key: cord-0974671-ibdbwwi4 authors: Raj, V. Stalin; Lamers, Mart M.; Smits, Saskia L.; Demmers, Jeroen A. A.; Mou, Huihui; Bosch, Berend-Jan; Haagmans, Bart L. title: Identification of Protein Receptors for Coronaviruses by Mass Spectrometry date: 2014-12-18 journal: Coronaviruses DOI: 10.1007/978-1-4939-2438-7_15 sha: acd1a32ec59a8f74b5f478b42cef098b101eafd0 doc_id: 974671 cord_uid: ibdbwwi4 As obligate intracellular parasites, viruses need to cross the plasma membrane and deliver their genome inside the cell. This step is initiated by the recognition of receptors present on the host cell surface. Receptors can be major determinants of tropism, host range, and pathogenesis. Identifying virus receptors can give clues to these aspects and can lead to the design of intervention strategies. Interfering with receptor recognition is an attractive antiviral therapy, since it occurs before the viral genome has reached the relative safe haven within the cell. This chapter describes the use of an immunoprecipitation approach with Fc-tagged viral spike proteins followed by mass spectrometry to identify and characterize the receptor for the Middle East respiratory syndrome coronavirus. This technique can be adapted to identify other viral receptors. The fi rst step of the infection cycle of a virus is characterized by the interaction between the viral particle and the cell surface receptor. This interaction is followed by a series of events that lead to the delivery of the viral genome inside the cytoplasm. Viruses can use diverse types of molecules to bind and enter cells. The presence of a receptor is the principal determinant of cell, tissue and organ tropism, host range, and virulence. Therefore, identifying a receptor can give clues on pathogenesis, mode of transmission, zoonotic transmission potential and can lead to the design of targeted intervention strategies. For the last three decades the identifi cation of virus receptors has been a major goal in virology. A group of viruses of which many receptors are known are coronaviruses (CoVs). Coronaviruses infect a wide range of avian and mammalian hosts and they are known for their ability to cross the species barrier [ 1 ] . This is exemplifi ed by the 2003 severe acute respiratory syndrome (SARS) pandemic that was caused by the SARS-CoV [ 2 ] . In 2012, a novel zoonotic CoV was identifi ed from a patient from Saudi Arabia that presented with a severe pneumonia [ 3 ] . This virus belongs to the same genus as SARS-CoV and was named Middle East respiratory syndrome coronavirus (MERS-CoV). For CoVs, the viral Spike (S) protein primarily determines host and cell tropism. It is a type I membrane glycoprotein that is assembled in trimers in the viral envelope. The S protein can be functionally divided into two distinct subunits, S1 and S2. The S1 subunit binds to a cell surface receptor, whereas S2 facilitates fusion with cellular membranes. Although virus receptors can be identifi ed using several methods [ 4 -8 ] , we identifi ed the MERS-CoV receptor using Fc-tagged S1 proteins in an immunoprecipitation assay followed by mass spectrometry [ 9 ] . This assay is basically similar to the method described by Li et al., for the identifi cation of the SARS-CoV receptor [ 10 ] . In this assay, the S1 subunit of MERS-CoV is ligated into a fusion vector to generate an S1-Fc fusion protein, for expression in HEK-293T cells and purifi cation using protein A-sepharose beads. Incubation of the S1-Fc proteins with whole cell lysate of virus-susceptible cells allows the precipitation of the virus receptor with the tagged S1. This complex can then be pulled down from the lysate using protein A-sepharose beads. Subsequently, mass spectrometry is employed to identify candidate protein receptors (Fig. 1 ) . These candidates must be evaluated functionally, which is done using fl ow-cytometric binding assays, infection blocking experiments using antibodies against the candidate receptor, and fi nally by attempting to infect non-susceptible cells that have been transfected with the candidate receptor. This method has been successfully employed for the rapid identifi cation of the SARS-CoV and MERS-CoV receptor [ 9 , 10 ] and is suitable for identifi cation of protein receptors with reasonable affi nity. Glycan receptors cannot be identifi ed using the described method; treatment of susceptible cells with glycosidases prior to infection can give an insight into the type of viral receptor. Success of the protein receptor pulldown using the S1-Fc as bait depends on the affi nity of S1-receptor interaction. A FACS-based S1-Fc cell-binding assay provides good insight in the strength of this interaction. The FACS-based S1-Fc assay is also instrumental to identify cell lines with high levels of receptor expression that can be used as a source for receptor affi nity-isolation. Alternatively, in the absence of a suitable cell line, homogenates of tissue targeted by the virus can also be used for immunoprecipitation of the receptor. 20. SpeedVac. 21. EASY-nLC coupled to a Q Exactive mass spectrometer (both Thermo Scientifi c). 22. ReproSil C18 reversed-phase column (Dr. Maisch GmbH). 1. Cos-7 cells. 2. Cos-7 cell growth medium: DMEM, 10 % FCS, 100 U/ml penicillin, and 100 mg/ml streptomycin. 3. Trypsin-EDTA: 0.25 % w/v trypsin, 0.02 % w/v EDTA in PBS. 4. Hemocytometer or cell counting chamber. 5. Anti-DPP4 or antibody against other protein of interest. 6. Flow cytometer. 7. 4 % formaldehyde. 8. 10 % normal goat serum or serum corresponding species from which secondary antibody is raised. 9. Anti-SARS nsp4 or antibody against other viral protein. 10. Goat anti-rabbit FITC or other suitable secondary antibody. 11. Fluorescence microscope. The virus used in this method, MERS-CoV EMC, was described previously [ 12 ] and is used as an example. 1. Isolate viral RNA from 140 µl of virus stock at 10 7 TCID 50 / ml using the viral RNA isolation kit, following manufacturer's instructions. The tissue RNA isolation kit was used to isolate RNA from 2 × 10 7 Huh-7 cells, following manufacturer's instructions ( see Note 1 ). To convert RNA into cDNA we use SuperScript II reverse transcriptase but other reverse transcriptase enzymes can also be used. 6. Store at 4 °C. We strongly recommend the use of Pfu Ultra II Fusion HS DNA polymerase, although other enzymes may be used. The PCR instructions in this protocol are optimized for the use of this polymerase. 5. Analyze the PCR products by standard agarose gel electrophoresis. 6. If a single PCR product of the expected size is detected, remove polymerase, dNTPs, and primers using a standard PCR cleanup kit. 7. If multiple products are detected, separate PCR products by gel electrophoresis, remove an agarose slice containing the required product and use a gel extraction kit to isolate the DNA from the agarose slice. 8. Elute the required PCR product from the cleanup or gel purifi cation kit column in dH 2 O. 9. Analyze the PCR product using an appropriate restriction enzyme or enzymes followed by standard agarose gel electrophoresis to confi rm that the PCR product is as expected. 10. Quantify the nucleic acid concentration of the PCR product using NanoDrop 1000 or similar spectrophotometer. 11. Store the PCR product at −20 °C. 4. Electrophorese the restriction digests of the vector and S1 PCR product in a agarose gel, identify the products and cut out the gel slices containing the digested products. 5. Purify the DNA products from the agarose slices using a gel extraction kit, elute the DNA into H 2 O and quantify the nucleic acid concentrations using a NanoDrop. 6. Ligate the digested S1 PCR product into the pCAGGS-Fc vector by adding the following to a 1. 8. Next day, extract plasmid DNA from the bacteria using a maxi prep DNA kit, according to manufacturer's instructions. 9. Perform a restriction digest and analyze the products by standard agarose gel electrophoresis to confi rm the plasmid DNA is correct. 10. Determine DNA concentration using a NanoDrop or spectrophotometer and prepare a DNA stock of 1 µg/µl. 10. Prepare 50 % (w/v) protein-A sepharose beads: Add 0.25 g of protein-A sepharose CL-4B to a tube, add 10 ml PBS to form a slurry, centrifuge for 2 min at 2,000 × g , remove supernatant, and repeat two more times. Pellet the beads for 2 min at 2,000 × g and resuspend in 1.4 ml PBS per tube (50 % w/v), the fi nal volume will be ~2.8 ml. 11. Collect the expression medium from the transfected HEK-293T cells into 50 ml tubes and centrifuge at 2,850 × g for 10 min to remove cell debris. 12. Transfer medium to new 50 ml tubes and centrifuge again at 2,850 × g for 15 min. 13. Transfer cleared medium to new 50 ml tubes and keep it on ice; take a 100 µl aliquot and store at −20 °C. 14. Add 0.5 ml of washed protein-A sepharose beads (50 % w/v) and 800 µl of 1 M Tris-HCl pH 8.0 to each 40 ml supernatant to neutralize the pH and incubate overnight, rotating at 4 °C ( see Note 5 ). 15. Collect the protein-A sepharose beads by centrifugation at 2,000 × g for 15 min (see Note 6 ). 16 . Pool all the protein-A sepharose beads together in a 50 ml tube and wash three times with 10 ml PBS. 17. After the fi nal centrifugation, resuspend the protein-A sepharose beads in 1 ml of 0.5 M acetic acid pH 3 elution buffer and incubate for 1 min at room temperature. 18. Centrifuge the protein-A sepharose beads at 14,000 × g for 10 min and transfer the supernatant to a 1.5 ml tube. 19. Repeat steps 17 and 18 twice more to elute any remaining S1-Fc protein from the protein-A sepharose beads. 20. To remove any remaining protein-A sepharose beads in the supernatant repeat step 18 once and transfer supernatant to a fresh tube. To neutralize the pH of the eluted S1-Fc protein, add 200 µl of 3 M Tris-HCl pH 8.8 (fi nal pH 7.5). 22. Quantify the protein concentration using a NanoDrop at 280 nm. 23. To analyze the size of the eluted S1-Fc protein, run 2 µg of the protein in a standard 10 % SDS-PAGE gel. 24. Aliquot the S1-Fc protein and store at −80 °C. 1. Seed 5 × 10 7 Huh-7 cells in 100 mm dishes with growth medium and incubate at 37 °C for 24 h to allow the cells to become confl uent. 2. Wash the adherent cells twice with ice-cold PBS and allow the PBS to drain off. 3. Add 1 ml of DDM lysis buffer onto the cells and gently rock the dish to cover the entire cell sheet. 4. Scrape adherent cells off the dish with either a rubber policeman or a plastic cell scraper and transfer the cell suspension into a fresh centrifuge tube. Gently rock the suspension on either a rocker or an orbital shaker at 4 °C for 15 min to lyse the cells. Centrifuge the lysate at 14,000 × g in a precooled microcentrifuge for 1 min. 6. Immediately transfer the supernatant to a fresh centrifuge tube and discard the pellet. 1. Prepare a 50 % (w/v) protein-A sepharose bead slurry as in Subheading 3.6 , step 10 . 2. Add 100 µl of the protein A-sepharose bead slurry to every 1.5 ml of cell lysate and incubate at 4 °C for 10 min on a rocker or orbital shaker ( see Note 7 ). 3. Remove the beads by centrifugation at 14,000 × g at 4 °C for 1 min and carefully transfer supernatant to a fresh tube. 3 1. Add 2.5 µg of the purifi ed S1-Fc fusion protein (Subheading 3.6 , step 24 ) to 1.5 ml of the Huh-7 precleared lysate and incubate for 1 h at room temperature on a rocker or an orbital shaker. 2. Use 1.5 ml of the Huh-7 precleared lysate, without the purifi ed S1-Fc fusion protein, as a negative control and incubate as described in step 1 . 3. Capture any immunocomplexes between the S1-Fc fusion protein and the precleared Huh-7 cell lysate by adding 150 µl of the protein A-sepharose 50 % bead slurry to 1.5 ml of the lysates in Subheading 3.7 , step 2 , gently mix overnight at 4 °C on either a rocker or an orbital shaker. 4. Collect the protein A-sepharose beads by pulse centrifugation (i.e., 5 s in the microcentrifuge at 14,000 × g ). Discard the supernatant and wash the protein A-sepharose beads twice with DDM lysis buffer and once in PBS alone. Discard the supernatants. 4. Centrifuge at 14,000 × g for 1 min 5. Load 15 µl of supernatant on a 10 % pre-cast Tris-Glycine SDS-PAGE gel, electrophorese the sample for 40 min at 100 V; Alternatively, the supernatant can be transferred to a fresh 1.5 ml tube and stored frozen at −20 °C for later use, frozen supernatants should be reboiled for 5 min directly prior to loading on a gel. 6. Transfer SDS-PAGE gel to a clean cell culture dish or other plastic container and cover with Coomassie blue staining solution. Incubate at room temperature for 45 min while shaking. 7. Destain the gel with destaining solution until bands can clearly be seen and leave the gel in dH 2 O in a clean cell culture dish. 8. Cut out the lane of interest using a clean razor blade and tweezers and put the complete lane onto two dH 2 O-wetted fi lter papers (1.5 × 10 cm). 9. Clean the razor blade of the Mickle gel slicer with methanol and then dH 2 O. 3. Add 1 ml of PBS and mix it by pipetting up and down. 4. Transfer the cell suspension in to a new tube and add additional 5 ml of PBS. 5. Centrifuge at 400 × g for 5 min. 6. Resuspend the cells in 2 ml PBS and count the cells using the counting chamber. 7. Place 5 × 10 5 cells in a 96 well "v" bottom plate and add S1-Fc or 5 µg/ml antibody against protein under investigation such as goat anti-DPP4 polyclonal antibodies in this example, or without any protein as a control, in volume of 50 µl. 8. Incubate on ice for 30 min. 9. Wash the cells three times with PBS containing 0.5 % BSA. 10 . Add 50 µl of FITC-labelled goat anti-human IgG or FITClabelled rabbit anti goat serum (5 µg/ml) or any other labelled secondary antibody depending on the origin of the antibody in step 7 . 11. Incubate on ice for 30 min and wash the cells three times with PBS. 12. Resuspend the cells in 190 µl of PBS. 13. Analyze the cells for any fl uorescence by fl ow cytometry (Fig. 2 ) . Add 500 µl of 70 % ethanol and keep the plate at 4 °C until immunofl uorescent staining Wash the cells three times with PBS Add 200 µl of 10 % normal goat serum or serum corresponding to the species from which the secondary antibody in step 14 is derived Incubate the cells at 37 °C for 30 min Remove the 10 % normal goat serum and add 200 µl of any antibody to a specifi c virus protein, for example rabbit-anti-SARS-CoV nsp4 (5 µg/ml) is cross-reactive for Add 200 µl of goat anti-rabbit serum conjugated with FITC (5 µg/ml) Incubate the cells at 37 °C for 1 h Wash the cells three times with PBS and analyze using a fl uorescent microscope (Fig. 3 ) Notes 1. When using another RNA isolation kit, please refer to the recommendations of the respective manufacturer Cross-species virus transmission and the emergence of new epidemic diseases Identifi cation of a novel coronavirus in patients with severe acute respiratory syndrome Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus The major human rhinovirus receptor is ICAM-1 Identifi cation of the integrin VLA-2 as a receptor for echovirus 1 Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily Human aminopeptidase N is a receptor for human coronavirus 229E Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus Structure of coronavirus hemagglutinin-esterase offers insight into corona and infl uenza virus evolution Genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans This work was supported by a grant from the Dutch Scientifi c Research (NWO; no. 40-00812-98-13066) granted to BJB and BLH. SLS is partly employed by Viroclinics Biosciences.