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Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

2022; Elsevier BV; Volume: 298; Issue: 11 Linguagem: Inglês

10.1016/j.jbc.2022.102500

1083-351X

Norihiro Kotani, Takanari Nakano, Ryusuke Kuwahara,

Receptor Mechanisms and Signaling

Coronavirus disease represents a real threat to the global population, and understanding the biological features of the causative virus, that is, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is imperative for mitigating this threat. Analyses of proteins such as primary receptors and coreceptors (cofactors), which are involved in the entry of SARS-CoV-2 into host cells, will provide important clues to help control the virus. Here, we identified host cell membrane protein candidates present in proximity to the attachment sites of SARS-CoV-2 spike proteins, using proximity labeling and proteomic analysis. The identified proteins represent key candidate factors that may be required for viral entry. We found SARS-CoV-2 host protein DPP4, cell adhesion protein Cadherin 17, and glycoprotein CD133 colocalized with cell membrane–bound SARS-CoV-2 spike proteins in Caco-2 cells and thus showed potential as candidate factors. Additionally, our analysis of the experimental infection of HEK293T cells with a SARS-CoV-2 pseudovirus indicated a 2-fold enhanced infectivity in the CD133-ACE2-coexpressing HEK293T cells compared to that in HEK293T cells expressing ACE-2 alone. The information and resources regarding these coreceptor labeling and analysis techniques could be utilized for the development of antiviral agents against SARS-CoV-2 and other emerging viruses. Coronavirus disease represents a real threat to the global population, and understanding the biological features of the causative virus, that is, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is imperative for mitigating this threat. Analyses of proteins such as primary receptors and coreceptors (cofactors), which are involved in the entry of SARS-CoV-2 into host cells, will provide important clues to help control the virus. Here, we identified host cell membrane protein candidates present in proximity to the attachment sites of SARS-CoV-2 spike proteins, using proximity labeling and proteomic analysis. The identified proteins represent key candidate factors that may be required for viral entry. We found SARS-CoV-2 host protein DPP4, cell adhesion protein Cadherin 17, and glycoprotein CD133 colocalized with cell membrane–bound SARS-CoV-2 spike proteins in Caco-2 cells and thus showed potential as candidate factors. Additionally, our analysis of the experimental infection of HEK293T cells with a SARS-CoV-2 pseudovirus indicated a 2-fold enhanced infectivity in the CD133-ACE2-coexpressing HEK293T cells compared to that in HEK293T cells expressing ACE-2 alone. The information and resources regarding these coreceptor labeling and analysis techniques could be utilized for the development of antiviral agents against SARS-CoV-2 and other emerging viruses. Coronavirus disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first reported in Wuhan and now represents a global threat. SARS-CoV-2 is a member of Coronaviridae, and many of these coronaviruses have long been known to cause severe respiratory failure (1Zhu N. Zhang D. Wang W. Li X. Yang B. Song J. et al.A novel coronavirus from patients with pneumonia in China, 2019.New Engl. J. Med. 2020; 382: 727-733Crossref PubMed Scopus (17405) Google Scholar). Therefore, it is inferred that cells within the respiratory organs are the growth sites for SARS-CoV-2 virions, and analyses of virus receptors within these host cells have been performed. In addition to its ability to infect respiratory organs, SARS-CoV-2 can also infect vascular endothelium and the intestinal tract (2Lamers M.M. Beumer J. van der Vaart J. Knoops K. Puschhof J. 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Other membrane proteins involved in virus entry are also of importance in understanding disease pathogenesis. The chemokine receptors CCR5 and CXCR4 have been reported as coreceptors involved in human immunodeficiency virus infection (9Bandres J.C. Wang Q.F. O'Leary J. Baleaux F. Amara A. Hoxie J.A. et al.Human immunodeficiency virus (HIV) envelope binds to CXCR4 independently of CD4, and binding can Be enhanced by interaction with soluble CD4 or by HIV envelope deglycosylation.J. Virol. 1998; 72: 2500-2504Crossref PubMed Google Scholar, 10Wu L. Gerard N.P. Wyatt R. Choe H. Parolin C. Ruffing N. et al.CD4-induced interaction of primary HIV-1 gp120 glycoproteins with the chemokine receptor CCR-5.Nature. 1996; 384: 179-183Crossref PubMed Scopus (1085) Google Scholar, 11Trkola A. Dragic T. Arthos J. Binley J.M. Olson W.C. 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Host cell membrane proteins involved in SARS-CoV-2 attachment and entry can be broadly considered as crucial key factors and therapeutic targets for SARS-CoV-2 infection. For example, TMPRSS2 protease is an important infectious factor in host cell membrane that cleaves SARS-CoV-2 spike proteins to allow virus entry into host cell (8Hoffmann M. Kleine-Weber H. Schroeder S. Krüger N. Herrler T. Erichsen S. et al.SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.Cell. 2020; 181: 271-280.e8Abstract Full Text Full Text PDF PubMed Scopus (12453) Google Scholar). In the case of SARS-CoV-2, various host cell membrane proteins other than ACE2 and TMPRSS2 have also been reported as factors that mediate viral entry (14Xia P. Dubrovska A. Tumor markers as an entry for SARS-CoV-2 infection?.FEBS J. 2020; 287: 3677-3680Crossref PubMed Scopus (20) Google Scholar, 15Qi F. Qian S. Zhang S. Zhang Z. Single cell RNA sequencing of 13 human tissues identify cell types and receptors of human coronaviruses.Biochem. Biophys. Res. Commun. 2020; 526: 135-140Crossref PubMed Scopus (577) Google Scholar); however, the detailed mechanisms underlying their functions remain unknown. It is speculated that these factors are membrane proteins located in proximity to the viral attachment point (binding site of SARS-CoV-2 spike protein) in the host cell membrane. The proximity labeling method (16Bar D.Z. Atkatsh K. Tavarez U. Erdos M.R. Gruenbaum Y. Collins F.S. Biotinylation by antibody recognition—a method for proximity labeling.Nat. Met. 2017; 15: 127-133Crossref PubMed Scopus (74) Google Scholar, 17Bar D.Z. Atkatsh K. Tavarez U. Erdos M.R. Gruenbaum Y. Collins F.S. Addendum: biotinylation by antibody recognition—a method for proximity labeling.Nat. Met. 2018; 15: 749Crossref PubMed Scopus (4) Google Scholar, 18Kim D.I. Roux K.J. Filling the void: proximity-based labeling of proteins in living cells.Trends Cell Biol. 2016; 26: 804-817Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 19Rees J.S. Li X.-W. Perrett S. Lilley K.S. Jackson A.P. Protein neighbors and proximity proteomics.Mol. Cell Proteomics. 2015; 14: 2848-2856Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) has recently been used to analyze the physiological protein interactions. We developed a simple physiological method, termed Enzyme-Mediated Activation of Radical Source (EMARS) (20Kotani N. Gu J. Isaji T. Udaka K. Taniguchi N. Honke K. Biochemical visualization of cell surface molecular clustering in living cells.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 7405-7409Crossref PubMed Scopus (119) Google Scholar), that uses horseradish peroxidase (HRP)-induced radicals derived from arylazide or tyramide compounds (21Miyagawa-Yamaguchi A. Kotani N. Honke K. Each GPI-anchored protein species forms a specific lipid raft depending on its GPI attachment signal.Glycoconjugate J. 2015; 32: 531-540Crossref PubMed Scopus (20) Google Scholar). The radicals produced through EMARS attack and form covalent bonds with the proteins in proximity to HRP [e.g., radicals from arylazide: approximately 200–300 nm (20Kotani N. Gu J. Isaji T. Udaka K. Taniguchi N. Honke K. Biochemical visualization of cell surface molecular clustering in living cells.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 7405-7409Crossref PubMed Scopus (119) Google Scholar); from tyramide: approximately 20 nm (22Sato S. Hatano K. Tsushima M. Nakamura H. 1-Methyl-4-aryl-urazole (MAUra) labels tyrosine in proximity to ruthenium photocatalysts.Chem. Commun. (Camb). 2018; 54: 5871-5874Crossref PubMed Google Scholar)]. The labeled proteins can subsequently be analyzed using an antibody array and/or a typical proteome strategy (23Jiang S. Kotani N. Ohnishi T. Miyagawa-Yamguchi A. Tsuda M. Yamashita R. et al.A proteomics approach to the cell-surface interactome using the enzyme-mediated activation of radical sources reaction.Proteomics. 2012; 12: 54-62Crossref PubMed Scopus (55) Google Scholar). The EMARS method has been applied for various studies on molecular complexes in the cell membrane (24Hashimoto N. Hamamura K. Kotani N. Furukawa K. Kaneko K. Honke K. et al.Proteomic analysis of ganglioside-associated membrane molecules: substantial basis for molecular clustering.Proteomics. 2012; 12: 3154-3163Crossref PubMed Scopus (35) Google Scholar, 25Ishiura Y. Kotani N. Yamashita R. Yamamoto H. Kozutsumi Y. Honke K. Anomalous expression of Thy1 (CD90) in B-cell lymphoma cells and proliferation inhibition by anti-Thy1 antibody treatment.Biochem. Biophys. Res. Commun. 2010; 396: 329-334Crossref PubMed Scopus (28) Google Scholar, 26Iwamaru Y. Kitani H. Okada H. Takenouchi T. Shimizu Y. Imamura M. et al.Proximity of SCG10 and prion protein in membrane rafts.J. Neurochem. 2016; 136: 1204-1218Crossref PubMed Scopus (10) Google Scholar, 27Kaneko K. Ohkawa Y. Hashimoto N. Ohmi Y. Kotani N. Honke K. et al.Neogenin, defined as a GD3-associated molecule by enzyme-mediated activation of radical sources, confers malignant properties via intracytoplasmic domain in melanoma cells.J. Biol. Chem. 2016; 291: 16630-16643Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 28Kotani N. Ishiura Y. Yamashita R. Ohnishi T. Honke K. Fibroblast growth factor receptor 3 (FGFR3) associated with the CD20 antigen regulates the rituximab-induced proliferation inhibition in B-cell lymphoma cells.J. Biol. Chem. 2012; 287: 37109-37118Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 29Ohkawa Y. Momota H. Kato A. Hashimoto N. Tsuda Y. Kotani N. et al.Ganglioside GD3 enhances invasiveness of gliomas by forming a complex with platelet-derived growth factor receptor α and yes kinase.J. Biol. Chem. 2015; 290: 16043-16058Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 30Yamashita R. Kotani N. Ishiura Y. Higashiyama S. Honke K. Spatiotemporally-regulated interaction between β1 integrin and ErbB4 that is involved in fibronectin-dependent cell migration.J. Biochem. 2011; 149: 347-355Crossref PubMed Scopus (28) Google Scholar, 31Kotani N. Yamaguchi A. Ohnishi T. Kuwahara R. Nakano T. Nakano Y. et al.Proximity proteomics identifies cancer cell membrane cis-molecular complex as a potential cancer target.Cancer Sci. 2019; 110: 2607-2619Crossref PubMed Scopus (6) Google Scholar). Proximity labeling typically analyzes intracellular molecular interactions to provide a measure of the proximity between free proteins. In contrast, EMARS is a tool for analyzing proximity between molecules on the cell surface; this method facilitates the labeling of key factor proteins in proximity to the virus-binding protein on the cell membrane, at the initial stages of infection. Therefore, we speculated that this method would provide a useful tool for identifying key candidate molecules responsible for SARS-CoV-2 infection. Herein, we identified protein molecules that exist in close proximity to virus spike proteins bound to host cells in the early stage of SARS-CoV-2 infection, using the EMARS method and proteomic analysis. The EMARS reaction was performed in A549 lung cancer cells and Caco-2 cells using the HRP-conjugated recombinant SARS-CoV-2 spike protein (S1-RBD). Caco-2 cells are a commonly used culture cell type that allow for efficient replication of SARS-CoV-2 virions. The labeled proteins identified as candidate membrane proteins were analyzed using proteomics technology. Following infection experiments using a SARS-CoV-2 pseudovirus (pSARS-CoV-2), we generated HEK293T cells coexpressing the identified candidate molecules, together with ACE2, and observed the effects of these candidate molecules on virus infection. To perform an EMARS reaction, an EMARS probe with HRP conjugated to a given molecule that recognizes a target molecule is required (20Kotani N. Gu J. Isaji T. Udaka K. Taniguchi N. Honke K. Biochemical visualization of cell surface molecular clustering in living cells.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 7405-7409Crossref PubMed Scopus (119) Google Scholar). The HRP-conjugated SARS-CoV-2 spike protein corresponds to an EMARS probe in this study (Fig. 1). As ACE2 is listed as the primary receptor for SARS-CoV-2, we examined the expression of ACE2 in Caco-2 and A549 cells. From Western blot analysis, a clear band was observed at a molecular weight of approximately 100,000 Da in Caco-2 cells; however, this signal was almost undetectable in A549 cells (Fig. 2A). The cells exhibited positive staining for ACE2 antibody, which demonstrated the presence of ACE2 in both cell lines; however, Caco-2 cells exhibited a higher expression of ACE2 than A549 cells (Fig. 2B). Additionally, Caco-2 cells exhibited strong staining of portions of the cell membrane, while A549 cells showed a homogeneous staining pattern (Fig. 2B). The spike protein used in this study was a receptor-binding domain (RBD) that is present in the S1 protein of SARS-CoV-2 and possesses a mouse immunoglobulin Fc region (mFc) at the C-terminus (total 457 a. a.). We used the mFc sequence to conjugate HRP. We initially examined whether the spike protein binds to membrane receptors on Caco-2 and A549 cells. Caco-2 cells were treated with Alexa Fluor 488 fluorescent dye-conjugated monovalent spike proteins created using a commercial labeling kit, and labeling was then observed using a fluorescent microscope. We simultaneously prepared the sample that was created through incubation with intact spike protein-mFc, followed by incubation with anti-mouse IgG-Alexa Fluor 488. Both samples exhibited binding of spike protein-mFc to the cells; however, the latter two-step staining procedure was found to be more effective (Fig. 2C). Based on these results, a method for EMARS in this study was adopted wherein the SARS-CoV-2 spike protein was directly applied to living cells and then treated with HRP-conjugated anti-mouse IgG. Similar results were obtained using A549 cells; however, binding of the SARS-CoV-2 spike protein was weaker than that observed in Caco-2 cells (Fig. 2C). The EMARS reaction was performed in these cells using the EMARS probe described above. Following the EMARS reaction, the labeled molecules were subjected to SDS-PAGE. The gels used for electrophoresis can be analyzed directly using a fluorescence image analyzer when the labeled molecules are present in large amounts; however, the bands for the EMARS sample prepared using the SARS-CoV-2 spike protein could not be clearly detected. The labeled molecules were therefore observed with Western blot analysis using the anti-fluorescein antibody. The EMARS reaction was first performed in Caco-2 cells using HRP-conjugated cholera toxin subunit B as the positive control, as this conjugate is known to bind to a lipid raft structure on the cell membrane and can subsequently label numerous cell surface proteins (Fig. 3A). We then performed the EMARS reaction, using the SARS-CoV-2 spike protein, in addition to negative control experiments. Although weak bands were observed in the negative control (treated with anti-mouse IgG-HRP antibody alone; hereinafter referred to as spike [-] sample), the combination of both SARS-CoV-2 spike protein and anti-mouse IgG-HRP antibody (hereinafter referred to as spike [+] sample) yielded significant bands (Fig. 3A). As the signal was weak, compared to that obtained using the cholera toxin probe, it was possible that certain specific cell membrane proteins were labeled in a limited manner in the samples prepared using the SARS-CoV-2 spike protein probe. For A549 cells, a number of labeled proteins were detected in the spike [+] sample; however, the band pattern was different from that obtained from Caco-2 cells (Fig. 3B). These labeled proteins were subjected to proteomic analysis. There was a possibility that identification may not be sufficiently achieved using mass spectrometry, as the number of labeled molecules was likely to be lesser than that obtained using the HRP-conjugated Cholera Toxin B Subunit B probe. Therefore, multiple independent experiments were performed (twice for Caco-2 cells and three times for A549 cells), and the samples were combined and used for analysis. Moreover, this experiment was performed in duplicate. Spike [+] samples and spike [-] samples (used as the negative control) were both prepared from each cell line. For shotgun analysis using mass spectrometry, the labeled molecules were purified via immunoprecipitation with an anti-fluorescein antibody. A total of 181 (first MS analysis) and 315 (second MS analysis) proteins were detected in the spike [+] samples from Caco-2 cells, whereas 59 and 230 proteins were detected from the spike [−] samples (Tables S1 and S2). A total of 184 and 263 proteins were detected in the spike [+] samples from A549 cells, and 186 and 150 proteins were detected from the spike [−] samples (Tables S3 and S4). The molecules detected in the spike [-] sample may be proteins that were nonspecifically adsorbed during the purification process. In particular, for unknown reasons, some spike [−] samples contained many suspected nonspecific-binding proteins. In this study, membrane proteins that were present in the spike [+] sample but not in the spike [-] sample, for each cell line, were preferentially categorized as the most likely candidates in this study. In Caco-2 cells, 65 types of cell surface membrane proteins, including the known SARS-CoV-2 host factors ACE2, DPP4, integrin, and CEACAM, were identified using combined data from duplicate experiments (Table S5). Among these candidates, we listed other less implicated proteins in SARS-CoV-2 infection, such as Cadherin 17 (Table 1). In A549 cells, 18 types of membrane proteins, including known SARS-CoV-2 host factors (Table S5), and less reported proteins, such as Contactin-1 (Table 2), were identified.Table 1Selected candidates for proximal membrane proteins around the SARS-CoV-2 spike protein in Caco-2 cell surfaceAccession no.Protein nameScoreaFrom the search engine (Proteome Discoverer 2.4 software).PeptideQ12864Cadherin 1742211O43490Prominin-1 (CD133)2747Q9HBB8Cadherin-related family member 52734Q5ZPR3CD276 antigen1504P21796Voltage-dependent anion-selective channel protein 11454Q9BYE9Cadherin-related family member 21094Q9NZU0Leucine-rich repeat transmembrane protein FLRT31055P13987CD59 glycoprotein982P50895Basal cell adhesion molecule882O00592Podocalyxin733P05026Sodium/potassium-transporting ATPase subunit beta-1722P15328Folate receptor alpha711Q9BY67Cell adhesion molecule 1702Q86SQ4Adhesion G-protein coupled receptor G6582P78310Coxsackievirus and adenovirus receptor561P04156Major prion protein551Q8WW52Protein FAM151A531Q9Y3Q0N-acetylated-alpha-linked acidic dipeptidase 2511P08174Complement decay-accelerating factor482Q53RT3Retroviral-like aspartic protease 1481Q9Y277Voltage-dependent anion-selective channel protein 3481O75915PRA1 family protein 3441P16444Dipeptidase 1411P081954F2 cell surface antigen heavy chain382P0C7N4Transmembrane protein 191B381Q16651Prostasin351Q8NFZ8Cell adhesion molecule 4321P48960Adhesion G protein-coupled receptor E5271Q9P0L0Vesicle-associated membrane protein-associated protein A261Q08174Protocadherin-1251Q14118Dystroglycan 1251P05023Sodium/potassium-transporting ATPase subunit alpha-1231Q13641Trophoblast glycoprotein221P13796Plastin-2212Q12913Receptor-type tyrosine-protein phosphatase eta211O60449Lymphocyte antigen 7501Q9H251Cadherin-2301Q9H6A9Pecanex-like protein 301a From the search engine (Proteome Discoverer 2.4 software). Open table in a new tab Table 2Selected candidates for proximal membrane proteins around the SARS-CoV-2 spike protein in A549 cell surfaceAccession no.Protein nameScoreaFrom the search engine (Proteome Discoverer 2.4 software).PeptideQ12860Contactin-12315P01833Polymeric immunoglobulin receptor2133Q86UN3Reticulon-4 receptor-like 2774Q53RT3Retroviral-like aspartic protease 1331P21796Voltage-dependent anion-selective channel protein 1261Q6YHK3CD109 antigen221O00398Putative P2Y purinoceptor 1001Q12864Cadherin 1702a From the search engine (Proteome Discoverer 2.4 software). Open table in a new tab We next examined whether the identified membrane proteins colocalized with the SARS-CoV-2 spike protein bound to Caco-2 cell membrane. Caco-2 cells were treated with SARS-CoV-2 spike proteins, followed by staining with antibodies against ACE2, CD133, Cadherin 17, DPP4, and VAPA candidates. Representative images of these proteins are shown in Figure 4. It was found that all these proteins (red signals) were at least expressed in Caco-2 cells and colocalized with the SARS-CoV-2 spike protein (green signals). Interestingly, despite the observation that these proteins were expressed abundantly, the colocalized area was limited to specific membrane sites. These colocalizations were subsequently examined via transmission electron microscopy. DPP4, CD133, CDH17, and VAPA were labeled with 10 nm gold particles (yellow arrowhead), and the spike protein was labeled with 20 nm gold particles (red arrow). CD133 (Fig. 5A), DPP4 (Fig. 5B), CDH17 (Fig. 5C), and VAPA (Fig. 5D) localized close to the binding site of the spike proteins, and this finding was consistent with the results of confocal microscopy analysis. To elucidate whether the candidate proteins affect the efficacy of SARS-CoV-2 infection, HEK293T cells expressing ACE2 and/or candidate proteins were prepared for the infection assay of SARS-CoV-2. We first prepared two types of ACE2-expressing HEK293T cells using a PCMV3 expression vector system (P-ACE2; Fig. S1A) or a lentivirus expression system (L-ACE2; Fig. S1B). Flow cytometric analysis revealed sufficient expression levels of ACE2 in both cell lines (Fig. S1C). As ACE2 is a primary receptor for SARS-CoV-2, we confirmed the binding capacity of the SARS-CoV-2 spike protein, and strong binding was observed in both cell lines (Fig. S1D). The binding amount was slightly higher in L-ACE2 (Fig. S1E), demonstrating a correlation with ACE2 expression. Furthermore, we prepared single transfectant HEK293T cells–expressing candidate proteins (CD133, CDH17, and VAPA), using a lentivirus expression system (Fig. S2). Binding of the SARS-CoV-2 spike protein was not detected in these cells (Fig. S2), indicating that these candidate proteins are not the primary receptors of SARS-CoV-2. To perform cotransfection of CD133, CDH17, and VAPA with ACE2-expressing cells, the lentivirus system used in the above-mentioned experiment was subsequently applied to P-ACE2 cells (Fig. S3A). Glypican-3 (GPC3), which was detected in both the spike [+] and spike [-] samples in MS analysis, was also cotransfected in P-ACE2 cells as the negative control (Fig. S3A). Western blot analysis was also used to confirm the expression of each protein in the transfected cells (Fig. 6A).Figure 6In vitro infection assay of SARS-CoV-2 pseudovirus. A, expression of ACE2 and candidate membrane proteins in transfectant HEK293 cells. Western blot analysis of transfectant cell lysates; each cell lysates were subjected to SDS-PAGE (on 6–10% gels) and stained with antibodies recognizing ACE2 or candidate membrane proteins. Arrows indicate bands of the target proteins. The Coomassie Brilliant Blue-staining image indicates load control. Asterisks indicate predicted nonspecific bands. B, schematic illustration of the assay procedure using HEK293T transfectant host cells. C, representative images of GFP-positive P-ACE2 cells after pSARS-CoV-2 infection. ACE2-expressing HEK293T cells were treated (pSARS-CoV-2 (+)) or not treated (pSARS-CoV-2 (−)) with pSARS-CoV-2, followed by fluorescein microscopic observation. Two independent experiments were carried out. White bar represents 100 μm. D-F, flow cytometric analysis of pSARS-CoV-2-infected cells. P-ACE2 cells (D), candidate protein-single expressing cells (E), and candidate protein-coexpressing P-ACE2 cells (F) were analyzed using BD FACS Canto II. GFP-positive cells were defined as the infected cells with a GFP fluorescence intensity of 103 or higher (P3 area). Two (E) or five (D and F) independent replications were carried out in each experiment. G, increase in pSARS-CoV-2 infection in candidate protein-coexpressing P-ACE2 cells. The number of GFP-positive cells in each cell was quantified using flow cytometry. The number of infected cells (GFP-positive) in P-ACE2–CD133, P-ACE2–CDH17, and P-ACE2–VAPA was significantly higher than that in P-ACE2 cells (p < 0.05 or p < 0.005; Dunnett's test), but not in P-ACE2-GPC3 (N.D.) as the negative control. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6In vitro infection assay of SARS-CoV-2 pseudovirus. A, expression of ACE2 and candidate membrane proteins in transfectant HEK293 cells. Western blot analysis of transfectant cell lysates; each cell lysates were subjected to SDS-PAGE (on 6–10% gels) and stained with antibodies recognizing ACE2 or candidate membrane proteins. Arrows indicate bands of the target proteins. The Coomassie Brilliant Blue-staining image indicates load control. Asterisks indicate predicted nonspecific bands. B, schematic illustration of the assay procedure using HEK293T transfectant host cells. C, representative images of GFP-positive P-ACE2 cells after pSARS-CoV-2 infection. ACE2-expressing HEK293T cells were treated (pSARS-CoV-2 (+)) or not treated (pSARS-CoV-2 (−)) with pSARS-CoV-2, followed by fluorescein microscopic observation. Two independent experiments were carried out. White bar represents 100 μm. D-F, flow cytometric analysis of pSARS-CoV-2-infected cells. P-ACE2 cells (D), candidate protein-single expressing cells (E), and candidate protein-coexpressing P-ACE2 cells (F) were analyzed using BD FACS Canto II. GFP-positive cells were defined as the infected cells with a GFP fluorescence intensity of 103 or higher (P3 area). Two (E) or five (D and F) independent replications were carried out in each experiment. G, increase in pSARS-CoV-2 infection in candidate protein-coexpressing P-ACE2 cells. The number of GFP-positive cells in each cell was quantified using flow cytometry. The number of infected cells (GFP-positive) in P-ACE2–CD133, P-ACE2–CDH17, and P-ACE2–VAPA was significantly higher than that in P-ACE2 cells (p < 0.05 or p < 0.005; Dunnett's test), but not in P-ACE2-GPC3 (N.D.) as the negative cont