Ebola virus entry requires the host-programmed recognition of an intracellular receptor
2012; Springer Nature; Volume: 31; Issue: 8 Linguagem: Inglês
10.1038/emboj.2012.53
ISSN1460-2075
AutoresEmily Happy Miller, Gregor Obernosterer, Matthijs Raaben, Andrew S. Herbert, Maïka S. Deffieu, Anuja Krishnan, Esther Ndungo, Rohini G. Sandesara, Jan E. Carette, Ana I. Kuehne, Gordon Ruthel, Suzanne R. Pfeffer, John M. Dye, Sean P. J. Whelan, Thijn R. Brummelkamp, Kartik Chandran,
Tópico(s)Hepatitis B Virus Studies
ResumoArticle6 March 2012free access Ebola virus entry requires the host-programmed recognition of an intracellular receptor Emily Happy Miller Emily Happy Miller Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Gregor Obernosterer Gregor Obernosterer Department of Biochemistry, Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Matthijs Raaben Matthijs Raaben Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Andrew S Herbert Andrew S Herbert Virology Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, USA Search for more papers by this author Maika S Deffieu Maika S Deffieu Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Anuja Krishnan Anuja Krishnan Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USAPresent address: Institute of Molecular Medicine, New Delhi, India Search for more papers by this author Esther Ndungo Esther Ndungo Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Rohini G Sandesara Rohini G Sandesara Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Jan E Carette Jan E Carette Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Ana I Kuehne Ana I Kuehne Virology Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, USA Search for more papers by this author Gordon Ruthel Gordon Ruthel Virology Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, USA Search for more papers by this author Suzanne R Pfeffer Suzanne R Pfeffer Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author John M Dye Corresponding Author John M Dye Virology Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, USA Search for more papers by this author Sean P Whelan Corresponding Author Sean P Whelan Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Thijn R Brummelkamp Corresponding Author Thijn R Brummelkamp Department of Biochemistry, Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Kartik Chandran Corresponding Author Kartik Chandran Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Emily Happy Miller Emily Happy Miller Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Gregor Obernosterer Gregor Obernosterer Department of Biochemistry, Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Matthijs Raaben Matthijs Raaben Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Andrew S Herbert Andrew S Herbert Virology Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, USA Search for more papers by this author Maika S Deffieu Maika S Deffieu Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Anuja Krishnan Anuja Krishnan Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USAPresent address: Institute of Molecular Medicine, New Delhi, India Search for more papers by this author Esther Ndungo Esther Ndungo Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Rohini G Sandesara Rohini G Sandesara Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Jan E Carette Jan E Carette Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Ana I Kuehne Ana I Kuehne Virology Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, USA Search for more papers by this author Gordon Ruthel Gordon Ruthel Virology Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, USA Search for more papers by this author Suzanne R Pfeffer Suzanne R Pfeffer Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author John M Dye Corresponding Author John M Dye Virology Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, USA Search for more papers by this author Sean P Whelan Corresponding Author Sean P Whelan Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Thijn R Brummelkamp Corresponding Author Thijn R Brummelkamp Department of Biochemistry, Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Kartik Chandran Corresponding Author Kartik Chandran Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Author Information Emily Happy Miller1,‡, Gregor Obernosterer2,‡, Matthijs Raaben3,‡, Andrew S Herbert4,‡, Maika S Deffieu5, Anuja Krishnan1, Esther Ndungo1, Rohini G Sandesara1, Jan E Carette6, Ana I Kuehne4, Gordon Ruthel4, Suzanne R Pfeffer5, John M Dye 4, Sean P Whelan 3, Thijn R Brummelkamp 2 and Kartik Chandran 1 1Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA 2Department of Biochemistry, Netherlands Cancer Institute, Amsterdam, The Netherlands 3Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, USA 4Virology Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, USA 5Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA 6Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA ‡These authors contributed equally to this work *Corresponding authors: Virology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, MD 21702, USA. Tel.: +1 301 619 8782; Fax: +1 301 619 2290; E-mail: [email protected] of Microbiology and Immunobiology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA. Tel.: +1 617 432 1923; Fax: +1 617 432 7664; E-mail: [email protected] Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Tel.: +31 20 512 1891; Fax: +31 20 512 9070; E-mail: [email protected] of Microbiology and Immunology, Albert Einstein College of Medicine, 403 Golding Building, 1300 Morris Park Avenue, Bronx, NY 10461, USA. Tel.: +1 718 430 8851; Fax: +1 718 430 8850; E-mail: [email protected] The EMBO Journal (2012)31:1947-1960https://doi.org/10.1038/emboj.2012.53 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Ebola and Marburg filoviruses cause deadly outbreaks of haemorrhagic fever. Despite considerable efforts, no essential cellular receptors for filovirus entry have been identified. We showed previously that Niemann-Pick C1 (NPC1), a lysosomal cholesterol transporter, is required for filovirus entry. Here, we demonstrate that NPC1 is a critical filovirus receptor. Human NPC1 fulfills a cardinal property of viral receptors: it confers susceptibility to filovirus infection when expressed in non-permissive reptilian cells. The second luminal domain of NPC1 binds directly and specifically to the viral glycoprotein, GP, and a synthetic single-pass membrane protein containing this domain has viral receptor activity. Purified NPC1 binds only to a cleaved form of GP that is generated within cells during entry, and only viruses containing cleaved GP can utilize a receptor retargeted to the cell surface. Our findings support a model in which GP cleavage by endosomal cysteine proteases unmasks the binding site for NPC1, and GP–NPC1 engagement within lysosomes promotes a late step in entry proximal to viral escape into the host cytoplasm. NPC1 is the first known viral receptor that recognizes its ligand within an intracellular compartment and not at the plasma membrane. Introduction Ebola virus (EBOV) and Marburg virus (MARV) are members of the family Filoviridae of enveloped viruses with non-segmented negative-strand RNA genomes (Kuhn et al, 2010). Filovirus entry is mediated by the membrane glycoprotein, GP, which is organized into trimeric spikes at the viral surface (Lee et al, 2008; White et al, 2008). GP consists of a receptor-binding subunit, GP1, and a membrane fusion subunit, GP2. Following attachment to host cells (Becker et al, 1995; Alvarez et al, 2002; Kondratowicz et al, 2011), viral particles are internalized and delivered to late endosomes (Nanbo et al, 2010; Saeed et al, 2010). Endosomal cysteine proteases then cleave GP1 to remove heavily glycosylated C-terminal sequences, generating an entry intermediate comprising an N-terminal GP1 fragment and GP2 (Supplementary Figure S1A; Chandran et al, 2005; Schornberg et al, 2006; Lee et al, 2008; Hood et al, 2010). An unknown trigger signal acts on this 'primed' GP, inducing GP1–GP2 dissociation and driving GP2-mediated membrane fusion (Chandran et al, 2005; Schornberg et al, 2006; Lee et al, 2008; Dube et al, 2009; Wong et al, 2010; Brecher et al, 2012). Current evidence indicates that an interaction between GP and a cellular protein receptor is required for filovirus entry (Takada et al, 1997; Wool-Lewis and Bates, 1998; Yang et al, 1998; Manicassamy et al, 2005; Kuhn et al, 2006; Brindley et al, 2007; Kaletsky et al, 2007; Dube et al, 2009; Ou et al, 2010). Furthermore, the near-universal tropism of filoviruses for mammalian cell types strongly suggests that this host molecule is widely distributed (Van den Groen et al, 1978; Takada et al, 1997; Wool-Lewis and Bates, 1998). However, none of the candidate receptors proposed to date are required in all cell types susceptible to viral infection, or allow filoviruses to overcome species barriers to infection, suggesting that a critical filovirus receptor remains to be identified (Becker et al, 1995; Chan et al, 2001; Alvarez et al, 2002; Shimojima et al, 2006; Kondratowicz et al, 2011). We and others recently identified Niemann-Pick C1 (NPC1) to be an essential host factor for filovirus entry and infection in all studied cell types (Carette et al, 2011; Côté et al, 2011). Moreover, we showed that NPC1 is required for pathogenesis in mouse models of filovirus infection (Carette et al, 2011). NPC1 is a large polytopic membrane protein that resides in the late endosomes and lysosomes of all cells and is involved in transport of lysosomal cholesterol to the endoplasmic reticulum and other cellular sites (Supplementary Figure S1B; Carstea et al, 1997; Cruz et al, 2000; Davies and Ioannou, 2000). Mutation of NPC1 in humans causes Niemann-Pick type C1 disease, a rare but fatal disorder associated with lysosomal storage of cholesterol and sphingolipids in the brain and other tissues (Patterson et al, 2001; Walkley and Suzuki, 2004). Analysis of NPC1 mutations that cause Niemann-Pick type C1 disease has revealed key roles for the three large luminal 'loop' domains, A, C, and I, and for the 'sterol-sensing domain', comprising transmembrane domains 3–7, in lysosomal cholesterol transport by NPC1 (Supplementary Figure S1B; Ioannou, 2000; Ory, 2004; Infante et al, 2008a). While a substantial body of information about the housekeeping functions of NPC1 is available, its specific role in filovirus entry remains unknown. Previous findings suggest that the cholesterol transport function of NPC1 is dispensable for its viral host factor function, and that GP can bind to cellular membranes by associating directly or indirectly with full-length NPC1 (Carette et al, 2011; Côté et al, 2011). However, they do not fully distinguish among three mechanisms of action: NPC1 might (i) play an indirect role in viral entry by regulating endosomal/lysosomal morphology or membrane composition; (ii) participate in trafficking of viral particles to the sites of membrane fusion; or (iii) act directly as a filovirus receptor. In this study, we demonstrate that filovirus entry does not require the full-length NPC1 protein. Instead, we provide multiple lines of evidence that a single luminal domain of NPC1 mediates filovirus entry by binding specifically and directly to the viral glycoprotein. Our work reveals that the NPC1-binding site within the GP1 subunit is recessed beneath the heavily glycosylated mucin and glycan cap GP1 subdomains, and explains our observation that proteolytic removal of these sequences is a prerequisite for direct GP–NPC1 interaction. We exploit our new findings to engineer a synthetic NPC1 analogue that is targeted to the cell surface and that mediates cell attachment and entry only by cleaved viruses containing exposed NPC1-binding sites. Furthermore, we provide evidence that human NPC1 can render cells from a non-permissive species susceptible to filovirus entry and infection. Cumulatively, our results indicate that NPC1 is an essential intracellular receptor for EBOV and MARV that promotes a late step in viral entry by binding to a proteolytically primed form of the viral glycoprotein within the host endosomal/lysosomal pathway. Results Human NPC1 renders non-permissive reptilian cells susceptible to filovirus entry and infection Mammalian cells are broadly susceptible to filoviruses, but reptilian and amphibian cells are reported to be refractory to infection (Van den Groen et al, 1978; Takada et al, 1997). Consistent with these previous findings, wild-type (WT) EBOV and Sudan virus (SUDV) did not infect VH-2 cells derived from the Russell's viper (Daboia russellii). The recent discovery of NPC1 as a critical host factor for filovirus entry and infection (Carette et al, 2011; Côté et al, 2011) led us to speculate that this protein could determine the species tropism of filoviruses. Accordingly, we engineered VH-2 cells to stably express human NPC1 (Figure 1A), and challenged them with WT EBOV and SUDV (Figure 1B). Remarkably, human NPC1 rendered these cells highly susceptible to filovirus infection. The block to infection in VH-2 cells and its rescue by ectopic expression of human NPC1 were recapitulated both with recombinant vesicular stomatitis viruses bearing EBOV or MARV GP (rVSV-GP-EBOV/MARV) (Figure 1C) and with VSV pseudotypes (Figure 1D), showing that VH-2 cells resist filovirus infection at the level of viral entry. Human NPC1 was dispensable for viral attachment and internalization, since fluorescently labelled rVSV-GP particles were delivered to perinuclear sites in both VH-2 cells lacking or expressing human NPC1 (Supplementary Figure S2A). Furthermore, VH-2 cells were replete with endosomal cysteine protease activities that could mediate viral entry upon provision of human NPC1 (Figure 1E), strongly suggesting that the entry block in these cells does not arise from the failure to generate a proteolytically primed GP intermediate within endosomes and/or lysosomes. Instead, ectopic expression of human NPC1 was associated with a reduction in virus-positive intracellular puncta (Supplementary Figure S2B and C), suggesting that this protein facilitates viral escape from the endosomal/lysosomal pathway of Russell's viper VH-2 cells, as observed previously in human and rodent cells (Carette et al, 2011). Finally, the NPC1-dependent entry block in VH-2 cells was not a consequence of defective lysosomal cholesterol transport, since the aberrant accumulation of cholesterol in lysosomes was only observed when these cells were exposed to U18666A, a small molecule that mimics the cellular effects of NPC1 deficiency (Supplementary Figure S2D; Liscum and Faust, 1989). These results demonstrate that the transmembrane protein NPC1 possesses a cardinal property of viral receptors: it allows filoviruses to overcome a species barrier to cell entry and infection. Figure 1.Human NPC1 confers susceptibility to filovirus entry and infection. (A) Viper VH-2 cells were engineered to express empty vector or human NPC1. Expression of human NPC1 was determined by immunoblotting (IB) with an anti-NPC1 antibody. Proteins non-specifically detected by this antibody were used as a loading control. Samples for IB of NPC1 were resolved on one gel and loading controls were resolved on another. (B, C) Infection of control and NPC1-expressing VH-2 cells by wild-type filoviruses (B), or recombinant VSVs bearing VSV or filovirus glycoproteins (C). Infected cells (green) and counter-stained nuclei (blue) were visualized by fluorescence microscopy. Scale bars, 20 μm. (D, E) Infectivity of VSV pseudotypes bearing VSV or filovirus glycoproteins in control and NPC1-expressing VH-2 cells. (E) NPC1-expressing VH-2 cells were pretreated with the cysteine protease inhibitor E-64 (300 μM) for 4 h at 37°C and then exposed to virus. (D, E) Results (n=3) are representative of two independent experiments. Error bars indicate s.d. Asterisk indicates values below the limit of detection. Figure source data can be found in Supplementary data. Download figure Download PowerPoint Filovirus entry requires the luminal domain C of NPC1, but not the full-length protein All experiments to date examining the role of NPC1 in filovirus entry have been carried out with the full-length protein. To determine if viral entry requires the entire protein or can instead be attributed to a discrete region within it, we expressed NPC1 deletion mutants individually lacking the large luminal loop domains A, C, and I in an NPC1-null cell line (Chinese hamster ovary (CHO) CT43) (Cruz et al, 2000; Figure 2A), and examined their capacity to mediate lysosomal cholesterol transport and viral infection (Figure 2B–D). CT43 cells accumulated lysosomal cholesterol (Cruz et al, 2000), and they were completely resistant to infection by WT EBOV/MARV and rVSV-GP-EBOV/MARV (Carette et al, 2011). As we showed previously, expression of Flag epitope-tagged WT NPC1 (NPC1–Flag) in these cells not only corrected their cholesterol transport defect but also rendered them highly susceptible to infection by WT filoviruses and rVSVs bearing filovirus GPs (Carette et al, 2011). All three 'loop-minus' NPC1 mutants were inactive at lysosomal cholesterol transport (Figure 2B), despite their significant localization to LAMP1-positive late endosomal/lysosomal compartments (Supplementary Figure S3), confirming that this cellular activity of NPC1 requires all three luminal domains A, C, and I. However, the mutants differed in their capacity to support filovirus GP-mediated entry (Figure 2B and C). Both NPC1-ΔA–Flag and NPC-ΔI–Flag could mediate entry, albeit at reduced levels relative to WT NPC1–Flag. In striking contrast, NPC1-ΔC–Flag was unable to rescue viral entry (Figure 2B and C) even though it resembled the other mutants in expression level and intracellular distribution (Figure 2A; Supplementary Figure S3). Similar results were obtained in infection assays with WT MARV (Figure 2D). These findings unequivocally separate NPC1's functions in lysosomal cholesterol transport and filovirus entry. More importantly, they demonstrate that a discrete region within NPC1, the luminal domain C, is essential for EBOV and MARV entry. Figure 2.NPC1 luminal loop domain C is required for filovirus entry, but full-length NPC1 is dispensable. (A) NPC1-null CHO CT43 cells were engineered to express mutant forms of human NPC1–Flag lacking domains A, C, or I. NPC1 expression was determined by IB with an anti-Flag antibody. Cyclin-dependent kinase 4 (CDK4) in each sample was detected by IB (Abcam) and provided a loading control. Samples for IB of each NPC1 mutant and its paired WT NPC1 control were resolved on the same gel. NPC1 and CDK4 were detected on separate gels. (B) Capacity of mutant NPC1 proteins to rescue viral entry and transport lysosomal cholesterol. (Left) Infection of NPC1-null CHO CT43 cells expressing mutant NPC1–Flag proteins by recombinant VSVs bearing VSV G or filovirus glycoproteins. Infected cells (green) were visualized by fluorescence microscopy. (Right) Cholesterol clearance by mutant NPC1–Flag proteins in CT43 cells was determined by filipin staining and fluorescence microscopy. Images were inverted for clarity. Scale bars, 20 μm. (C, D) Infectivity of VSV pseudotypes bearing VSV or filovirus glycoproteins (C) and wild-type MARV (D) in CT43 cells expressing mutant NPC–Flag proteins. SUDV, Sudan virus. Results in (C) (n=4–6) are from two independent experiments. Results in (D) (n=3) are from a representative experiment. Error bars indicate s.d. Asterisks indicate values below the limit of detection. Figure source data can be found in Supplementary data. Download figure Download PowerPoint NPC1 binds specifically and directly to a proteolytically cleaved form of EBOV GP A viral receptor mediates entry by binding specifically and directly to a viral surface protein. Recent work indicated that proteolytically cleaved GP could associate with endosomal membranes derived from WT but not NPC1-deficient cells, suggesting that GP and NPC1 interact (Côté et al, 2011). To examine this hypothesis, we first tested if EBOV GP could bind to NPC1 in a cell- and membrane-free system. Concentrated rVSV-GP-EBOV particles were solubilized in a non-ionic detergent-containing buffer, and GP in these extracts was captured by magnetic beads coated with the GP-specific monoclonal antibody KZ52 (Maruyama et al, 1999; Lee et al, 2008). These GP-decorated beads did not retrieve NPC1–Flag from CT43 detergent extracts in a co-immunoprecipitation (co-IP) assay (Figure 3A). We next incubated rVSV-GP-EBOV with the bacterial metalloprotease thermolysin to generate a GP intermediate (GPCL) that resembles the product of endosomal/lysosomal GP cleavage (Chandran et al, 2005; Schornberg et al, 2006). GPCL could capture NPC1–Flag at both neutral and acid pH (Figure 3A). Similar results were obtained in a reciprocal co-IP experiment: magnetic beads displaying NPC1–Flag captured GPCL but not GP (Figure 3B). Figure 3.NPC1 binds specifically and directly to a cleaved form of the Ebola virus glycoprotein. (A) Co-immunoprecipitation (co-IP) of NPC1 by EBOV GP. Magnetic beads coated with GP-specific monoclonal antibody KZ52 were incubated with detergent extracts containing no virus (None), uncleaved rVSV-GP, or cleaved rVSV-GPCL. The resulting control or glycoprotein-decorated beads were mixed with cell lysates containing human NPC1–Flag at pH 7.5 or 5.1 and 4°C. Beads were then retrieved and NPC1–Flag in the immune pellets and supernatants was detected by IB with an anti-Flag antibody. Pellets and supernatants were resolved on separate gels but exposed simultaneously to the same piece of film. (B) Reciprocal co-IP of GP by NPC1. Cell lysates lacking (Ctrl) or containing NPC1–Flag were incubated with anti-Flag antibody-coated magnetic beads. The resulting control or NPC1-decorated beads were mixed with detergent extracts of rVSV-GP or rVSV-GPCL at pH 7.5 and 4°C. Beads were then retrieved and GP or GPCL in the immune pellets and supernatants was detected by IB with an anti-GP antiserum. Pellets and supernatants were resolved on separate gels but exposed simultaneously to the same piece of film. Asterisks indicate bands detected non-specifically by the antiserum. (C) GPCL captures NPC1 but not NPC1-like1 (NPC1L1) in an ELISA. Plates coated with rVSV-GP or rVSV-GPCL were incubated with cell extracts containing NPC1–Flag or NPC1L1–Flag, and bound Flag-tagged proteins were detected with an anti-Flag antibody. Results (n=3) are representative of at least four independent experiments. (D) Cell extracts used in (C) were incubated with plates coated with an anti-Flag antibody or an isotype-matched control, and captured proteins were eluted and detected by IB with the anti-Flag antibody. Samples were resolved on the same gel. (E, F) NPC1L1 cannot support filovirus entry and infection. CT43 cells expressing NPC1L1–Flag were exposed to wild-type EBOV or MARV (E) or to recombinant VSVs (F), and infected cells were visualized and enumerated by fluorescence microscopy. Asterisks in (E) indicate values below the limit of detection. Scale bar, 20 μm. (G, H) GPCL but not GP captures affinity-purified NPC1–Flag in an ELISA. (G) NPC1–Flag was purified from CT43 CHO cell lysates by Flag affinity chromatography and visualized by SDS–PAGE and staining with Krypton infrared protein stain. Samples were resolved on the same gel. (H) ELISA plates coated with rVSV-GP or rVSV-GPCL were incubated with NPC1–Flag purified in (G), and bound Flag-tagged proteins were detected with an anti-Flag antibody. Results (n=3) are representative of at least four independent experiments. Error bars indicate s.d. Figure source data can be found in Supplementary data. Download figure Download PowerPoint To confirm these findings, we examined the capacity of rVSV-derived GP and GPCL to capture NPC1–Flag from 293T human embryonic kidney cell extracts using an enzyme-linked immunosorbent assay (ELISA). GP and GPCL were captured onto antibody KZ52-coated ELISA plates, and then incubated with CT43 extracts containing NPC1–Flag. We found that NPC1–Flag bound saturably to wells coated with GPCL but not with GP, consistent with our results from the co-IP assay (Figure 3C). To establish the specificity of GPCL–NPC1 association, we used the ELISA to determine if GPCL could capture NPC1-like1 (NPC1L1), a cholesterol transport protein that resembles NPC1 in topology and sequence (∼50% similarity) (Davies et al, 2000; Wang et al, 2009). NPC1L1–Flag did not detectably bind to wells coated with either GP or GPCL (Figure 3C), despite its greater abundance in cell extracts relative to NPC1–Flag (Figure 3D). Consistent with these binding results, NPC1L1 could not substitute for NPC1 in mediating infection by WT filoviruses (Figure 3E) and rVSVs bearing filovirus GPs (Figure 3F). Therefore, NPC1 associates specifically with GPCL and is specifically required for filovirus entry. Finally, affinity-purified NPC1–Flag bound saturably to wells coated with GPCL but not with GP in the ELISA, providing evidence that GPCL directly interacts with NPC1 (Figure 3G and H). Cumulatively, these findings demonstrate that the proteolytic priming of EBOV GP creates, or unmasks, a specific and direct binding site for NPC1. NPC1 luminal domain C is necessary and sufficient for GP–NPC1 binding and viral entry mediated by filovirus GPs To begin to map the GP binding site within NPC1, we tested the loop-minus NPC1 mutants for GPCL-binding activity in the co-IP and ELISA assays, as described above (Figure 4A and B). NPC1-ΔA–Flag and NPC1-ΔI–Flag in detergent extracts of CT43 cells were fully competent to bind to GPCL, but little or no binding was obtained with NPC1-ΔC–Flag. Therefore, the same region of NPC1, the luminal domain C, is absolutely required for both EBOV GP–NPC1 binding and NPC1-mediated filovirus entry. Figure 4.NPC1 luminal domain C is necessary and sufficient for GP–NPC1 binding. (A, B) Binding of NPC1 mutants lacking domains A, C, or I to EBOV GP. (A) Co-IP of mutant NPC1–Flag proteins from CT43 cell extracts by rVSV-GP was carried out as described in Figure 3A. Pellet samples for each NPC1 construct were resolved on the same gel. Pellets and supernatants were resolved on separate gels but exposed simultaneously to the same piece of film. (B) Capture of mutant NPC1–Flag proteins by rVSV-GP in an ELISA was carried out as described in Figure 3C. (C, D) CT43 cells were engineered to express synthetic single-pass membrane proteins comprising individual luminal domains of NPC1 fused to the first transmembrane domain of NPC1, the NPC1 cytoplasmic tail, and a Flag tag. Binding of these NPC1 mutants to EBOV GP was determined by co-IP (C) and ELISA (D) as described above. Results in (B) and (D) (n=3) are representative of at least three independent experiments. Error bars indicate s.d. Figure source data can be found in Supplementary data. Download figure Download PowerPoint We then used multiple approaches to test if domain C is not only necessary but also sufficient to mediate EBOV GPCL–NPC1 binding. First, we engineered synthetic single-pass membrane proteins comprising each luminal domain fused to the first transmembrane domain of NPC1, the NPC1 cytoplasmic tail (which contains a lysosomal targeting signal; Scott et al, 2004), and a Flag tag, and expressed them in CT43 cells. All three proteins were expressed at similar levels (Supplementary Figure S4A), and domain A-Flag and domain C-Flag localized significantly to late endosomes and/or lysosomes (Supplementary Figure S4B). However, domain I-Flag appeared to be entirely restricted to the endoplasmic reticulum, suggesting that it misfolds (Supplementary Figure S4B); accord
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