producer cells. The retrovirus is assembled by its Gag protein, and it buds from an infected cell membrane that expresses the virus envelope protein¹. Therefore, it is possible to obtain high titer influenza pps using the retrovirus Gag protein to produce buds on a cellular membrane expressing influenza HA and NA. In our previous studies, HAs/NAs in all combinations were functional and able to perform their corresponding functions in the viral life cycle^{16,17,18,20,21}. These pps are used to investigate influenza biological characteristics, including hemagglutination, neuraminidase activity, HA-receptor binding tropism, and infectivity. Because HA and NA are both important surface functional proteins in the viral life cycle, mismatched HAs and NAs derived from different strains of influenza can partly demonstrate reassortment between them. Here, we generate eight types of influenza pps by combining two HAs and two NAs (derived from the HPAI H5N1 strain and the H7N9 stain), using a three-plasmid pseudotyping system. These eight types of pps include two native pps, H5N1pp, H7N9pp; two mismatched pps, (H5+N9)pp, (H7+N1)pp; and four pps only harboring a single glycoprotein (HA or NA), H5pp, N1pp, H7pp, N9pp. Studies on the influenza virus, such as H5N1 and H7N9, are limited by biosafety requirements. All studies of the wild influenza virus strains should be performed in a biosafety level 3 (BSL-3) laboratory. Pseudotyped viral particle technology can be used to package an artificial virion in a biosafety level 2 (BSL-2) setting. Therefore, pps represent a safer and useful tool to study the influenza virus processes depending on its two major glycoproteins: hemagglutinin (HA) and neuraminidase (NA).

This protocol describes the generation of these pps with a three-plasmid cotransfection strategy (overviewed in Figure 1), how to quantify pps, and infectivity detection. The pp production involves three kinds of plasmids (Figure 1). The gag-pol gene, which encodes the retrovirus Gag-Pol protein, was cloned from a retrovirus packaging kit and inserted into the pcDNA 3.1 plasmid and named pcDNA-Gag-Pol. The enhanced green fluorescent protein (eGFP) gene, which encodes Green Fluorescent Protein, was cloned from pTRE-EGFP vector, inserted into the pcDNA 3.1 plasmid, and called pcDNA-GFP. During cloning, a packaging signal (ψ) sequence was added via a primer. The HA and NA genes were cloned into a pVRC plasmid, named pVRC-HA and pVRC-NA, respectively. The last plasmid encodes the fusion protein and can be replaced with any other fusion protein of interest. Our pseudotyping platform includes two glycoprotein expression plasmids: pVRC-HA and pVRC-NA. This can simplify the research on reassortment between different virus strains in a BSL-2 setting.

Protocol

1. Day 1: Cell Culture and Seeding

1. Cultivate human embryonic kidney (HEK) 293T/17 cells in 60 mm dishes with Dulbecco's modified essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin-streptomycin (DMEM Complete Medium, DCM) in a 37 °C, 5% carbon dioxide (CO₂) incubator until about 80% confluent.
   NOTE: HEK 293T/17 low passage cells are recommended.
2. Carefully wash the cells with 5 mL of phosphate buffered saline (PBS) 1x.
   NOTE: Manual handling of the HEK 293T/17 cells must be very gentle, because they easily detach.
3. Remove PBS and dissociate the cells with 1 mL of 0.25% trypsin-ethylene diamine tetraacetic acid (EDTA) solution. Place the dish in a 37 °C, 5% CO₂ incubator for no more than 5 min until the cells are dissociated.
4. Deactivate trypsin by adding 5 mL of DCM. Disperse the cells into a single-cell suspension by pipetting up and down several times.
5. Transfer the cell suspensions to a prechilled 15 mL centrifuge tube. Collect the cells by centrifugation at 250 x g for 5 min at 4 °C.
6. Decant as much of the supernatant as possible. Resuspend the cell pellet with 6 mL of DCM medium and count the cells. Dilute the cells to 1 x 10⁶ cells/mL with DCM medium.
7. Seed the cells into a 6 well plate with 1 mL of cell suspension per well. Gently pat the plate to evenly distribute the cells. Incubate the plate overnight (14–16 h) in a 37 °C, 5% CO₂ incubator.

2. Day 2: Four-plasmid Cotransfection Mediated by Lipofection

1. Check the cell morphology and density under an inverted light microscope. Ideally, cells should be approximately 85% confluent at transfection. Replace the medium with 1 mL of serum-free DMEM medium per well, and then put the plate back into the incubator.
   NOTE: Manual handling of the HEK 293T/17 cells must be very gentle, because they easily detach.
2. For each well of cultured cells to be transfected, dilute 8 µL of the transfection reagent to a volume of 150 µL with Reduced Serum Medium (tube 1). Mix gently and incubate for 5 min at room temperature (RT, approximately 20 °C).
   NOTE: For each transfection sample, prepare two 1.5 mL microcentrifuge tubes, numbered 1 and 2.
3. In tube 2, dilute 2.5 µg of plasmid DNA into 150 µL of Reduced Serum Medium.
   NOTE: In tube 2, dilute each plasmid DNA as shown in Table 1. A plasmid encoding vesicular stomatitis virus (VSV) G glycoprotein (plasmid pLP-VSVG) was used as a positive control, because VSV is able to infect a wide range of cells. Negative control particles that lack influenza envelope glycoproteins (Δenv pps) were generated using pcDNA-Gag-Pol and pcDNA-GFP plasmids.
4. After a 5 min incubation, combine the diluted DNA with diluted transfection reagent. Mix gently and incubate for another 15 min at RT.
5. Add the DNA-lipid complex to the corresponding well containing the cells and serum-free medium. Mix gently by rocking the plate back and forth.
6. After incubation for 4–6 h in a 37 °C, 5% CO₂ incubator, remove the medium, and replace with 2 mL of DMEM. Incubate for another 36–48 h in a 37 °C, 5% CO₂ incubator.
   NOTE: Replace with serum-free and antibody-free DMEM. In this protocol, two HAs and two NAs can be used to generate eight types of pps (shown in Table 1).

3. Day 3: Susceptible Cells Seeding

1. For the infectivity assay, seed each type of susceptible cells at 1 x 10⁴ cells per well in a 96 well plate.
   NOTE: Use two types of target cell to perform the infectivity assay in this article: an alveolar-derived human cell line (A549 cells) and the Madin-Darby Canine Kidney (MDCK) cells. MDCK cells are widely used in influenza research and can be a good control. This step is flexible. Any other target cell lines can be used according to the research requirements.
2. Incubate the plate overnight (14–16 h) in a 37 °C, 5% CO₂ incubator.

4. Day 4: Pseudotyped Viral Particle Collection, Quantification, and Infectivity Assay

1. Pseudotyped viral particle collection
   1. Check the color of the medium. Ideally, it should be light pink or slightly orange. Examine the cells with an inverted fluorescent biological microscope under 440–460 nm.
   2. At 36–48 h posttransfection, harvest the pps by passaging through a 0.45 µm polyvinylidene fluoride (PVDF) membrane filter to eliminate cell debris.
   3. Divide the pps into small volume aliquots.
   4. Store the pps at -80 °C.
      NOTE: The protocol can be paused here. However, this is not encouraged, because the infectivity of the pps will sharply decline after freezing and thawing.
2. Pseudotyped viral particle quantification
   1. Transfer 20 µL of purified pps to a 1.5 mL ribonuclease (RNase)-free microcentrifuge tube.
   2. Add 1 µL of 0.24 U/mL benzonase nuclease. Incubate at 37 °C for 1 h to eliminate any DNA and RNA contamination.
      NOTE: Target RNA, commonly downregulated cytomegalovirus (CMV)-GFP RNA, is packaged in the pps and can avoid being degraded by benzonase nuclease.
   3. Freeze the sample at -70 °C to inactivate the benzonase nuclease.
   4. Add 2 µL of proteinase K. Incubate at 50 °C for 30 min to digest the envelope proteins and release the CMV-GFP RNA. Inactivate the proteinase K at 100 °C for 3 min.
   5. Quantify the pps by real-time quantitative reverse-transcription (qRT)-PCR with a Universal Probe One-Step RT-qPCR Kit, using the forward primer 5'-AACAAAAGCTGGAGCTCGTTTAA-3', the reverse primer 5'-GGGTCTCCTCAGAGTGATTGACTAC-3', and the probe 5'-FAM-CCCCCAAATGAAAGACCCCCGAG-TAM-3', on a Real-Time PCR thermocycler. Normalize the pps for RNA copy number before infectivity.
3. Infectivity assay
   1. Dilute each type of pps in terms of the qRT-PCR data to 4 x 10⁵ copies/mL (pp normalization).
   2. Add Tosyl-Phenylalanine Chloromethyl-Ketone (TPCK)-trypsin to a final concentration of 40 µg/mL into pps that harbor H7N9 HA. Incubate at 37 °C for 1 h to form its functional subunits HA1 and HA2.
      NOTE: There is no need to treat the pps that harbor H5 with TPCK-trypsin, because they have multiple arginine and lysine residues at the HA1-HA2 cleavage site. This multi-basic cleavage site can be cleaved by ubiquitous cellular proteases.
   3. Mix normalized pps with DMEM medium (serum-free) at a 1:1 ratio (volume/volume).
   4. Bring the plate containing susceptible cells to the biosafety cabinet.
   5. Aspirate the supernatant and wash the cells once with 0.1 mL of prewarmed PBS.
   6. Add 0.1 mL of pps-DMEM mixture to one well. Triplicate the infectivity tests of each type of pps to one susceptible cell line (overviewed in Figure 2).
   7. Incubate the 96 well plate for 4–6 h in a 37 °C, 5% CO₂ incubator.
   8. Aspirate the supernatant and replace with 0.1 mL of DCM.
   9. Incubate the 96 well plate for another 24–36 h in a 37 °C, 5% CO₂ incubator.

5. Day 5 or 6: Infectivity Detection

1. Bring the 96 well plate to the biosafety cabinet.
2. Aspirate the supernatant and wash the cells 1x with 0.2 mL of prewarmed PBS.
3. Remove PBS and dissociate the cells with 0.1 mL of 0.25% trypsin-EDTA solution.
4. Place the dish in a 37 °C, 5% CO₂ incubator for 3 min until the cells are dissociated.
   NOTE: Avoid incubating for more than 5 min, because this will lead to cell clumping.
5. Deactivate trypsin by adding 0.4 mL of DCM.
6. Disperse the cells into single-cell suspension by pipetting up and down several times.
7. Transfer the cell suspensions to a chilled 1.5 mL microcentrifuge tube.
8. Determine the GFP reporter-positive cells with Fluorescence Activated Cell Sorting (FACS).
   NOTE: To determine the ratio of GFP reporter-positive target cells, set flow cytometer gates using the control samples (pps-untreated A549 cells or MDCK cells), and then count the GFP reporter-positive cells of 10,000 cells per sample.

Results

Depending on the general procedure described above, we have generated 10 types of pps combining two group HAs/NAs or VSV-G glycoprotein or no-envelope glycoproteins (shown in Table 1). Seven of them are infectious. The pps that harbor no-envelope glycoprotein or only harbor NA did not show any infectivity here. The influenza pp production procedure is overviewed in Figure 1. Transmission electron micrographs of pps (e.g., H5N1pp) are shown in Figure 3

Discussion

In this protocol, we describe a method to produce influenza virus pseudotyped particles (pp) in a BSL-2 setting. The reporter plasmid pcDNA-GFP is incorporated into the pps and can be used to quantify pps by FACS in an infectivity assay. We chose two types of susceptible cell lines because they are widely used in influenza research. MDCK cells would provide a good control to the variable immortalized human cells used in these studies.

This protocol is based on the retrovirus MLV, which can inc...

Materials

Name	Company	Catalog Number	Comments
Benzonase Nuclease	Millipore	70664	Effective viscosity reduction and removal of nucleic acids from protein solutions
Clear Flat Bottom Polystyrene TC-treated Microplates (96-well)	Corning	3599	Treated for optimal cell attachment Sterilized by gamma radiation and certified nonpyrogenic Individual alphanumeric codes for well identification
Clear TC-treated Multiple Well Plates (6-wells)	Costar	3516	Individual alphanumerical codes for well identification Treated for optimal cell attachment Sterilized by gamma irradiation
Dulbecco's modified essential medium (DMEM)	Gibco	11965092	A widely used basal medium for supporting the growth of many different mammalian cells
Fetal bovine serum	Excell	FND500	fetal bovine sera that can offer excellent value for basic cell culture, specialty research, and specific assays
Fluorescence Activated Cell Sorting (FACS)	Beckman coulter	cytoflex
Human alveolar adenocarcinoma A549 cells	ATCC	CRM-CCL-185
Human embryonic kidney (HEK) HEK-293T/17 cells	ATCC	CRL-11268	A versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines
Inverted fluorescent biological microscope	Olympus	BX51-32P01-FLB3
Inverted light microscope	Olympus	CKX31-12PHP
Lipofectamine 2000 Transfection Reagent	Invitrogen	11668019	Rapid, sensitive and precise probe-based qPCR detection and quantitation of target RNA targets.
Luna Universal Probe One-Step RT-qPCR Kit	NEB	E3006L	Will withstand up to 14,000 RCF RNase-/DNase-free Nonpyrogenic
Madin-Darby Canine Kidney (MDCK) cells	ATCC	CCL-34
MaxyClear Snaplock Microcentrifuge Tube (1.5 mL)	Axygen	MCT-150-C	33 mm, gamma sterilized
Millex-HV Syringe Filter Unit, 0.45 µm, PVDF	Millipore	SLHV033RS	an improved Minimal Essential Medium (MEM) that allows for a reduction of Fetal Bovine Serum supplementation by at least 50% with no change in cell growth rate or morphology. Opti-MEM I medium is also recommended for use with cationic lipid transfection reagents, such as Lipofectamine reagent.
Opti-MEM I Reduced Serum Medium	Gibco	11058021	The antibiotics penicillin and streptomycin are used to prevent bacterial contamination of cell cultures due to their effective combined action against gram-positive and gram-negative bacteria.
penicillin-streptomycin	Gibco	15140122	Maximum RCF is 12,500 xg Temperature range from -80 °C to 120 °C RNase-/DNase-free Sterile
PP Centrifuge Tubes (15 mL)	Corning	430791	a stable and highly reactive serine protease
Proteinase K	Beyotime	ST532	Treated for optimal cell attachment Sterilized by gamma radiation and certified nonpyrogenic
TC-treated Culture Dish (60mm)	Corning	430166	Trypsin from bovine pancreas TPCK Treated, essentially salt-free, lyophilized powder, ≥10,000 BAEE units/mg protein
TPCK-trypsin	Sigma	T1426	This liquid formulation of trypsin contains EDTA and phenol red. Gibco Trypsin-EDTA is made from trypsin powder, an irradiated mixture of proteases derived from porcine pancreas. Due to its digestive strength, trypsin is widely used for cell dissociation, routine cell culture passaging, and primary tissue dissociation. The trypsin concentration required for dissociation varies with cell type and experimental requirements.
Trypsin-EDTA (0.25%), phenol red	Gibco	25200056