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• W2783129997 abstract "•3.2 Å structure of mAb MR191 complexed with trimeric marburgvirus surface glycoprotein•MR191 neutralizes by occupying the conserved receptor-binding site•MR191 competes with the host receptor Niemann-Pick C1•Escape mutants map outside the MR191 footprint, suggesting effect on quaternary structure Since their first identification 50 years ago, marburgviruses have emerged several times, with 83%–90% lethality in the largest outbreaks. Although no vaccines or therapeutics are available for human use, the human antibody MR191 provides complete protection in non-human primates when delivered several days after inoculation of a lethal marburgvirus dose. The detailed neutralization mechanism of MR191 remains outstanding. Here we present a 3.2 Å crystal structure of MR191 complexed with a trimeric marburgvirus surface glycoprotein (GP). MR191 neutralizes by occupying the conserved receptor-binding site and competing with the host receptor Niemann-Pick C1. The structure illuminates previously disordered regions of GP including the stalk, fusion loop, CX6CC switch, and an N-terminal region of GP2 that wraps about the outside of GP1 to anchor a marburgvirus-specific “wing” antibody epitope. Virus escape mutations mapped far outside the MR191 receptor-binding site footprint suggest a role for these other regions in the GP quaternary structure. Since their first identification 50 years ago, marburgviruses have emerged several times, with 83%–90% lethality in the largest outbreaks. Although no vaccines or therapeutics are available for human use, the human antibody MR191 provides complete protection in non-human primates when delivered several days after inoculation of a lethal marburgvirus dose. The detailed neutralization mechanism of MR191 remains outstanding. Here we present a 3.2 Å crystal structure of MR191 complexed with a trimeric marburgvirus surface glycoprotein (GP). MR191 neutralizes by occupying the conserved receptor-binding site and competing with the host receptor Niemann-Pick C1. The structure illuminates previously disordered regions of GP including the stalk, fusion loop, CX6CC switch, and an N-terminal region of GP2 that wraps about the outside of GP1 to anchor a marburgvirus-specific “wing” antibody epitope. Virus escape mutations mapped far outside the MR191 receptor-binding site footprint suggest a role for these other regions in the GP quaternary structure. Filoviruses cause severe disease and have been responsible for multiple outbreaks among both humans and non-human primates. Within the filovirus family are three genera: Ebolavirus (which includes Ebola virus [EBOV], Sudan virus [SUDV], Bundibugyo virus, Taï Forest virus, and Reston virus), Marburgvirus (which includes Marburg virus [MARV] and Ravn virus [RAVV]), and Cuevavirus. Ebola virus disease (EVD) and Marburg virus disease (MVD) are clinically similar. MARV was the first filovirus identified, and has re-emerged multiple times since its 1967 discovery. The largest outbreak was nearly 90% lethal (CDC, 2005CDC. (2005). Brief report: outbreak of Marburg virus hemorrhagic fever –- Angola. October 1, 2004 – March 29, 2005. CDC. Available at: http://www.cdc.gov/mmwr/preview/mmwrhtml/mm54d330a1.htm.Google Scholar). Antibody therapies are a promising avenue for prophylaxis, post-exposure prophylaxis, and therapeutic treatment of emerging viral diseases (Chanock et al., 1993Chanock R.M. Crowe Jr., J.E. Murphy B.R. Burton D.R. Human monoclonal antibody Fab fragments cloned from combinatorial libraries: potential usefulness in prevention and/or treatment of major human viral diseases.Infect. Agent Dis. 1993; 2: 118-131PubMed Google Scholar, Zeitlin et al., 1999Zeitlin L. Cone R.A. Whaley K.J. Using monoclonal antibodies to prevent mucosal transmission of epidemic infectious diseases.Emerg. Infect. Dis. 1999; 5: 54-64Crossref PubMed Scopus (83) Google Scholar, Lachmann, 2012Lachmann P.J. The use of antibodies in the prophylaxis and treatment of infections.Emerg. Microbes Infect. 2012; 1: e11Crossref PubMed Scopus (16) Google Scholar, Burton and Saphire, 2015Burton D.R. Saphire E.O. Swift antibodies to counter emerging viruses.Proc. Natl. Acad. Sci. USA. 2015; 112: 10082-10083Crossref PubMed Scopus (4) Google Scholar). One antibody-based therapy, ZMapp (Qiu et al., 2014Qiu X. Wong G. Audet J. Bello A. Fernando L. Alimonti J.B. Fausther-Bovendo H. Wei H. Aviles J. Hiatt E. et al.Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp.Nature. 2014; 514: 47-53Crossref PubMed Scopus (773) Google Scholar), appeared to be beneficial during the 2013–2016 EVD outbreak, although the results did not reach the threshold of statistical significance (PREVAIL II Writing Group et al., 2016Davey Jr., R.T. Dodd L. Proschan M.A. Neaton J. Neuhaus Nordwall J. Koopmeiners J.S. Beigel J. Tierney J. PREVAIL II Writing GroupMulti-National PREVAIL II Study TeamA randomized, controlled trial of ZMapp for Ebola virus infection.N. Engl. J. Med. 2016; 375: 1448-1456Crossref PubMed Scopus (341) Google Scholar). None of the antibodies in ZMapp reacts with marburgviruses, and at this time there are no approved treatments available for MVD. Filoviruses produce enveloped virions that express a single glycoprotein (GP) on the surface. GP is responsible for attachment to and entry of target cells, and is the primary target for antibodies and vaccines (Dye et al., 2012Dye J.M. Herbert A.S. Kuehne A.I. Barth J.F. Muhammad M.A. Zak S.E. Ortiz R.A. Prugar L.I. Pratt W.D. Postexposure antibody prophylaxis protects nonhuman primates from filovirus disease.Proc. Natl. Acad. Sci. USA. 2012; 109: 5034-5039Crossref PubMed Scopus (229) Google Scholar). Filovirus GPs share a common core fold and trimeric organization, but are antigenically distinct. Marburgvirus GPs are only 30% identical to EBOV GP in primary amino acid sequence. The two marburgvirus GPs, however, MARV and RAVV GP, are quite similar to each other in sequence, and likely structure, with 78% amino acid identity overall and 90% identity outside the mucin-like domain. Filovirus GPs are 676–681 amino acids in length and are cleaved in the producer cell to yield two subunits, GP1 and GP2, which remain anchored by a single disulfide bond (Volchkov et al., 1998Volchkov V.E. Feldmann H. Volchkova V.A. Klenk H.D. Processing of the Ebola virus glycoprotein by the proprotein convertase furin.Proc. Natl. Acad. Sci. USA. 1998; 95: 5762-5767Crossref PubMed Scopus (408) Google Scholar, Volchkov et al., 2000Volchkov V.E. Volchkova V.A. Ströher U. Becker S. Dolnik O. Cieplik M. Garten W. Klenk H.D. Feldmann H. Proteolytic processing of Marburg virus glycoprotein.Virology. 2000; 268: 1-6Crossref PubMed Scopus (95) Google Scholar). The larger GP1 subunit harbors a receptor-binding core, a “glycan cap” subdomain, and a C-terminal, heavily glycosylated mucin-like domain. GP2 contains the membrane fusion machinery, including the internal fusion loop (IFL), two heptad repeat regions (HR1 and HR2), and a transmembrane domain to anchor the protein in the viral membrane (Bukreyev et al., 1993Bukreyev A. Volchkov V.E. Blinov V.M. Netesov S.V. The GP-protein of Marburg virus contains the region similar to the 'immunosuppressive domain' of oncogenic retrovirus P15E proteins.FEBS Lett. 1993; 323: 183-187Crossref PubMed Scopus (44) Google Scholar, Feldmann et al., 1993Feldmann H. Klenk H.D. Sanchez A. Molecular biology and evolution of filoviruses.Arch. Virol. Suppl. 1993; 7: 81-100Crossref PubMed Scopus (130) Google Scholar, Lee et al., 2008Lee J.E. Fusco M.L. Hessell A.J. Oswald W.B. Burton D.R. Saphire E.O. Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor.Nature. 2008; 454: 177-182Crossref PubMed Scopus (541) Google Scholar). After attachment, filoviruses enter cells via macropinocytosis (Nanbo et al., 2010Nanbo A. Imai M. Watanabe S. Noda T. Takahashi K. Neumann G. Halfmann P. Kawaoka Y. Ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner.PLoS Pathog. 2010; 6: e1001121Crossref PubMed Scopus (324) Google Scholar, Saeed et al., 2010Saeed M.F. Kolokoltsov A.A. Albrecht T. Davey R.A. Cellular entry of Ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes.PLoS Pathog. 2010; 6: e1001110Crossref PubMed Scopus (323) Google Scholar, Aleksandrowicz et al., 2011Aleksandrowicz P. Marzi A. Biedenkopf N. Beimforde N. Becker S. Hoenen T. Feldmann H. Schnittler H.J. Ebola virus enters host cells by macropinocytosis and clathrin-mediated endocytosis.J. Infect. Dis. 2011; 204: S957-S967Crossref PubMed Scopus (180) Google Scholar, Mulherkar et al., 2011Mulherkar N. Raaben M. de la Torre J.C. Whelan S.P. Chandran K. The Ebola virus glycoprotein mediates entry via a non-classical dynamin-dependent macropinocytic pathway.J. Virol. 2011; 419: 72-83Crossref Scopus (102) Google Scholar). Once in the endosome, ebolavirus GPs are cleaved by cathepsins B and/or L (Chandran et al., 2005Chandran K. Sullivan N.J. Felbor U. Whelan S.P. Cunningham J.M. Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection.Science. 2005; 308: 1643-1645Crossref PubMed Scopus (672) Google Scholar). Cleavage removes the heavily glycosylated glycan cap and mucin-like domains from ebolavirus GP1, and is required to expose the binding site for the entry receptor, Niemann-Pick C1 (NPC1) (Chandran et al., 2005Chandran K. Sullivan N.J. Felbor U. Whelan S.P. Cunningham J.M. Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection.Science. 2005; 308: 1643-1645Crossref PubMed Scopus (672) Google Scholar, Schornberg et al., 2006Schornberg K. Matsuyama S. Kabsch K. Delos S. Bouton A. White J. Role of endosomal cathepsins in entry mediated by the Ebola virus glycoprotein.J. Virol. 2006; 80: 4174-4178Crossref PubMed Scopus (343) Google Scholar, Hood et al., 2010Hood C.L. Abraham J. Boyington J.C. Leung K. Kwong P.D. Nabel G.J. Biochemical and structural characterization of cathepsin L-processed Ebola virus glycoprotein: implications for viral entry and immunogenicity.J. Virol. 2010; 84: 2972-2982Crossref PubMed Scopus (93) Google Scholar, Brecher et al., 2012Brecher M. Schornberg K.L. Delos S.E. Fusco M.L. Saphire E.O. White J.M. Cathepsin cleavage potentiates the Ebola virus glycoprotein to undergo a subsequent fusion-relevant conformational change.J. Virol. 2012; 86: 364-372Crossref PubMed Scopus (111) Google Scholar, Marzi et al., 2012Marzi A. Reinheckel T. Feldmann H. Cathepsin B & L are not required for Ebola virus replication.PLoS Negl. Trop. Dis. 2012; 6: e1923Crossref PubMed Scopus (55) Google Scholar). Interestingly, marburgviruses use the same NPC1 receptor but do not share the dependence on cathepsins B and L of ebolaviruses (Gnirss et al., 2012Gnirss K. Kühl A. Karsten C. Glowacka I. Bertram S. Kaup F. Hofmann H. Pöhlmann S. Cathepsins B and L activate Ebola but not Marburg virus glycoproteins for efficient entry into cell lines and macrophages independent of TMPRSS2 expression.Virology. 2012; 424: 3-10Crossref PubMed Scopus (75) Google Scholar). The shared NPC1 receptor is an endosomal/lysosomal 13-pass transmembrane protein with three large luminal domains, A, C, and I, of which domain C (NPC1-C) is necessary and sufficient for filovirus binding (Carette et al., 2011Carette J.E. Raaben M. Wong A.C. Herbert A.S. Obernosterer G. Mulherkar N. Kuehne A.I. Kranzusch P.J. Griffin A.M. Ruthel G. et al.Ebola virus entry requires the cholesterol transporter Niemann-Pick C1.Nature. 2011; 477: 340-343Crossref PubMed Scopus (896) Google Scholar, Miller et al., 2012Miller E.H. Obernosterer G. Raaben M. Herbert A.S. Deffieu M.S. Krishnan A. Ndungo E. Sandesara R.G. Carette J.E. Kuehne A.I. et al.Ebola virus entry requires the host-programmed recognition of an intracellular receptor.EMBO J. 2012; 31: 1947-1960Crossref PubMed Scopus (245) Google Scholar, Gong et al., 2016Gong X. Qian H. Zhou X. Wu J. Wan T. Cao P. Huang W. Zhao X. Wang X. Wang P. et al.Structural insights into the Niemann-Pick C1 (NPC1)-mediated cholesterol transfer and Ebola infection.Cell. 2016; 165: 1467-1478Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Recent work has shown that the interaction between EBOV GP and NPC1-C is mediated by two protruding loops of NPC1-C, which engage a hydrophobic pocket on the surface of cleaved GP (GPcl) (Wang et al., 2016Wang H. Shi Y. Song J. Qi J. Lu G. Yan J. Gao G.F. Ebola viral glycoprotein bound to its endosomal receptor Niemann-Pick C1.Cell. 2016; 164: 258-268Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Prior to cleavage, an 86 amino acid “glycan cap” occupies the NPC1-binding site on EBOV GP (Lee et al., 2008Lee J.E. Fusco M.L. Hessell A.J. Oswald W.B. Burton D.R. Saphire E.O. Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor.Nature. 2008; 454: 177-182Crossref PubMed Scopus (541) Google Scholar). Hence, uncleaved ebolavirus GPs do not bind to NPC1-C (Miller et al., 2012Miller E.H. Obernosterer G. Raaben M. Herbert A.S. Deffieu M.S. Krishnan A. Ndungo E. Sandesara R.G. Carette J.E. Kuehne A.I. et al.Ebola virus entry requires the host-programmed recognition of an intracellular receptor.EMBO J. 2012; 31: 1947-1960Crossref PubMed Scopus (245) Google Scholar). Potent antibodies against marburgvirus recently were isolated from circulating B cells in the blood of a human survivor of natural MVD (Flyak et al., 2015Flyak A.I. Ilinykh P.A. Murin C.D. Garron T. Shen X. Fusco M.L. Hashiguchi T. Bornholdt Z.A. Slaughter J.C. Sapparapu G. et al.Mechanism of human antibody-mediated neutralization of Marburg virus.Cell. 2015; 160: 893-903Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Among these, antibody MR191 was shown to protect non-human primates against a lethal marburgvirus challenge when treatment was initiated as late as 5 days post-exposure (Mire et al., 2017Mire C.E. Geisbert J.B. Borisevich V. Fenton K.A. Agans K.N. Flyak A.I. Deer D.J. Steinkellner H. Bohorov O. Bohorova N. et al.Therapeutic treatment of Marburg and Ravn virus infection in nonhuman primates with a human monoclonal antibody.Sci. Transl. Med. 2017; 9https://doi.org/10.1126/scitranslmed.aai8711Crossref PubMed Scopus (49) Google Scholar). In that study, MR191 provided protection superior to that of two other antibodies in the same competition group, MR78 and MR82 (Mire et al., 2017Mire C.E. Geisbert J.B. Borisevich V. Fenton K.A. Agans K.N. Flyak A.I. Deer D.J. Steinkellner H. Bohorov O. Bohorova N. et al.Therapeutic treatment of Marburg and Ravn virus infection in nonhuman primates with a human monoclonal antibody.Sci. Transl. Med. 2017; 9https://doi.org/10.1126/scitranslmed.aai8711Crossref PubMed Scopus (49) Google Scholar). Here, we present the crystal structure at 3.2 Å resolution, of trimeric, uncleaved, pre-fusion RAVV GP in complex with antibody MR191 (PDB: 6BP2). This structure is higher in resolution than the marburgvirus GP structure previously available. The higher-resolution map reveals that the N-terminal region of marburgvirus GP2 wraps around the outside of the GP core, to occupy a position that, in ebolaviruses, is instead held by GP1. This newly visualized subdomain of GP2 anchors a marburgvirus-specific “wing” epitope: the only other epitope that has been yet shown to elicit protective antibodies against MVD (Fusco et al., 2015Fusco M.L. Hashiguchi T. Cassan R. Biggins J.E. Murin C.D. Warfield K.L. Li S. Holtsberg F.W. Shulenin S. Vu H. et al.Protective mAbs and cross-reactive mAbs raised by immunization with engineered Marburg virus GPs.PLoS Pathog. 2015; 11: e1005016Crossref PubMed Scopus (31) Google Scholar). This structure also now illustrates the complete IFL, GP1-GP2 disulfide anchor, CX6CC switch region, and HR2 stalk of RAVV GP, all of which were disordered in structures obtained previously. Fundamental differences between marburgviruses and ebolaviruses in the organization of the GP2 wing and the glycosylated regions in GP1 help explain why marburgvirus entry is cathepsins B- and L-independent, and why it elicits a different pattern of antibody reactivity than ebolaviruses. Further, the crystal structure illustrates that the potent therapeutic antibody MR191 binds into the receptor-binding site near the apex of GP1. In ebolaviruses, this site is inaccessible due to the position of the glycan cap in the absence of cathepsin cleavage. However, in the marburgvirus GP, the polypeptide region equivalent to the ebolavirus glycan cap appears to be flexible and does not as effectively block antibody access to the receptor-binding site. MR191 competes with NPC1-C for binding of GP and appears to mimic NPC1-C (Wang et al., 2016Wang H. Shi Y. Song J. Qi J. Lu G. Yan J. Gao G.F. Ebola viral glycoprotein bound to its endosomal receptor Niemann-Pick C1.Cell. 2016; 164: 258-268Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) in its attachment. Contact residues for MR191 are also essential for receptor binding and are highly conserved among related filoviruses. The conservation and importance of the MR191 binding site suggests that the footprint of MR191 may be somewhat more resistant to mutagenic escape than other, less-conserved antibody epitopes. Interestingly, the escape mutations that arose during cell culture passage occurred in distant sites, suggesting an allosteric communication or an as-yet unknown role of these regions in maintenance of GP quaternary structure. Recombinant RAVV GP ectodomain (residues 1–636, with 257–425 deleted to remove the mucin-like domain) was expressed in Drosophila S2 cells, purified, and complexed with Fab fragments of the human MR191 antibody for crystallization. Data to 3.2 Å were collected from cryo-protected crystals at beamline 23-ID-D of the Advanced Photon Source. Residues 33–180 of GP1, 469–629 of GP2, and glycans attached to N94, N171, and N564 were visible. Residues 2–216 of the light chain and 2–227 of the heavy chain of MR191 also were visible. One GP protomer-Fab complex is contained in the asymmetric unit of the P321 crystals, with the biologically relevant trimer formed about a crystallographic 3-fold axis (Figure 1 and Table S1). The structure illuminates functionally critical regions of marburgvirus GP (Figure 2) that were disordered in the previous marburgvirus GP structure. First, the IFL of GP2 (residues 514–551) can now be traced in its entirety (Figure 2A). The IFL is anchored to the protein core via an anti-parallel β strand scaffold, with a 20-residue loop containing the hydrophobic fusion peptide. The IFL rests in the GP1/GP2 interface of the adjacent protomer in the GP trimer, and is secured by several hydrophobic interactions and hydrogen bonds. Second, we can now visualize an N-terminal region of GP2, beginning 34 residues downstream of the furin cleavage site and including residues 469–478 and 487–498. These residues anchor the marburgvirus-specific “wing” domain, residues 436–501. This site is targeted by marburgvirus-specific protective antibodies (Fusco et al., 2015Fusco M.L. Hashiguchi T. Cassan R. Biggins J.E. Murin C.D. Warfield K.L. Li S. Holtsberg F.W. Shulenin S. Vu H. et al.Protective mAbs and cross-reactive mAbs raised by immunization with engineered Marburg virus GPs.PLoS Pathog. 2015; 11: e1005016Crossref PubMed Scopus (31) Google Scholar), and is the only major recognition site of antibodies against marburgviruses yet described, beyond the receptor-binding site. We were able to place the GP2 wing anchor at the base of GP unambiguously, packing against the GP1 core directly beneath the C terminus of the fusion loop and the start of heptad repeat 1 (HR1) (Figure 2B). This observation was unexpected, since this same site on the GP1 core of EBOV or SUDV is not bound by any part of GP2, but instead by residues 32–45 at the N terminus of GP1 (Figure S5). The wing domain is unique to marburgviruses and results from a 66-residue N-terminal shift in the position of the furin cleavage event (R501 in EBOV, but R435 in MARV), which separates the GP1 and GP2 segments. Therefore, residues at the equivalent sequence region are included in the ebolavirus mucin-like domain and attach to the top of GP1 in ebolaviruses, not to GP2 as in marburgviruses. The crystal structure also revealed the structure of the heptad repeat 2 (HR2) region, which forms the “stalk” between the GP core and the viral membrane (Figure 2C). Here, HR2 forms a three-helix bundle with five hydrophobic residues from each helix facing inward to form a hydrophobic core, likely stabilizing the trimer. Although these residues differ in sequence from those of ebolaviruses, the hydrophobic packing is conserved (Zhao et al., 2016Zhao Y. Ren J. Harlos K. Stuart D.I. Structure of glycosylated NPC1 luminal domain C reveals insights into NPC2 and Ebola virus interactions.FEBS Lett. 2016; 590: 605-612Crossref PubMed Scopus (30) Google Scholar). Further, an N-linked glycosylation sequon is present in the HR2 of all known filoviruses. This glycan has been visualized for EBOV (Zhao et al., 2016Zhao Y. Ren J. Harlos K. Stuart D.I. Structure of glycosylated NPC1 luminal domain C reveals insights into NPC2 and Ebola virus interactions.FEBS Lett. 2016; 590: 605-612Crossref PubMed Scopus (30) Google Scholar), and likely shields a portion of this conserved site from antibody recognition. Although an NXS glycosylation sequon is present in the sequence of all marburgvirus isolates, a glycan is not visible in this structure, and there does not appear to be enough space for a glycan attached at this site to fit within in the crystal packing. It is unknown if this site is glycosylated on authentic marburgvirus particles. Immediately prior to HR2, the CX6CC disulfide-bearing switch region also can be seen in its entirety. The first and second cysteines in this motif (Cys-602 and Cys-609) form an intra-GP2 disulfide bond that anchors the switch region in a 360° turn as it descends downward to the membrane. The third cysteine in this motif (Cys-610) forms the disulfide anchor to GP1 (Cys-37). In all structures of uncleaved, mucin-deleted EBOV or SUDV GP, a glycan cap subdomain of GP1 occupies the hydrophobic NPC1-C binding pocket (Lee et al., 2008Lee J.E. Fusco M.L. Hessell A.J. Oswald W.B. Burton D.R. Saphire E.O. Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor.Nature. 2008; 454: 177-182Crossref PubMed Scopus (541) Google Scholar, Dias et al., 2011Dias J.M. Kuehne A.I. Abelson D.M. Bale S. Wong A.C. Halfmann P. Muhammad M.A. Fusco M.L. Zak S.E. Kang E. et al.A shared structural solution for neutralizing ebolaviruses.Nat. Struct. Mol. Biol. 2011; 18: 1424-1427Crossref PubMed Scopus (102) Google Scholar, Bale et al., 2012Bale S. Dias J.M. Fusco M.L. Hashiguchi T. Wong A.C. Liu T. Keuhne A.I. Li S. Woods Jr., V.L. Chandran K. et al.Structural basis for differential neutralization of ebolaviruses.Viruses. 2012; 4: 447-470Crossref PubMed Scopus (55) Google Scholar, Misasi et al., 2016Misasi J. Gilman M.S. Kanekiyo M. Gui M. Cagigi A. Mulangu S. Corti D. Ledgerwood J.E. Lanzavecchia A. Cunningham J. et al.Structural and molecular basis for Ebola virus neutralization by protective human antibodies.Science. 2016; 351: 1343-1346Crossref PubMed Scopus (134) Google Scholar, Pallesen et al., 2016Pallesen J. Murin C.D. de Val N. Cottrell C.A. Hastie K.M. Turner H.L. Fusco M.L. Flyak A.I. Zeitlin L. Crowe Jr., J.E. et al.Structures of Ebola virus GP and sGP in complex with therapeutic antibodies.Nat. Microbiol. 2016; 1: 16128Crossref PubMed Scopus (86) Google Scholar, Zhao et al., 2016Zhao Y. Ren J. Harlos K. Stuart D.I. Structure of glycosylated NPC1 luminal domain C reveals insights into NPC2 and Ebola virus interactions.FEBS Lett. 2016; 590: 605-612Crossref PubMed Scopus (30) Google Scholar). The equivalent residues for marburgviruses (174–256) are included in proteins used for crystallization in this study, and the RAVV GP was intact and uncleaved. However, a glycan cap was not visible. Instead, these 83 residues, their five predicted N-linked glycans, and four predicted O-linked glycans are disordered, and likely occupy the ∼90 Å solvent channels between receptor-binding sites in the crystal packing. Marburgviruses and ebolaviruses possess little sequence identity in this region, and this domain of MARV GP is predicted to be more disordered than that of EBOV GP (Figure S2). These observations suggest that these residues of marburgvirus GP diverge structurally from the corresponding domain of ebolaviruses. A great many marburgvirus antibodies have been identified against the hydrophobic trough of the GP1, while no such antibodies are yet described for ebolaviruses, leading to speculation that this site is more exposed in marburgviruses than ebolaviruses. We note here, however, that NPC1-C is unable to bind uncleaved RAVV GP in vitro (Figure 4A). The RAVV glycan cap, although mobile in MR191- and MR78-bound structures, may still partially shield the receptor-binding site. The antibodies may simply better displace the cap than NPC1-C. MR191 binds in the NPC1-C binding site of RAVV GP, interacting with both the hydrophobic trough and the crest at the apex of GP1 (Figures 3 and S4). CDRs H3, H2, L3, and L1 participate in this interaction. CDR H3 extends 11 Å into the hydrophobic trough of GP, with antibody residue F100a (Kabat numbering) at its apex interacting with W70, F72, and M154 of RAVV GP (residues equivalent to W86, F88, and I170 in EBOV GP). Along the C-terminal side of the extended CDR H3, residues V100b and W100d of MR191 form additional hydrophobic interactions with the pocket. Further, residue E100c of MR191 CDR H3 forms a hydrogen bond with Q128 of the crest of GP1, which rises above the hydrophobic trough. Four residues of CDR H2 (S52, S54, N56, and Y58) also hydrogen bond to Q128 and to the main chain carbonyls of D99 and P100 of the GP1 crest (Figure 3). The light chain of MR191 forms a mixture of hydrophobic and hydrophilic contacts with the lower and outer rim of the GP trough. MR191 mimics the interaction made by NPC1-C loop 2 in which an extended loop bearing a Phe (F100a in MR191, F131 in NPC1-C) at its apex binds into the GP hydrophobic trough (Wang et al., 2016Wang H. Shi Y. Song J. Qi J. Lu G. Yan J. Gao G.F. Ebola viral glycoprotein bound to its endosomal receptor Niemann-Pick C1.Cell. 2016; 164: 258-268Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). A Phe also is employed by the related human mAb MR78 (Hashiguchi et al., 2015Hashiguchi T. Fusco M.L. Bornholdt Z.A. Lee J.E. Flyak A.I. Matsuoka R. Kohda D. Yanagi Y. Hammel M. Crowe Jr., J.E. et al.Structural basis for Marburg virus neutralization by a cross-reactive human antibody.Cell. 2015; 160: 904-912Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), and by the EBOV and SUDV glycan caps, which insert into and mask this region prior to cathepsin cleavage (Lee et al., 2008Lee J.E. Fusco M.L. Hessell A.J. Oswald W.B. Burton D.R. Saphire E.O. Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor.Nature. 2008; 454: 177-182Crossref PubMed Scopus (541) Google Scholar, Dias et al., 2011Dias J.M. Kuehne A.I. Abelson D.M. Bale S. Wong A.C. Halfmann P. Muhammad M.A. Fusco M.L. Zak S.E. Kang E. et al.A shared structural solution for neutralizing ebolaviruses.Nat. Struct. Mol. Biol. 2011; 18: 1424-1427Crossref PubMed Scopus (102) Google Scholar, Bale et al., 2012Bale S. Dias J.M. Fusco M.L. Hashiguchi T. Wong A.C. Liu T. Keuhne A.I. Li S. Woods Jr., V.L. Chandran K. et al.Structural basis for differential neutralization of ebolaviruses.Viruses. 2012; 4: 447-470Crossref PubMed Scopus (55) Google Scholar, Zhao et al., 2016Zhao Y. Ren J. Harlos K. Stuart D.I. Structure of glycosylated NPC1 luminal domain C reveals insights into NPC2 and Ebola virus interactions.FEBS Lett. 2016; 590: 605-612Crossref PubMed Scopus (30) Google Scholar). An aromatic residue appears to be essential for interaction with this conserved filovirus site: mutation of F100a in the MR191 heavy chain to a tyrosine (F100aY) maintained binding to the GP, while mutation to an alanine (F100aA) greatly reduced the strength of the interaction (Figure 3C). Interestingly, however, the other strongly hydrophobic, aromatic residue inserted into the pocket, W100d, did not appear to be as critical for binding (Figure 3C). Based on their binding sites, it is perhaps unsurprising that MR191 outcompetes NPC1-C when assayed in a competition-binding ELISA (Figure 4). These results suggest that MR191 sterically interferes with the binding of NPC1-C as a primary mechanism of neutralization. MR191 and NPC1-C appear to make similar interactions with the hydrophobic trough of filovirus GP, but only MR191 interacts with the crest (positioned above the receptor-binding site). We used mutagenesis to probe the antibody-GP interactions and determine which residues are critical for binding MR191 to marburgvirus GP. In MR191, in addition to the mutants discussed above, we mutated two residues that interact with the hydrophilic rim around the trough (Y91 and T93 in CDR L3), and four residues that interact with the crest above (S52, S54, N56, and Y58 in CDR H2 and E100c in CDR H3). Notably, no single mutation to any residue that makes hydrophilic interactions, whether to the hydrophilic rim or crest, significantly affected binding: T93A and Y91A mutations in the light chain of MR191, and S54A, N56A, Y58A, and Y58F mutations in the heavy chain, each resulted in binding of GP equivalent to that of wild-type GP (Figure S3). In contrast, MR191 bearing an F100a to Ala point mutation exhibited a 225-fold increase in a half maximal effective concentration (EC50) compared with that of wild-type MR1" @default.
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• W2783129997 date "2018-01-01" @default.
• W2783129997 modified "2023-10-16" @default.
• W2783129997 title "The Marburgvirus-Neutralizing Human Monoclonal Antibody MR191 Targets a Conserved Site to Block Virus Receptor Binding" @default.
• W2783129997 cites W1760776415 @default.
• W2783129997 cites W1863223069 @default.
• W2783129997 cites W1913208537 @default.
• W2783129997 cites W1971308430 @default.
• W2783129997 cites W1996037007 @default.
• W2783129997 cites W1999295452 @default.
• W2783129997 cites W2006614780 @default.
• W2783129997 cites W2016230098 @default.
• W2783129997 cites W2022339063 @default.
• W2783129997 cites W2028328803 @default.
• W2783129997 cites W2028463307 @default.
• W2783129997 cites W2031428754 @default.
• W2783129997 cites W2033650210 @default.
• W2783129997 cites W2043674872 @default.
• W2783129997 cites W2052956178 @default.
• W2783129997 cites W2055043387 @default.
• W2783129997 cites W2064369118 @default.
• W2783129997 cites W2067510101 @default.
• W2783129997 cites W2072075382 @default.
• W2783129997 cites W2072154238 @default.
• W2783129997 cites W2073539776 @default.
• W2783129997 cites W2075604706 @default.
• W2783129997 cites W2079526653 @default.
• W2783129997 cites W2084723039 @default.
• W2783129997 cites W2092220359 @default.
• W2783129997 cites W2094993149 @default.
• W2783129997 cites W2095036253 @default.
• W2783129997 cites W2102818658 @default.
• W2783129997 cites W2103114095 @default.
• W2783129997 cites W2103795849 @default.
• W2783129997 cites W2108168271 @default.
• W2783129997 cites W2110951885 @default.
• W2783129997 cites W2113132163 @default.
• W2783129997 cites W2114409495 @default.
• W2783129997 cites W2117671399 @default.
• W2783129997 cites W2123481741 @default.
• W2783129997 cites W2128963410 @default.
• W2783129997 cites W2144081223 @default.
• W2783129997 cites W2147874841 @default.
• W2783129997 cites W2154714625 @default.
• W2783129997 cites W2163341755 @default.
• W2783129997 cites W2180229411 @default.
• W2783129997 cites W2237639303 @default.
• W2783129997 cites W2266709709 @default.
• W2783129997 cites W2274022023 @default.
• W2783129997 cites W2274478712 @default.
• W2783129997 cites W2396625484 @default.
• W2783129997 cites W2396636851 @default.
• W2783129997 cites W2401876702 @default.
• W2783129997 cites W2487638977 @default.
• W2783129997 cites W2507513922 @default.
• W2783129997 cites W2544612685 @default.
• W2783129997 cites W2596908440 @default.
• W2783129997 cites W2604139512 @default.
• W2783129997 cites W2749679400 @default.
• W2783129997 cites W4291960446 @default.
• W2783129997 cites W807802234 @default.
• W2783129997 doi "https://doi.org/10.1016/j.chom.2017.12.003" @default.
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