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Journal of Visualized Experiments Copyright 2019 Journal of Visualized Experiments March 2019 145 e59010 Page 1 of 9 Video Article Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting Jean K Millet1 2 Tiffany Tang3 Lakshmi Nathan3 Javier A Jaimes4 Hung Lun Hsu3 5 Susan Daniel3 Gary R Whittaker1 1Department of Microbiology and Immunology College of Veterinary Medicine Cornell University 2INRA Virologie et Immunologie Mol culaires 3Robert Frederick Smith School of Chemical and Biomolecular Engineering Cornell University 4Department of Microbiology College of Agricultural and Life Sciences Cornell University 5Horae Gene Therapy Center University of Massachusetts Medical School Correspondence to Gary R Whittaker at gary whittaker cornell edu URL DOI doi 10 3791 59010 Keywords Cancer Research Issue 145 Pseudotyped particle pseudovirion coronavirus CoV spike protein severe acute respiratory syndrome coronavirus SARS CoV Middle East respiratory syndrome coronavirus MERS CoV murine leukemia virus MLV Date Published 3 1 2019 Citation Millet J K Tang T Nathan L Jaimes J A Hsu H L Daniel S Whittaker G R Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting J Vis Exp 145 e59010 doi 10 3791 59010 2019 Abstract The protocol aims to generate coronavirus CoV spike S fusion protein pseudotyped particles with a murine leukemia virus MLV core and luciferase reporter using a simple transfection procedure of the widely available HEK 293T cell line Once formed and released from producer cells these pseudovirions incorporate a luciferase reporter gene Since they only contain the heterologous coronavirus spike protein on their surface the particles behave like their native coronavirus counterparts for entry steps As such they are the excellent surrogates of native virions for studying viral entry into host cells Upon successful entry and infection into target cells the luciferase reporter gets integrated into the host cell genome and is expressed Using a simple luciferase assay transduced cells can be easily quantified An important advantage of the procedure is that it can be performed in biosafety level 2 BSL 2 facilities instead of BSL 3 facilities required for work with highly pathogenic coronaviruses such as Middle East respiratory syndrome coronavirus MERS CoV and severe acute respiratory syndrome coronavirus SARS CoV Another benefit comes from its versatility as it can be applied to envelope proteins belonging to all three classes of viral fusion proteins such as the class I influenza hemagglutinin HA and Ebola virus glycoprotein GP the class II Semliki forest virus E1 protein or the class III vesicular stomatitis virus G glycoprotein A limitation of the methodology is that it can only recapitulate virus entry steps mediated by the envelope protein being investigated For studying other viral life cycle steps other methods are required Examples of the many applications these pseudotype particles can be used in include investigation of host cell susceptibility and tropism and testing the effects of virus entry inhibitors to dissect viral entry pathways used Video Link The video component of this article can be found at Introduction Host cell entry constitutes the initial steps of the viral infectious life cycle For enveloped viruses this involves binding to a single host cell receptor or several receptors followed by fusion of viral and cellular membranes These essential functions are carried out by viral envelope glycoproteins1 2 The coronavirus envelope glycoprotein is called the spike S protein and is a member of the class I viral fusion proteins2 3 4 5 6 Studying viral envelope glycoproteins is critical for understanding many important characteristics of a given virus such as lifecycle initiation its host and cellular tropism interspecies transmission viral pathogenesis as well as host cell entry pathways Viral pseudotyped particles also named pseudovirions are powerful tools that enable us to easily study the function of viral fusion proteins Pseudotyped particles or pseudovirions are chimeric virions that consist of a surrogate viral core with a heterologous viral envelope protein at their surface The protocol s main purpose is to show how to obtain coronavirus spike pseudotyped particles that are based on a murine leukemia virus MLV core and contain a luciferase reporter gene As examples the method to produce pseudotyped particles with the spike proteins of the highly pathogenic severe acute respiratory syndrome SARS and Middle East respiratory syndrome MERS coronaviruses are presented The protocol describes the transfection procedure involved how to infect susceptible target cells and infectivity quantification by luciferase assay Since the entry steps of the pseudovirions are governed by the coronavirus S at their surface they enter cells in a similar fashion to native counterparts As such they are excellent surrogates of functional infectivity assays Pseudotyped particles are usually derived from parental model viruses such as retroviruses MLV7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 and the lentivirus human immunodeficiency virus HIV23 24 25 26 27 28 29 30 31 32 33 34 35 or rhabdoviruses vesicular stomatitis virus VSV36 39 40 41 42 43 44 45 46 47 When used in pseudotyping the parental viruses genomes are modified to remove essential genes rendering them defective for accomplishing a complete replication cycle This feature allows them to be used in intermediate biosafety level facilities BSL 2 and is an important advantage over using highly pathogenic native viruses that require higher biosafety facilities BSL 3 BSL 4 which are not as readily available when conducting virus entry studies Journal of Visualized Experiments Copyright 2019 Journal of Visualized Experiments March 2019 145 e59010 Page 2 of 9 Here the S proteins of risk group 3 pathogens SARS CoV and MERS CoV are used as examples of viral envelope proteins being incorporated into MLV pseudotyped particles generating SARS CoV S and MERS CoV S pseudovirions SARS Spp and MERS Spp respectively These pseudovirions have been successfully used in studies focusing on entry events of these viruses48 49 50 51 Another advantage is that the technique described here is not limited to pseudotyping coronavirus S proteins it is very flexible and can be used to incorporate representatives of all three classes of viral fusion proteins Examples include influenza hemagglutinin HA class I 52 Ebola virus glycoprotein GP class I E1 protein of the alphavirus Semliki Forest virus SFV class II and VSV glycoprotein G class III 53 In addition more than one kind of viral glycoprotein can be co incorporated into a pseudotyped particle as in the case of influenza HA and NA pseudotyped particles51 Based on the work performed by Bartosch et al 20 this protocol describes the generation of MLV pseudotyped particles with a three plasmid co transfection strategy using the widely available and highly transfection competent HEK 293T cell line54 The first plasmid encodes the MLV core genes gag and pol but lacks the MLV envelope env gene The second plasmid is a transfer vector that encodes a firefly luciferase reporter gene an MLV RNA packaging signal along with 5 and 3 flanking MLV long terminal repeat LTR regions The third plasmid encodes the fusion protein of interest in this case either the SARS CoV S or MERS CoV S protein Upon co transfection of the three plasmids using a transfection reagent viral RNA and proteins get expressed within transfected cells allowing generation of pseudotyped particles Since MLV is used as pseudovirion backbone this occurs at the plasma membrane the RNAs containing the luciferase gene reporter and packaging signal get encapsulated into nascent particles that also incorporate plasma membrane expressed coronavirus spike proteins The particles that bud out from cells contain the coronavirus S protein at their surface and are harvested for use in infectivity assays Because pseudotyped particles harbor the coronavirus S protein and not the MLV envelope protein when used for infecting cells they behave like their native coronavirus counterparts for entry steps The viral RNA containing the luciferase reporter and flanking LTRs is then released within the cell and the retroviral polymerase activities enable its reverse transcription into DNA and integration into the host cell genome Quantification of the infectivity of viral pseudotyped particles in infected cells is then performed with a simple luciferase activity assay Because the DNA sequence that gets integrated into the host cell genome only contains the luciferase gene and none of the MLV or coronavirus protein encoding genes they are inherently safer to use than replication competent native viruses In addition to being safer surrogates and highly adaptable to allow incorporation of various kinds of envelope glycoproteins the pseudotyped particles described here are also highly versatile and can be used to study many aspects of virus entry This includes but is not limited to testing host cell susceptibility to virus infection analyzing the cellular entry pathways an enveloped virus uses studying the effects of pharmacological inhibitors and drug screenings conducting neutralization antibody assays characterizing host cell entry of enveloped viruses that cannot be cultured and generating viral vectors for gene delivery stable cellular expression of genes of interest or gene silencing Protocol 1 Cell Seeding for Pseudotyped Particle Production NOTE Perform this step in the biosafety cabinet 1 By standard cell culture techniques obtain an 80 90 confluent 75 cm2 flask of HEK 293T 17 cells passaged in complete Dulbecco s Modified Eagle s Medium DMEM C containing 10 vol vol fetal bovine serum FBS 10 mM 4 2 hydroxyethyl 1 piperazineethanesulfonic acid HEPES 100 IU mL penicillin and 100 g mL streptomycin Prepare DMEM T medium for transfections its composition is the same as DMEM C but without antibiotics 2 Wash cells with 10 mL of pre warmed 37 C Dulbecco s Phosphate Buffered Saline DPBS twice NOTE Handle HEK293T 17 cells with care as they easily detach 3 Aspirate the supernatant and detach cells with 1 mL of 0 25 trypsin solution pre warmed at 37 C Place the flask of cells at 37 C 5 CO2 incubator for 3 5 min or until cells start detaching NOTE Avoid incubating cells with trypsin for more than 5 min as this typically leads to cell clumping 4 Deactivate trypsin by adding 4 mL of DMEM C medium and count cells using a cell counting slide and light microscope NOTE To avoid having to count too many cells an additional dilution step may be required beforehand Remember to factor in this dilution when calculating the actual cell density of trypsinized cells 5 Dilute cells to 5 x 105 cells mL with DMEM C 6 Seed 6 well tissue culture plate with 2 mL of cell solution per well and gently move the plate back and forth and side to side to evenly distribute cells avoiding circular motion NOTE This is a key step Evenly distributed cells will ensure that cells do not clump at the center of wells In turn this will ensure good transfection efficiencies and pseudotyped particle production 7 Incubate the plate overnight 16 18 h in a 37 C 5 CO2 cell culture incubator 2 Three plasmid Co transfection NOTE Perform this step in the biosafety cabinet 1 Observe cells under an inverted light microscope to check for the cell morphology and density NOTE Ideally cell density should be in the 40 60 confluency range It is critical that cells are neither too confluent 80 90 confluent nor too sparsely distributed 20 30 confluent in each well A cell density of 40 60 confluency will ensure good pseudotyped particles production 2 Plasmids mix 1 Calculate the plasmid mix for each envelope glycoprotein following the quantities for one well of a 6 well plate shown in Table 1 Multiply quantities if transfecting several wells and include an extra well to avoid running out of mix NOTE Along with the SARS CoV S and MERS CoV S encoding plasmids include empty vector control for the generation of negative control particles which lack viral envelope glycoproteins env particles along with a positive control glycoprotein such as vesicular Journal of Visualized Experiments Copyright 2019 Journal of Visualized Experiments March 2019 145 e59010 Page 3 of 9 stomatitis virus VSV G glycoprotein that is known to robustly infect a very wide range of cells VSV Gpp Plasmids are available upon request 2 Mix calculated volumes of plasmids and reduced serum cell culture medium see Table of Materials in a microcentrifuge tube 3 Lipid based transfection reagent mix see Table of Materials 1 Calculate the volumes for the transfection reagent mix from the quantities shown in Table 2 for one well 1 3 transfection ratio multiply quantities as needed Include extra wells to avoid running out of transfection reagent mix 2 Mix calculated volumes of lipid based transfection reagent 3 L per well and reduced serum cell culture medium 47 L per well in a microcentrifuge tube making sure to add the transfection reagent into the reduced serum cell culture medium and not the other way around 4 Incubate both mixes for one well 50 L of plasmids mix and 50 L of lipid based transfection reagent mix separately for 5 min at room temperature 5 Add the contents of the transfection reagent mix to the plasmids mix at a 1 1 ratio for 1 well 50 L of each mix 6 Perform thorough up down pipetting of the resulting mix 7 Incubate the mix for at least 20 min at room temperature 8 Aspirate the spent medium of cells 9 Add gently 1 mL of pre warmed 37 C reduced serum cell culture medium per well 10 Add dropwise 100 L of transfection mix to each well NOTE Exercise care when adding the transfection mix to wells of HEK 293T as they detach easily 11 Incubate cells in a 37 C 5 CO2 cell culture incubator for 4 6 h 12 Add 1 mL per well of pre warmed 37 C DMEM T medium which does not contain antibiotics NOTE This is a key step Transfection reagents increase cell permeability and increase sensitivity to antibiotics To ensure good transfection efficiency and pseudotyped particle production it is important to avoid using cell culture medium containing antibiotics 13 Incubate cells in a 37 C 5 CO2 cell culture incubator for 48 h 3 Pseudotyped Particles Collection NOTE Perform this step in the biosafety cabinet 1 Observe cells under inverted light microscope to check for cell morphology and general condition Also check the color of the medium which should be light pink slightly orange NOTE This is an important step If there is too much cell death associated with the transfection or the medium color turned orange yellow acidic pH this will typically be associated with lower yields in infectious pseudotyped particles 2 Transfer supernatants of transfected cells to 50 mL conical centrifuge tubes 3 Centrifuge tubes at 290 x g for 7 min to remove cell debris 4 Filter clarified supernatants through a sterile 0 45 m pore sized filter 5 Make small volume aliquots e g 500 L or 1 mL of pseudotyped virus solution in cryovials 6 Store at 80 C NOTE The protocol can be paused here Pseudotyped particles are stable at 80 C for many months but once thawed avoid re freezing them as they will lose infectivity 4 Pseudotyped Particle Infection of Susceptible Cells NOTE Perform this step in the biosafety cabinet 1 Cell seeding of susceptible cells in 24 well plate 1 Obtain by standard cell culture techniques 80 90 confluent 75 cm2 flask of susceptible cells Vero E6 cells for SARS CoV pseudotyped particles SARS Spp and Huh 7 cells for MERS CoV S pseudotyped particles MERS Spp NOTE To confirm whether the pseudotyped particles that have been produced are infectious it is important to carefully choose an appropriate susceptible cell line for pseudovirion infectivity assays Using poorly permissive cells will lead to low infectivity 2 Wash cells twice with 10 mL of pre warmed 37 C DPBS 3 Aspirate the supernatant and detach cells with 1 mL of 0 25 trypsin solution pre warmed at 37 C Place the flask of cells at 37 C 5 CO2 incubator for 3 5 min or until cells start detaching NOTE Avoid incubating cells with trypsin for more than 5 minutes as this typically leads to cell clumping Huh 7 cells are especially sensitive to this effect 4 Deactivate trypsin by adding DMEM C medium and count cells using a cell counting slide and light microscope 5 Dilute cells to 5 x 105 cells mL with DMEM C 6 Seed wells of a 24 well plate with 0 5 mL of cell solution per well and gently move the plate back and forth and side to side to evenly distribute cells avoiding circular motion NOTE This is a key step Evenly distributed cells will ensure that cells do not clump at the center of wells In turn this will ensure good infectivity assays For each pseudotyped particle SARS Spp MERS Spp and condition prepare three wells for three experimental replicates Include wells for the non infected N I empty vector env particles and positive control particles such as VSV Gpp 7 Incubate the plate overnight 16 18 h in a 37 C 5 CO2 cell culture incubator 2 Pseudotyped particle infection 1 Observe cells under light microscope and visually confirm that there is a confluent carpet of cells 2 Bring cryovials of pseudotyped virus to thaw on ice 3 Wash cells three times with 0 5 mL pre warmed 37 C DPBS Journal of Visualized Experiments Copyright 2019 Journal of Visualized Experiments March 2019 145 e59010 Page 4 of 9 NOTE This is a key step Cells that are not properly rinsed prior to infection typically lead to poor infectivity readouts 4 Aspirate the supernatants of cells 5 Inoculate cells with 200 L of thawed pseudotyped particle solution 6 Incubate cells in a 37 C 5 CO2 cell culture incubator for 1 2 h 7 Add 300 L of pre warmed 37 C DMEM C medium 8 Incubate cells in a 37 C 5 CO2 cell culture incubator for 72 h 5 Infectivity Quantification by Luciferase Assay Readout NOTE Perform initial steps in the biosafety cabinet 1 Thaw luciferin substrate stored at 80 C and 5x luciferase assay lysis buffer stored at 20 C until they reach room temperature 2 Dilute luciferase assay lysis buffer to 1x with sterile water 3 Aspirate supernatants of cells infected with pseudotyped particles 4 Add 100 L of 1x luciferase assay lysis buffer to each well 5 Place the plate on a rocker and incubate for 15 min with rocking at room temperature from this point onwards the plate can be handled outside of a biosafety cabinet 6 Prepare microcentrifuge tubes for each well by adding 20 L of luciferin substrate in each tube 7 Turn on the luminometer 8 Perform luciferase activity measurement one well at a time by transferring 10 L of lysate to one tube containing 20 L of luciferin substrate 9 Flick the tube gently to mix contents but avoid displacing the liquid on walls of tube 10 Place the tube in device and close lid 11 Measure the luminescence value of the tube by using the luminometer 12 Record the relative light unit s measurement 13 Repeat steps 5 8 5 12 until all wells are analyzed NOTE With the appropriate equipment such as a plate reading luminometer this process can be performed automatically The assay will need to be scaled to the plate format e g 96 well plate format 6 Data Analysis 1 Calculation and plotting of relative luciferase units averages and standard deviations 1 Use a graph plotting software to calculate luciferase assay measurement averages and standard deviations of experimental and biological replicates 2 Plot data as bar chart with standard deviations NOTE When performing statistical analyses on data make sure to include at least three biological replicates in data sets Representative Results Representative results of infectivity assays of SARS CoV S and MERS CoV S pseudotyped particles are shown in Figure 1 As expected for both Figure 1A and 1B the VSV G pseudotyped positive control particles VSV Gpp gave very hig