key: cord-0787195-ebr8jy1e authors: Yang, Chee-Hing; Li, Hui-Chun; Hung, Cheng-Huei; Lo, Shih-Yen title: Studying Coronavirus–Host Protein Interactions date: 2014-12-18 journal: Coronaviruses DOI: 10.1007/978-1-4939-2438-7_17 sha: c6f25c5eafbaa2b783621ab22b6672ab9613d85f doc_id: 787195 cord_uid: ebr8jy1e To understand the molecular mechanisms of viral replication and pathogenesis, it is necessary to establish the virus–host protein interaction networks. The yeast two-hybrid system is a powerful proteomic approach to study protein–protein interactions. After the identification of specific cellular factors interacting with the target viral protein using the yeast two-hybrid screening system, co-immunoprecipitation and confocal microscopy analyses are often used to verify the virus–host protein interactions in cells. Identification of the cellular factors required for viral survival or eliminating virus infected cells could help scientists develop more effective antiviral drugs. Here we summarize a standard protocol used in our lab to study the coronavirus–host protein interactions, including yeast two-hybrid screening, co-immunoprecipitation, and immunofluorescence microscopy analyses. Virus-host interactions have long been studied since the fi rst discovery of Tobacco Mosaic Virus in 1898. However, due to the limitation of experimental tools to investigate the mass interacting networks was not always simple. The investigation became more productive upon the technical development of protein biochemistry, nucleic acid sequencing, and several high-throughput screening systems. Three common methods used for high-throughput protein interaction analysis are yeast two-hybrid (Y2H) system, affi nity purifi cation, and protein chip [ 1 ] . Y2H was fi rst described in 1989 for identifying and analyzing various protein-protein interactions in the yeast model [ 2 ] . The GAL4 based Y2H system relies on the GAL4 transcription activator, which consists of a DNA binding domain (BD) and a transcription activating domain (AD). The yeast strain used in this system contains several nutrient gene mutations and without adding these nutrients into the culturing media, these yeast cells will not grow. Successful introduction of separate bait and prey plasmids into the mutant yeast strain will provide the lacking nutrients hence the yeast cells will survive. One or more reporter genes will be cloned under the control of the UAS-GAL4 promoter. If the BD-bait fusion protein interacts with the AD-prey fusion protein, the GAL4 activator becomes functional and binds to the GAL UAS promoter region, then activates the reporter gene(s). The expression of one or more reporter genes thus indicates that there are protein-protein interactions between the target protein (bait) and the others (prey). The activation of reporter genes provides a platform to select the yeast clones that have protein-protein interactions. Most reporter genes used in this system are the complements of the mutated nutrient genes, e.g., Histidine, or an enzyme that catalyzes a color change reaction, e.g., LacZ. Figure 1 gives a schematic view of the yeast two-hybrid system. High-throughput screening of cDNA library by Y2H system provides a fast and comprehensive way to identify the possible proteins in the library that could interact with the bait protein. The yeast clones containing the interacting proteins will survive in a nutrient lacking medium due to the activation of the reporter gene. The identifi cation of virus-host interactions could facilitate the understanding of viral strategy to manipulate cellular functions for its survival or to know how the host controls and eliminates the pathogens. Several studies have used Y2H techniques to study the protein-protein interactions between viruses and host cells [ 3 -6 ] . A list of the interacting cellular proteins can be established after screening using a viral protein as the bait, providing a framework for further study on the relationship between the virus and the host. However, there are several limitations in this strategy that must be considered. First, due to the modular nature, the fusion proteins are sometimes not folded in a native form; second, the posttranslational modifi cations of some proteins in mammalian cells are not present in the yeast system, if the interactions between proteins are dependent on these modifi cations, false-negative results would be observed. In Y2H systems, both the BD-fused Fig. 1 A schematic view of yeast two-hybrid system. The bait protein fused to the DNA binding domain (BD) of GAL4 protein will bind to the GAL4 UAS. If the GAL4 activating domain (AD)-fused prey protein can interact with the bait protein, then the reporter genes (e.g., LacZ or H is3) will be activated and AD-fused proteins must be targeted to the nucleus; therefore, extracellular proteins or organelle targeted proteins may not work in this system. The protocol written in this chapter is used to study protein-protein interactions of cytosolic proteins. Protocols used to study interactions between membrane proteins were depicted previously [ 7 , 8 ] . We have used the protocol to identify many cellular factors interacting with different viral proteins [ 9 -11 ] , including SARS-CoV nucleocapsid protein [ 12 ] . To obtain a more reliable result of the protein-protein interactions, further experiments should be carried out to verify these interactions, e.g., co-immunoprecipitation and confocal microscopy analyses. After Y2H screening, co-immunoprecipitation is a biochemical method often used to verify whether the identifi ed prey protein physically interacts with the bait protein in cells. Figure 2 shows a schematic summary of the co-immunoprecipitation assay. Specifi c antibodies against X protein or tag 1 peptide will fi rst be coupled to Protein A/G beads, if the X protein is immunoprecipitated, then the Y protein which interacts with the X protein should be precipitated along with it. If the paired proteins of interest (X-Y) are indeed interacting with each other in cells, then these two proteins should also be co-localized in cells. Confocal microscopy analysis is used to verify the co-localization of the two interacting proteins. The co-immunoprecipitation and confocal microscopy analysis protocols written in this chapter have been used in our lab to study several protein-protein interactions [ 9 , 11 -16 ] . All the media are prepared with ddH 2 O. All reagents are autoclaved and stored at room temperature. 1. YEPD liquid medium for general yeast growth: 20 g/L peptone, 10 g/L yeast extract, 0.64 g/L L -tryptophan, add glucose to 2 % (50 ml of a sterile 40 % stock solution). 2. YEPD agar for general yeast growth: 20 g/L peptone, 10 g/L yeast extract, 0.64 g/L L -tryptophan, 2 % agar, add glucose to 2 % (50 ml of a sterile 40 % stock solution). Interaction of native X and Y Binding of antibody to Protein A/G beads (A) Antibody againts X protein (B) Antibody againts tag 1 protein Removal of non-specific binding proteins by washing Elution of the target protein and its associated proteins Interaction of X-tag 1 and Y-tag 2 proteins fusion proteins a b x Anti-X antibody from virus-infected cells will be immunoprecipitated by the antibody against viral protein X. If the cellular protein Y interacts with protein X, it will be co-immunoprecipitated by the anti-X antibody in the presence but not absence of protein X. ( b ) 48 h after transfection of two plasmids encoding the viral X-tag 1 and the cellular Y-tag 2 fusion proteins separately, lysates derived from transfected cells will be immunoprecipitated by the antibody against tag 1. If the cellular protein Y interacts with viral protein X, Y-tag 2 fusion protein will be co-immunoprecipitated by the anti-tag 1 antibody in the presence but not absence of the X-tag 1 fusion protein 3. YNP-selection medium (minimal medium necessary for the selection of nutritional mutants): 3 g/L yeast nitrogen base without amino acids and ammonium sulfate, 10 g/L ammonium sulfate, and 0.043 g/L inositol to a volume of 1 L. Prepare different selection media or agar plates by adding different amino acids or 3AT as listed in 3. X-GAL working solution: Dilute the X-GAL stock solution to 0.2 mg/ml with Z buffer. 4. Sterile fi lter paper. 5. Liquid nitrogen in suitable container. 6. Hybond-N nylon membrane. 9. Analyze expression of BD-bait fusion protein by standard SDS-PAGE and Western blot using an anti-Gal-BD antibody. Before the library screen, it is important to confi rm that the bait protein does not activate the reporter genes without the presence of any prey protein ( see Note 6 ). 1. Pick a colony of yeast cells with BD-bait fusion protein and transfer the cells to a selection plate with −W−H+G. −W−H+G plates do not contain tryptophan and histidine for the screening of self-activation of bait protein. 2. Incubate the plates at 30 °C for 4-5 days. 3. If BD-bait fusion proteins do not have self-activation activities, the yeast cells will not grow on −W−H+G selection plates. On the other hand, yeast cells will grow if BD-bait fusion proteins have the self-activation activities. Transfer these yeast cells grown on −W−H+G selection plates to a −W−H+G+3AT plate to confi rm the BD-bait fusion proteins do have selfactivation activities. 4. BD-bait fusion proteins with self-activation activities are not recommended for further library screening. Deletion mapping analysis should be conducted to remove the domain with the self-activation activity. 3. Incubate the cells at 30 °C, observe the growth of yeast colonies after 3 days (Fig. 3 ) . 4 . Only the positive clones will produce histidine and allow the yeast cells to grow on −W−L−H+G selection plates. 3AT acts as a competitive inhibitor of the product of His3 gene, Imidazoleglycerol-phosphate dehydratase, which is an enzyme catalyzing the production of histidine. Higher expression of histidine in yeast cells will allow the cells survive in the media that contain higher concentration of 3AT ( see Note 7 ). 1. Place a sterile nylon membrane on top of yeast colonies. 2. Remove the nylon membrane and place it on a container with the colony side up. 3. Put the nylon membrane with yeast cells into liquid nitrogen for 15 s and then allow it to thaw at room temperature 4. Repeat the freezing-thawing step three times. 5. Wet the fi lter paper with 4 ml X-gal working solution and place the nylon membrane on top of the wetted fi lter paper (prevent bubble in between). 6. Incubate the fi lter paper at 37 °C for 2.5 h, until colonies turn blue, but no more than 5 h. 1. Pick one yeast colony into 3 ml −W−L−H+G medium, incubate the cells with rotation overnight (16-18 h) at 30 °C. 2. Centrifuge at 15,000 × g for 1 min and then remove the supernatant. 3. Resuspend the pellet in 200 µl solution I. i. Plate 7 × 10 5 cells in 60 mm culture dish with 3 ml culture medium. Cell density should reach 60-80 % confl uent before viral infection (usually, it takes 16-24 h). ii. Remove culture medium and wash three times with 1× PBS. iii. Add 3 ml serum-free medium containing virus to the plates. More than 2.1 × 10 6 pfu (plaque forming unit) of virus (M.O.I. >3) should be used to infect the cells. Mix gently by rocking the plates and incubate at 37 °C incubator. iv. After 2 h, add serum to the medium to a fi nal concentration of 10 %. v. Incubate cells for 16-24 h for further analysis. (B) Transfect protein expression plasmids into the cells and incubate for 2 days. i. Plate 7 × 10 5 cells in 60 mm culture dish with 3 ml culture medium. Cell density should reach 60-80 % confl uent before DNA transfection (usually, it takes 16-24 h). ii. Dilute 4 µg plasmid with 300 µl serum-free medium in an eppendorf. iii. Add 10 µl of 1 mg/ml PEI. Vortex to mix and incubate at room temperature for 15 min. iv. Remove medium from cells and wash three times with 1× PBS. v. Add 3 ml serum-free medium and premixed DNA-PEI ( step ii ) to the cells. Mix gently by rocking and incubate at 37 °C. vi. After 2 h, add serum to the medium to a fi nal concentration of 10 %. vii. Incubate cells for 30-48 h for further analysis. 2. Cells (from A or B), after the removal of culture medium, were washed with 1× PBS three times at room temperature. Then, add 50 µl modifi ed RIPA buffer to the cells and incubate on ice for 5 min. 3. Scrape cells into buffer and centrifuge at 15,000 × g for 10 min. 4. Remove supernatant to a new tube. 5. Add 80 µl of Protein A magnetic beads into a 1.5 ml microtube, place the microtube into the magnetic rack and remove the storage buffer. 6. Add 0.5 ml Co-IP binding buffer to equilibrate the magnetic beads, resuspend and remove the buffer with the magnetic rack. 7. After the equilibration, add 0.6 ml of Co-IP binding buffer to resuspend the beads. 8. Add suitable amount of antibody diluted following the manufacturer's instructions (anti-X or anti-tag 1 in Fig. 2 ) to the magnetic beads and mix them with end-over-end rotation for 1 h at room temperature. 9. Place the microtube to the magnetic rack and remove the supernatant. 10. Wash the magnetic beads three times with 1 ml Co-IP binding buffer. After adding the buffer, fully mix the buffer with beads by inverting ten times at room temperature, using the magnetic rack to allow removal of buffer. 11. Add the protein samples from the step 4 to the beads and dilute with Co-IP binding buffer to a total volume of 0.6 ml ( see Note 9 ). 12. Incubate the protein samples with magnetic beads by endover-end rotation overnight at 4 °C. 13 . Wash the magnetic beads three times with 1 ml modifi ed RIPA buffer. After adding the buffer, fully mix the buffer with beads by inverting ten times at room temperature, then use the magnetic rack to allow removal of buffer. 14. Remove the supernatant using the magnetic rack and elute the proteins from the magnetic beads by using 30 µl Co-IP elution buffer. 15. Add 6 µl of 5× Laemmli sample buffer to the eluted sample protein. 16. Boil the sample at 95 °C for 5 min and centrifuge at 15,000 × g for 2 min. 17. Collect the samples and perform SDS-PAGE followed by Western blotting using standard procedures. Western blotting should be performed using antibodies specifi c for protein Y or tag 2 shown in Fig. 2 ( see Notes 8 and 10 ). 1. Saccharomyces cerevisiae is a single-cell eukaryote frequently used in scientifi c research. The laboratory yeast strains usually carry several nutrient gene mutations; therefore, complementation of exogenous amino acids could provide the nutrients for yeast cells to survive. Two most common media used in the Y2H system are the full medium (YEPD) which contains all the nutrients for yeast, and the selection medium which contains only several selective amino acids added to the minimal medium. 2. Yeast cells used for transformation should be in log phase. Transformation effi ciency in this phase is better than that in stationary phase. 3. Positive and negative controls should be included in all tests. 4. Addition of 10 % DMSO will increase the transformation effi ciency. 5. Whenever Western blotting analysis was conducted to verify protein expression, adding protease inhibitors in the samples could prevent the degradation of protein samples. 6. Self-activation should be tested before using the bait proteins for library screening to avoid false-positive clones. It is not necessary to test the self-activation of prey proteins. 7. In the GAL4 based two-hybrid system, LacZ and His 3 genes are usually used as reporter systems to verify the protein-protein interactions. In our lab, 3-Amino-1,2,4-triazole (3AT) is added to the selection plate as a competitive inhibitor of the His 3-gene product. Results of yeast growth in plates with different concentrations of 3AT are shown in Fig. 3 . X-Gal fi lter lift assay is also serves as a double confi rmation test to avoid false-positive clones [ 10 ] . 8. If Y protein interacts physically with X protein, Y protein will be immunoprecipitated by anti-X antibody in the presence but not absence of X protein. To avoid the cross-reaction of anti-X, a negative control should be included, i.e., Y protein in the cell lysate without X protein would not be precipitated by the anti-X antibody. 9. When co-IP is performed (Subheading 3.9 ), add 9/10 of the protein samples for the immunoprecipitation assay (the 12th step). 1/10 of the protein samples serve as an input control for Western blotting analysis. 10. It is annoying to have the heavy and light chains of immunoglobulins in the background of Western blotting analysis following immunoprecipitation, especially when the size of the target protein is close to that of the heavy chain or light chain. Crosslinking of the antibody to protein A/G beads using a crosslinker, e.g., disuccinmidyl suberate, should prevent the co-elution of heavy and light chains of immunoglobulins. Alternatively, a commercial immunoprecipitation kit, like EasyBlot (Genetex), provides a secondary antibody which specifi cally reacts with the native, non-reduced form of IgG. This will decrease the interference of heavy chain and light chain of IgG. 11. If the primary antibodies against two proteins (or peptide tags) are derived from the same animal species, it is necessary to conjugate a fl uorescent dye directly to one primary antibody. Incubate the other primary antibody without a conjugated fl uorescent dye fi rst, followed by the secondary antibody against this primary antibody. Then, add the primary antibody with a conjugated fl uorescent dye to the sample for incubation. 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