Dynamic Interaction of the Measles Virus Hemagglutinin with Its Receptor Signaling Lymphocytic Activation Molecule (SLAM, CD150)

The interaction of measles virus with its receptor signaling lymphocytic activation molecule (SLAM) controls cell entry and governs tropism. We predicted potential interface areas of the measles virus attachment protein hemagglutinin to begin the investigation. We then assessed the relevance of individual amino acids located in these areas for SLAM-binding and SLAM-dependent membrane fusion, as measured by surface plasmon resonance and receptor-specific fusion assays, respectively. These studies identified one hemagglutinin protein residue, isoleucine 194, which is essential for primary binding. The crystal structure of the hemagglutinin-protein localizes Ile-194 at the interface of propeller blades 5 and 6, and our data indicate that a small aliphatic side chain of residue 194 stabilizes a protein conformation conducive to binding. In contrast, a quartet of residues previously shown to sustain SLAM-dependent fusion is not involved in binding. Instead, our data prove that after binding, this quartet of residues on propeller blade 5 conducts conformational changes that are receptor-specific. Our study sets a structure-based stage for understanding how the SLAM-elicited conformational changes travel through the H-protein ectodomain before triggering fusion protein unfolding and membrane fusion. The interaction of measles virus with its receptor signaling lymphocytic activation molecule (SLAM) controls cell entry and governs tropism. We predicted potential interface areas of the measles virus attachment protein hemagglutinin to begin the investigation. We then assessed the relevance of individual amino acids located in these areas for SLAM-binding and SLAM-dependent membrane fusion, as measured by surface plasmon resonance and receptor-specific fusion assays, respectively. These studies identified one hemagglutinin protein residue, isoleucine 194, which is essential for primary binding. The crystal structure of the hemagglutinin-protein localizes Ile-194 at the interface of propeller blades 5 and 6, and our data indicate that a small aliphatic side chain of residue 194 stabilizes a protein conformation conducive to binding. In contrast, a quartet of residues previously shown to sustain SLAM-dependent fusion is not involved in binding. Instead, our data prove that after binding, this quartet of residues on propeller blade 5 conducts conformational changes that are receptor-specific. Our study sets a structure-based stage for understanding how the SLAM-elicited conformational changes travel through the H-protein ectodomain before triggering fusion protein unfolding and membrane fusion. Measles is a leading cause of childhood morbidity and mortality in developing countries (1Strebel, P. M., Papania, M. J., and Halsey, N. A. (2004) in Vaccines (Plotkin, S. A., and Orenstein, W. A., eds), Ed. 4, pp. 389-440, Saunders-Elsevier, Philadelphia, PAGoogle Scholar). The immune suppression that accompanies infection with wild-type measles viruses (MV) 2The abbreviations used are: MV, measles virus; SLAM, signaling lymphocyte activation molecule; H, hemagglutinin; F, fusion protein; NDV, Newcastle Disease Virus; HN, hemagglutinin neuraminidase; SVP, stereophysico-chemical variability plots; SPR, surface plasmon resonance; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; MOI, multiplicity of infection; CHO, chinese hamster ovary cells; FACS, fluorescent-activated cell sorting; TCID, tissue culture infective dose. exposes individuals to secondary infections causing most of the fatalities associated with the disease (2Borrow P. Oldstone M.B. Curr. Top Microbiol. Immunol. 1995; 191: 85-100PubMed Google Scholar). Live attenuated MV vaccines have effectively reduced the morbidity and mortality of measles world-wide (3Duclos P. Ward B.J. Drug Saf. 1998; 19: 435-454Crossref PubMed Scopus (64) Google Scholar). The difference in pathology between the wild-type and vaccine strains is in part explained by the receptors used for cell entry. Wild-type MV uses the signaling lymphocyte activation molecule (SLAM, CD150) (4Tatsuo H. Ono N. Tanaka K. Yanagi Y. Nature. 2000; 406: 893-897Crossref PubMed Scopus (863) Google Scholar, 5Ono N. Tatsuo H. Hidaka Y. Aoki T. Minagawa H. Yanagi Y. J. Virol. 2001; 75: 4399-4401Crossref PubMed Scopus (328) Google Scholar, 6Hsu E.C. Iorio C. Sarangi F. Khine A.A. Richardson C.D. Virology. 2001; 279: 9-21Crossref PubMed Scopus (160) Google Scholar, 7Erlenhoefer C. Wurzer W.J. Loffler S. Schneider-Schaulies S. ter Meulen V. Schneider-Schaulies J. J. Virol. 2001; 75: 4499-4505Crossref PubMed Scopus (168) Google Scholar), while the vaccine strain has gained the ability to also use the ubiquitous protein CD46 (8Naniche D. Varior-Krishnan G. Cervoni F. Wild T.F. Rossi B. Rabourdin-Combe C. Gerlier D. J. Virol. 1993; 67: 6025-6032Crossref PubMed Google Scholar, 9Dorig R.E. Marcil A. Chopra A. Richardson C.D. Cell. 1993; 75: 295-305Abstract Full Text PDF PubMed Scopus (878) Google Scholar, 10Schneider U. von Messling V. Devaux P. Cattaneo R. J. Virol. 2002; 76: 7460-7467Crossref PubMed Scopus (75) Google Scholar). SLAM-targeting accounts for lymphotropism and is central to understanding MV tropism and disease progression. Receptor attachment of MV and the other morbilliviruses is mediated by hemagglutinin (H) homodimers. Unlike other members of the Paramyxoviridae, the morbillivirus H-proteins do not have neuraminidase activity. Upon receptor attachment, H induces conformational changes in the trimeric fusion (F) protein, resulting in the fusion of viral and cellular membranes (11Cattaneo R. Rose J.K. J. Virol. 1993; 67: 1493-1502Crossref PubMed Google Scholar, 12Yin H.S. Wen X. Paterson R.G. Lamb R.A. Jardetzky T.S. Nature. 2006; 439: 38-44Crossref PubMed Scopus (343) Google Scholar). MV H is a 617-amino acid type II transmembrane glycoprotein, which is comprised of an N-terminal cytoplasmic tail, a membrane-spanning domain, and an extracellular stalk region connected to a C-terminal globular head (13Plemper R.K. Hammond A.L. Cattaneo R. J. Virol. 2000; 74: 6485-6493Crossref PubMed Scopus (58) Google Scholar). Previously, we generated a three-dimensional model of the extracellular domain of the MV H-protein based on the crystal structure of Newcastle Disease Virus (NDV) HN (hemagglutinin neuraminidase) protein (sequence identity of 12%), a related paramyxovirus (14Crennell S. Takimoto T. Portner A. Taylor G. Nat. Struct. Biol. 2000; 7: 1068-1074Crossref PubMed Scopus (350) Google Scholar, 15Vongpunsawad S. Oezgun N. Braun W. Cattaneo R. J. Virol. 2004; 78: 302-313Crossref PubMed Scopus (169) Google Scholar). This model predicts that the MV H ectodomain has a 6-blade propeller structure. We previously localized several residues specific for SLAM- or CD46-dependent fusion onto the H-protein model. Seven amino acids important for SLAM- and nine for CD46-dependent fusion function were located in contiguous areas in β-sheet 5 and 4, respectively. Especially interesting were four residues essential for SLAM-dependent fusion-support, amino acids Tyr-529, Asp-530, Arg-533, and Tyr-553, to be named the β-sheet 5 quartet (bs-5 quartet). To further characterize the mechanism of interaction of the MV H-protein with its receptor, SLAM, we extended H mutagenesis based on a suite of programs that allow the prediction of potential interacting surfaces. The prediction was based on the detailed analysis of sequence conservation in homologues of MV H by stereophysicochemical variability plots (SVP) and a recently developed computational method named InterProSurf (16Negi S.S. Braun W. J. Mol. Model. 2007; 13: 1157-1167Crossref PubMed Scopus (39) Google Scholar). A final assessment of potential receptor binding areas relied upon the combined predictions of InterProSurf and SVP. Screening of the library of H-protein mutants was based on a receptor-specific cell-to-cell fusion assay, and identified Ile-194 as a residue critical for SLAM-dependent fusion-support. A surface plasmon resonance (SPR) assay discriminated between residues involved in primary receptor binding, and those sustaining H-protein conformational changes subsequent to binding. The binding kinetics revealed that the Ile-194 mutations ablated SLAM binding, whereas the bs-5 quartet residues are involved in a post-binding event. Cells and Viruses—B95a, an Epstein-Barr virus-transformed marmoset B lymphoblastoid cell line (kindly provided by D. Gerlier), and Vero (African green monkey kidney) cells were maintained in Dulbecco modified Eagle's medium (DMEM) supplemented with 10 and 5% fetal bovine serum (FBS), respectively. CHO-SLAM (Chinese hamster ovary cells expressing human SLAM) cells were maintained in RPMI 1640 medium supplemented with 10% FBS and 0.5 mg/ml of G418. The DF-1 chicken cell line was maintained in DMEM supplemented with 10% FBS at 39 °C. The rescue helper cell line 293-3-46 (17Radecke F. Spielhofer P. Schneider H. Kaelin K. Huber M. Dotsch C. Christiansen G. Billeter M.A. EMBO J. 1995; 14: 5773-5784Crossref PubMed Scopus (572) Google Scholar) was grown in DMEM with 10% FBS and 1.2 mg/ml of G418. Prediction of Interaction Surface Regions with InterProSurf—Given a three-dimensional structure (either an experimental structure or a theoretical model) InterProSurf (18Negi S.S. Schein C.H. Oezguen N. Power T.D. Braun W. Bioinformatics. 2007; 23: 3397-3399Crossref PubMed Scopus (136) Google Scholar) calculates for each surface residue propensities for having this residue in a protein-protein interface region. In a second step, it clusters high residues and these clusters to the model of the MV which was used to of is and H-protein mutants were generated in the T. Spielhofer P. Cattaneo R. Virology. 1995; PubMed Scopus Google Scholar) by the mutagenesis The of the was by the H-protein in the of the the fusion assay, Vero cells or CHO-SLAM cells were on tissue culture and to to of the protein T. Spielhofer P. Cattaneo R. Virology. 1995; PubMed Scopus Google Scholar) and were the cells to the In of was with of medium and at for 5 of and were also in of and the and were and for to to Cells were and the fusion The of fusion was the on of in most of and the of the cells were in are based on the of of MV mutations in the H-protein sequence were generated by the the H the This MV has an green protein sequence in an located of the S. Billeter M.A. J. Virol. PubMed Google Scholar). the MV generated were of and were as previously (17Radecke F. Spielhofer P. Schneider H. Kaelin K. Huber M. Dotsch C. Christiansen G. Billeter M.A. EMBO J. 1995; 14: 5773-5784Crossref PubMed Scopus (572) Google Scholar, M. S. Cattaneo R. EMBO J. 2002; PubMed Scopus Google Scholar). Virus were by Vero cells at an of and then at a of the cells were in this cells were in and were by tissue culture infective on Vero To the of cells were with and at an of in for at °C. The of the infection was by at ectodomain of the H-protein residues was with the sequence and a and this was in a the The and its mutations in the sequence were cells to the were cells expressing the H-protein of were grown in medium for The cell was and through a and with were by and to have an with not The of the were with the of We were to and of the wild-type strain the extracellular domain of CD46 of the amino acids of CD46 protein was to amino acids of the region of which is part of the of M. J. Virol. PubMed Google Scholar). was DF-1 cells by the method CD46 was were through a membrane and to for with protein was with and with The and of was by residues of the SLAM protein were to human to allow for by protein A cell culture R. P. Mol. Cell. Biol. PubMed Scopus Google Scholar) cells were used for was used to the SLAM ectodomain and then to by a Receptor of MV interaction of the MV H-protein with and was by a and was used for protein and as the To the MV H-protein to the the was onto the surface the was in 10 and resonance were on the the MV H-protein was the at 10 for 5 to the H-protein to the To receptor binding, of and were the MV H-proteins at The receptors were then the or The as for receptor binding. The surface was by of for by 10 for with in the of the surface The function of the was used to the binding model to the experimental The was the or analysis of Virus virus Vero cells tissue culture were at an of and at of the cells were in this the cells were in and to a The cell were by at for 10 The cell was a and at for at °C. The virus was at the between the MV and the the were used for the binding cells were tissue culture by cells were with MV at for in DMEM supplemented with 5% FBS and Cells were with supplemented with 5% FBS, and Virus to cells was by cells with an by a secondary for Cells were in to analysis on a Prediction of H-protein and residues involved in we the MV H three-dimensional model to InterProSurf analysis S. Oezgun N. Braun W. Cattaneo R. J. Virol. 2004; 78: 302-313Crossref PubMed Scopus (169) Google Scholar, S.S. Schein C.H. Oezguen N. Power T.D. Braun W. Bioinformatics. 2007; 23: 3397-3399Crossref PubMed Scopus (136) Google Scholar). which clusters of residues for interaction with protein, predicted interacting of the protein of virus and the protein of human B virus (16Negi S.S. Braun W. J. Mol. Model. 2007; 13: 1157-1167Crossref PubMed Scopus (39) Google Scholar, S.S. A.A. Schein C.H. R.A. Braun W. J. Mol. Model. 2006; PubMed Scopus Google Scholar). The the surface of in clusters and calculates a for each as part of an interacting has that the high clusters a for the prediction (16Negi S.S. Braun W. J. Mol. Model. 2007; 13: 1157-1167Crossref PubMed Scopus (39) Google Scholar). on the of important residues on the surface of the H-protein was provided by the variability of of amino acids in a of protein onto a three-dimensional structure (16Negi S.S. Braun W. J. Mol. Model. 2007; 13: 1157-1167Crossref PubMed Scopus (39) Google Scholar, C.H. B. Braun W. J. PubMed Scopus Google Scholar). We that the H-proteins of morbilliviruses receptors by a and that residues that are part of the receptor binding are in residues of these binding and were used to this onto the three-dimensional model of H. residues were predicted by InterProSurf to be and to be part of an interacting These residues are in in Tyr-553, and were previously shown to be important for SLAM-dependent fusion S. Oezgun N. Braun W. Cattaneo R. J. Virol. 2004; 78: 302-313Crossref PubMed Scopus (169) Google Scholar). and are important for CD46-dependent fusion R. U. B. ter Meulen V. Schneider-Schaulies J. Virology. PubMed Scopus Google Scholar, E.C. Sarangi F. Iorio C. W. W.J. Richardson C.D. J. Virol. 1998; PubMed Google Scholar, V. J. M. J. R. Wild T.F. R. J. Virol. PubMed Google Scholar). other residues and not to be important for not The residues were To protein and small amino acids were to and and to for The mutations are shown in the and identity of the is the MV H-protein A of mutants were in of contiguous amino or residues contiguous were mutations were The were for the of and at were for each 194 for SLAM-dependent H-proteins were then for in receptor-specific cell fusion based on of the The cell fusion were in cells expressing SLAM not CD46 or in Vero a cell line which CD46 not A assessment of the of the H-proteins is shown in in SLAM-dependent fusion function while CD46-dependent fusion mutants and in second and CD46-dependent fusion while SLAM-dependent fusion-support. In one a CD46-dependent The and fusion in CHO-SLAM and Vero cells and were further of the H-protein mutants mutants identified receptor-specific residues and The Ile-194 in a further characterize the of residue 194 in SLAM-dependent fusion-support, mutations were these and the of the SLAM- and CD46-dependent fusion as or amino SLAM-dependent fusion changes also SLAM-dependent fusion isoleucine in the of a side is as as the the aliphatic amino acid also a in fusion the other isoleucine of the of the was the amino acid that SLAM-dependent fusion it a in of the on the also SLAM-dependent fusion. of the on CD46-dependent fusion as Vero cells the data indicate that Ile-194 is important for SLAM-dependent fusion and that mutants to SLAM-dependent of H-proteins with mutations at as as as in a Ile-194 for SLAM-dependent of Virus characterize the of Ile-194 in SLAM-dependent MV viruses with mutations of this residue were The and mutations were to an MV that the green protein, an S. Billeter M.A. J. Virol. PubMed Google Scholar). were to those of the virus in the Vero was then in cell that SLAM, not and a Vero cells expressing CD46 were the infection of the and the four viruses in CHO-SLAM and Vero cells after infection at an of MV the H-protein to on cell at the 194 an amino acid that SLAM-dependent fusion, also infection of the cell while having on the of the virus on Ile-194 mutations to and with SLAM-dependent fusion, also with of the viruses in the mutations at Ile-194 have on SLAM-dependent fusion and SLAM-dependent entry of the of in to assay we used residues that have on or SLAM-dependent fusion, between residues involved in primary receptor binding, and those sustaining H-protein conformational changes subsequent to binding. To which of these mutations has a on binding, we measured the binding kinetics of of H-protein mutants to the MV receptors by in a of the H-protein ectodomain were and that of the vaccine strain four at the 194 and one with mutations of the four bs-5 quartet amino acids The of the MV wild-type strain an shown to be important for CD46 interaction was also and To kinetics of receptor binding to of the MV SLAM or were and The of CD46 comprised the amino acids protein and SLAM comprised the extracellular of receptor and were receptor to as in The of the binding kinetics of the vaccine strain with and The binding kinetics of the with the four bs-5 residues and that of the with the with the and receptors are shown in the second and of respectively. and a specific interaction with that with receptor In contrast, interaction with at the receptor and interaction with the receptor, H-proteins with Ile-194 for of the receptor binding the in are in with those for the binding of four and A of the between the and that and have for SLAM and In contrast, the for the interaction between and is binding. This the of of the virus the in cell and for receptor binding to between H and the receptors and were measured with a and the binding between H and the receptors and were measured with a and the binding for the interaction of the the experimental 10 are for the interaction of the the experimental 10 are not and Interaction between H and the receptors and were measured with a and the binding The for the interaction The The The of the the experimental 10 are not and in a and have that of that Ile-194 is critical for H-protein binding to The that the for the CD46 interaction for these that this is and that the not the structure of the These data indicate that the receptor binding whereas the bs-5 quartet mutations a post-binding event. The between H and SLAM in our study is to that previously C. T. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, T. M. N. M. K. K. D. Yanagi Y. K. U. S. A. 2007; PubMed Scopus Google Scholar). The we between H and SLAM as with these studies be explained by the that we used a which comprised the H ectodomain as to a Wild-type H-protein to also the binding kinetics of the wild-type strain of The to be important for CD46-dependent fusion, was this and have for of In contrast, the wild-type strain H-protein has a for with that of of A analysis of the binding kinetics of not an interaction between and the wild-type H-protein T. M. N. M. K. K. D. Yanagi Y. K. U. S. A. 2007; PubMed Scopus Google Scholar). the the between the wild-type H-protein and the CD46 the is that for that other mutations are to the ability of the MV vaccine strain to use CD46 as a This is with a study a MV the was generated and to cells F. M. Minagawa H. Yanagi Y. J. Virol. 2006; PubMed Scopus Google Scholar). with an H-protein to analysis of binding to the MV receptors to a To study the of this on MV binding to we the of virus to cells by cells were with an of or virus and as by analysis of a of the viruses 4, virus was the virus with a and the virus by The in the with virus is with a in the is the of was with the the in the was that the with binding. to cells with as whereas binding to was be that were with cell is that the virus to other cell surface these are K. Ono N. Tatsuo H. Minagawa H. M. K. Yanagi Y. J. Virol. 2002; 76: PubMed Scopus Google Scholar). A MV H-protein residues involved in protein-protein mutagenesis that identified Ile-194 as a critical residue for SLAM-dependent fusion. Receptor binding studies that Ile-194 is involved in primary while the bs-5 quartet mutations Asp-530, Arg-533, and a post-binding to unfolding and membrane fusion. The of these residues is in 5 on the recently crystal structure of the H-protein ectodomain T. M. N. M. K. K. D. Yanagi Y. K. U. S. A. 2007; PubMed Scopus Google Scholar). In this structure the H the This propeller blades 5 and with the residues the cell A and SLAM residues are or of Tyr-529, Asp-530, Arg-533, Tyr-553, and as at in part These residues a binding on the of the H-protein We that these residues have as based on Tyr-529, Asp-530, Arg-533, Tyr-553, and were identified based on a fusion assay S. Oezgun N. Braun W. Cattaneo R. J. Virol. 2004; 78: 302-313Crossref PubMed Scopus (169) Google Scholar). and were shown to N. M. B. Wild T.F. R. J. Virol. 2004; 78: PubMed Scopus Google Scholar). We have and mutations in a receptor-specific cell-to-cell fusion assay and that cause a in SLAM-dependent fusion not identified by the sustain receptor binding or subsequent receptor-specific H-protein conformational in the H-protein conformation that of the Ile-194 surface is and for binding Tyr-529, Tyr-553, Asp-530, and (bs-5 quartet A and have 4, and of to respectively. most of the bs-5 quartet residues are to cellular an with of binding part in subsequent conformational changes for fusion to binding to by the side chain of the 194 were as in a that in the of binding. the membrane fusion that residues with side maintained and measured in the of side function and in and function and A of the amino acids that function and those that it at the as a of amino not involved in this sustain a protein conformation it The identity of residue the ability of MV H to use CD46 as a H-proteins MV wild-type strains have at this while vaccine strains have U. von Messling V. Devaux P. Cattaneo R. 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G. 2007; PubMed Scopus Google Scholar). the at on the H-protein the ability to previously it the interaction with SLAM, we have that H-protein mutants and membrane while the SLAM-binding of the protein to cell surface other SLAM for part of this In H be conducive to receptor binding, it is its ectodomain the H-protein be in an conformation conducive to SLAM binding the ectodomain Our analysis of the H-protein interaction with SLAM that it is based on H-protein conformational The SLAM interaction is by how this residue, located on one of propeller blade in with and on of propeller blade 6, as as with and on a of propeller blade 5 this of the Ile-194 surface is of this interface the of residue 194 mutations receptor binding. We that the H-protein crystal structure one of its the other the binding and fusion are based on H-proteins propeller blades and amino acid side are in as a of K. D. Nature. 2007; PubMed Scopus Google Scholar). SLAM to one of the that H and at residue 194 the of an H-protein conformation conducive to primary binding. We are the H-protein with to changes as a of Ile-194 of the four residues identified as essential for SLAM-dependent SLAM-binding S. Oezgun N. Braun W. Cattaneo R. J. Virol. 2004; 78: 302-313Crossref PubMed Scopus (169) Google Scholar). This that not binding, also subsequent H-protein conformational changes be receptor-specific. also that of H-protein conformational changes to unfolding and membrane fusion H.S. Wen X. Paterson R.G. Lamb R.A. Jardetzky T.S. Nature. 2006; 439: 38-44Crossref PubMed Scopus (343) Google Scholar). In the that on the H-protein fusion through receptors U. F. S. Cattaneo R. J. Virol. 2000; 74: PubMed Scopus Google Scholar, T. M. S. C.D. Nat. 23: PubMed Scopus Google Scholar, G. C. J. Cattaneo R. 2007; 67: PubMed Scopus Google Scholar) be by the of of H-protein conformational changes membrane fusion. In our and the MV H crystal structure T. M. N. M. K. K. D. Yanagi Y. K. U. S. A. 2007; PubMed Scopus Google Scholar) the stage for the of the of membrane fusion on the of other of the We and for and for the and for with