FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2139749521> ?p ?o ?g. }

• W2139749521 endingPage "15958" @default.
• W2139749521 startingPage "15951" @default.
• W2139749521 abstract "The GP1,2 envelope glycoproteins (GP) of filoviruses (marburg- and ebolaviruses) mediate cell-surface attachment, membrane fusion, and entry into permissive cells. Here we show that a 151-amino acid fragment of the Lake Victoria marburgvirus GP1 subunit bound filovirus-permissive cell lines more efficiently than full-length GP1. An homologous 148-amino acid fragment of the Zaire ebolavirus GP1 subunit similarly bound the same cell lines more efficiently than a series of longer GP1 truncation variants. Neither the marburgvirus GP1 fragment nor that of ebolavirus bound a nonpermissive lymphocyte cell line. Both fragments specifically inhibited replication of infectious Zaire ebolavirus, as well as entry of retroviruses pseudotyped with either Lake Victoria marburgvirus or Zaire ebolavirus GP1,2. These studies identify the receptor-binding domains of both viruses, indicate that these viruses utilize a common receptor, and suggest that a single small molecule or vaccine can be developed to inhibit infection of all filoviruses. The GP1,2 envelope glycoproteins (GP) of filoviruses (marburg- and ebolaviruses) mediate cell-surface attachment, membrane fusion, and entry into permissive cells. Here we show that a 151-amino acid fragment of the Lake Victoria marburgvirus GP1 subunit bound filovirus-permissive cell lines more efficiently than full-length GP1. An homologous 148-amino acid fragment of the Zaire ebolavirus GP1 subunit similarly bound the same cell lines more efficiently than a series of longer GP1 truncation variants. Neither the marburgvirus GP1 fragment nor that of ebolavirus bound a nonpermissive lymphocyte cell line. Both fragments specifically inhibited replication of infectious Zaire ebolavirus, as well as entry of retroviruses pseudotyped with either Lake Victoria marburgvirus or Zaire ebolavirus GP1,2. These studies identify the receptor-binding domains of both viruses, indicate that these viruses utilize a common receptor, and suggest that a single small molecule or vaccine can be developed to inhibit infection of all filoviruses. Filoviruses cause severe hemorrhagic fevers in human and nonhuman primates, with case fatality rates that reach 88%. The family Filoviridae contains two genera, Marburgvirus (species Lake Victoria marburgvirus) and Ebolavirus (species Côte d'Ivoire ebolavirus, Reston ebolavirus, Sudan ebolavirus, and Zaire ebolavirus) (1Feldmann H. Geisbert T.W. Jahrling P.B. Klenk H.-D. Netesov S.V. Peters C.J. Sanchez A. Swanepoel R. Volchkov V.E. Fauquet C.M. Mayo M.A. Maniloff J. Desselberger U. Ball L.A. Virus Taxonomy, Eighth Report of the International Committee on Taxonomy of Viruses. Elsevier/Academic Press, San Diego2005: 645-653Google Scholar). Like all mononegaviruses, filoviruses are enveloped and contain nonsegmented single-stranded RNA genomes of negative polarity (2Pringle C.R. Easton A.J. Semin. Virol. 1997; 8: 49-57Crossref Scopus (64) Google Scholar). Filoviral envelope glycoproteins (GP1,2) 3The abbreviations used are: GP, glycoprotein; CoV, coronavirus; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; HIV-1, human immunodeficiency virus, type 1; MARV-Ang, Lake Victoria marburgvirus strain Angola; MARV-Mus, Lake Victoria marburgvirus strain Musoke; MLV, Moloney murine leukemia virus; PBS, phosphate-buffered saline; RBD, receptor-binding domain; SARS, severe acute respiratory syndrome; VSV, vesicular stomatitis Indiana virus; ZEBOV-May, Zaire ebolavirus strain Mayinga; ORF, open reading frame. are type 1 transmembrane and class I viral fusion proteins that mediate cell association, fusion of viral and cellular membranes, and entry of the viral core into the cytosol (3Takada A. Robison C. Goto H. Sanchez A. Murti K.G. Whitt M.A. Kawaoka Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14764-14769Crossref PubMed Scopus (471) Google Scholar, 4Wool-Lewis R.J. Bates P. J. Virol. 1998; 72: 3155-3160Crossref PubMed Google Scholar, 5Chan S.Y. Speck R.F. Ma M.C. Goldsmith M.A. J. Virol. 2000; 74: 4933-4937Crossref PubMed Scopus (125) Google Scholar). The GP1,2 precursor assembles as a trimer and is modified by N-glycosylation in the endoplasmic reticulum. Trafficking of the trimeric GP1,2 precursor to the Golgi apparatus leads to refinement of N-glycosylation and addition of O-glycans (6Volchkov V.E. Feldmann H. Volchkova V.E. Klenk H.-D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5762-5767Crossref PubMed Scopus (410) Google Scholar, 7Volchkov V.E. Volchkova V.A. Ströher U. Becker S. Dolnik O. Cieplik M. Garten W. Klenk H.-D. Feldmann H. Virology. 2000; 268: 1-6Crossref PubMed Scopus (96) Google Scholar, 8Feldmann H. Volchkov V.E. Volchkova V.A. Ströher U. Klenk H.-D. J. Gen. Virol. 2001; 82: 2839-2848Crossref PubMed Scopus (98) Google Scholar, 9Becker S. Klenk H.-D. Mühlberger E. Virology. 1996; 225: 145-155Crossref PubMed Scopus (42) Google Scholar). Furin-like proteases cleave the polypeptide into the ectodomain GP1 and the transmembrane GP2 subunits, both of which remain connected through an intramolecular disulfide bond (GP1,2). Mature GP1,2 trimers are then incorporated into virions during budding (6Volchkov V.E. Feldmann H. Volchkova V.E. Klenk H.-D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5762-5767Crossref PubMed Scopus (410) Google Scholar, 7Volchkov V.E. Volchkova V.A. Ströher U. Becker S. Dolnik O. Cieplik M. Garten W. Klenk H.-D. Feldmann H. Virology. 2000; 268: 1-6Crossref PubMed Scopus (96) Google Scholar, 10Kolesnikova L. Berghöfer B. Bamberg S. Becker S. J. Virol. 2004; 78: 12277-12287Crossref PubMed Scopus (92) Google Scholar). The filoviral GP1 subunit mediates cell-surface receptor binding (8Feldmann H. Volchkov V.E. Volchkova V.A. Ströher U. Klenk H.-D. J. Gen. Virol. 2001; 82: 2839-2848Crossref PubMed Scopus (98) Google Scholar, 11Sanchez A. Kiley M.P. Holloway B.P. Auperin D.D. Virus Res. 1993; 29: 215-240Crossref PubMed Scopus (278) Google Scholar). Approximately half of the molecular weight of GP1 is because of N- and O-glycans, many of which are located at the C terminus of the subunit in a region described as the mucin-like domain (12Sanchez A. Yang Z.-Y. Xu L. Nabel G.J. Crews T. Peters C.J. J. Virol. 1998; 72: 6442-6447Crossref PubMed Google Scholar, 13Jeffers S.A. Sanders D.A. Sanchez A. J. Virol. 2002; 76: 12463-12472Crossref PubMed Scopus (199) Google Scholar). This domain contributes to cytopathicity observed in GP1,2-expressing cell lines and has been suggested to play a critical role in the pathogenesis of filoviral disease (14Simmons G. Wool-Lewis R.J. Baribaud F. Netter R.C. Bates P. J. Virol. 2002; 76: 2518-2528Crossref PubMed Scopus (179) Google Scholar, 15Takada A. Watanabe S. Ito H. Okazaki K. Kida H. Kawaoka Y. Virology. 2000; 278: 20-26Crossref PubMed Scopus (158) Google Scholar, 16Yang Z.-Y. Duckers H.J. Sullivan N.J. Sanchez A. Nabel E.G. Nabel G.J. Nat. Med. 2000; 6: 886-889Crossref PubMed Scopus (348) Google Scholar). However, its deletion enhances rather than decreases the efficiency of GP1,2-mediated infection (13Jeffers S.A. Sanders D.A. Sanchez A. J. Virol. 2002; 76: 12463-12472Crossref PubMed Scopus (199) Google Scholar, 16Yang Z.-Y. Duckers H.J. Sullivan N.J. Sanchez A. Nabel E.G. Nabel G.J. Nat. Med. 2000; 6: 886-889Crossref PubMed Scopus (348) Google Scholar, 17Manicassamy B. Wang J. Jiang H. Rong L. J. Virol. 2005; 79: 4793-4805Crossref PubMed Scopus (135) Google Scholar, 18Chandran K. Sullivan N.J. Felbor U. Whelan S.P. Cunningham J.M. Science. 2005; 308: 1643-1645Crossref PubMed Scopus (684) Google Scholar). Receptor binding is followed by endocytosis of the virions (19Geisbert T.W. Jahrling P.B. Virus Res. 1995; 39: 129-150Crossref PubMed Scopus (218) Google Scholar), acidification of the endocytotic vesicle (4Wool-Lewis R.J. Bates P. J. Virol. 1998; 72: 3155-3160Crossref PubMed Google Scholar, 5Chan S.Y. Speck R.F. Ma M.C. Goldsmith M.A. J. Virol. 2000; 74: 4933-4937Crossref PubMed Scopus (125) Google Scholar, 20Maryankova R.F. Glushakova S.E. Ryzhik E.V. Lukashevich I.S. Vopr. Virusol. 1993; 38: 74-76PubMed Google Scholar), and proteolytic processing of GP1 by endosomal cathepsins (18Chandran K. Sullivan N.J. Felbor U. Whelan S.P. Cunningham J.M. Science. 2005; 308: 1643-1645Crossref PubMed Scopus (684) Google Scholar, 21Schornberg K. Matsuyama S. Kabsch K. Delos S. Bouton A. White J. J. Virol. 2006; 80: 4147-4178Crossref PubMed Scopus (346) Google Scholar). Conformational changes in the filoviral GP2 subunit facilitate lipid mixing and fusion of the viral and cellular membranes, in a sequence of steps thought similar to those mediated by orthomyxoviral and retroviral transmembrane proteins (22Gallaher W.R. Cell. 1996; 85: 477-478Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 23Ito H.S. Watanabe S. Sanchez A. Whitt M.A. Kawaoka Y. J. Virol. 1999; 73: 8907-8912Crossref PubMed Google Scholar, 24Ruiz-Argüello M.B. Goñi F.M. Pereira F.B. Nieva J.L. J. Virol. 1998; 72: 1775-1781Crossref PubMed Google Scholar, 25Watanabe S. Takada A. Watanabe T. Ito H. Kida H. Kawaoka Y. J. Virol. 2000; 74: 10194-10201Crossref PubMed Scopus (114) Google Scholar). The host cell-surface receptor(s) for filoviruses have not yet been identified (26Simmons G. Rennekamp A.J. Chai N. Vandenberghe L.H. Riley J.L. Bates P. J. Virol. 2003; 77: 13433-13438Crossref PubMed Scopus (99) Google Scholar). However, the C-type lectin asialoglycoprotein receptor (27Becker S. Spiess M. Klenk H.-D. J. Gen. Virol. 1995; 76: 393-399Crossref PubMed Scopus (147) Google Scholar, 28Lin G. Simmons G. Pöhlmann S. Baribaud F. Ni H. Leslie G.J. Haggarty B.S. Bates P. Weissman D. Hoxie J.A. Doms R.W. J. Virol. 2003; 77: 1337-1346Crossref PubMed Scopus (223) Google Scholar), DC-SIGN (29Simmons G. Reeves J.D. Grogan C.C. Vandenbergh L.H. Baribaud F. Whitbeck J.C. Burke E. Buchmeier M.J. Soilleux E.J. Riley J.L. Doms R.W. Bates P. Pöhlmann S. Virology. 2003; 305: 115-123Crossref PubMed Scopus (313) Google Scholar, 30Marzi A. Gramberg T. Simmons G. Möller P. Rennekamp A.J. Krumbiegel M. Geier M. Eisemann J. Turza N. Saunier B. Steinkasserer A. Becker S. Bates P. Hofmann H. Pöhlmann S. J. Virol. 2004; 78: 12090-12095Crossref PubMed Scopus (305) Google Scholar), hMGL (31Takada A. Fujioka K. Tsuiji M. Morikawa A. Higashi N. Ebihara H. Kobasa D. Feldmann H. Irimura T. Kawaoka Y. J. Virol. 2004; 78: 2943-2947Crossref PubMed Scopus (211) Google Scholar), L-SIGN (29Simmons G. Reeves J.D. Grogan C.C. Vandenbergh L.H. Baribaud F. Whitbeck J.C. Burke E. Buchmeier M.J. Soilleux E.J. Riley J.L. Doms R.W. Bates P. Pöhlmann S. Virology. 2003; 305: 115-123Crossref PubMed Scopus (313) Google Scholar, 30Marzi A. Gramberg T. Simmons G. Möller P. Rennekamp A.J. Krumbiegel M. Geier M. Eisemann J. Turza N. Saunier B. Steinkasserer A. Becker S. Bates P. Hofmann H. Pöhlmann S. J. Virol. 2004; 78: 12090-12095Crossref PubMed Scopus (305) Google Scholar), and LSECtin (32Gramberg T. Hofmann H. Möller P. Lalor P.F. Marzi A. Geier M. Krumbiegel M. Winkler T. Kirchhoff F. Adams D.H. Becker S. Münch J. Pöhlmann S. Virology. 2005; 340: 224-236Crossref PubMed Scopus (166) Google Scholar), as well as other molecules, including folate receptor-α (33Chan S.Y. Empig C.J. Welte F.J. Speck R.F. Schmaljohn A. Kreisberg J.F. Goldsmith M.A. Cell. 2001; 106: 117-126Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar) and β1 integrins (15Takada A. Watanabe S. Ito H. Okazaki K. Kida H. Kawaoka Y. Virology. 2000; 278: 20-26Crossref PubMed Scopus (158) Google Scholar), have been shown or suggested to enhance filovirus cell entry. Subtle differences between marburgvirus and ebolavirus infection efficiencies in different cell lines or following glycosidase or protease treatment have led to the suggestion that these viruses utilize distinct receptors or entry mechanisms (5Chan S.Y. Speck R.F. Ma M.C. Goldsmith M.A. J. Virol. 2000; 74: 4933-4937Crossref PubMed Scopus (125) Google Scholar). Here we identify fragments of the Lake Victoria marburgvirus (Musoke strain; MARV-Mus) and Zaire ebolavirus (Mayinga stain; ZEBOV-May) GP1 subunit that efficiently bound cells permissive to filovirus infection but not a nonpermissive lymphocyte cell line. Each fragment inhibited infection of retroviruses pseudotyped with either marburgvirus or ebolavirus GP1,2. Both fragments also inhibited replication of infectious Zaire ebolavirus. Our data define homologous regions of otherwise divergent filoviruses that mediate association with a common receptor. Similarities in these receptor-binding domains may provide insight into the nature of this receptor and suggest vaccine and therapeutic approaches effective against all filoviruses. Cells and Culture Conditions—African green monkey kidney (Vero E6) cells and Jurkat lymphocytes were obtained from the American Type Culture Collection (ATCC numbers CRL-1586 and TIB-152, respectively). Human embryonic kidney 293T cells are a derivative of 293 cells (ATCC CRL1573) created by S. Haase and described originally as 293/tsA1609neo (34DuBridge R.B. Tang P. Hsia H.C. Leong P.M. Miller J.H. Calos M.P. Mol. Cell. Biol. 1987; 7: 379-387Crossref PubMed Scopus (922) Google Scholar). Adherent cells (Vero E6 and 293T) were maintained in Dulbecco's modified Eagle's medium (Invitrogen) and Jurkat lymphocytes in RPMI 1640 medium (Invitrogen). All media were supplemented with 10% fetal bovine serum (Sigma), 100 IU/ml penicillin, and 100 μg/ml streptomycin (Cellgro), and cell cultures were maintained at 37 °C in a humidified 5% CO2 atmosphere. Construction of Filovirus Envelope Glycoprotein-encoding Genes and Variants—Codon-optimized Lake Victoria marburgvirus strain Musoke (MARV-Mus) open reading frames (ORFs) encoding GP1 (amino acid residues 17-432) and GP1,2 (amino acid residues 17-681) lacking signal sequences were synthesized and amplified by de novo recursive PCR, using overlapping DNA oligomers based on the MARV-Mus GP1,2 protein sequence (GenBank™ accession number CAA781117). A codon-optimized Zaire ebolavirus strain Mayinga (ZEBOV-May) ORF encoding a mucin-like domain-deleted GP1 truncation variant (amino acid residues 33–308) (13Jeffers S.A. Sanders D.A. Sanchez A. J. Virol. 2002; 76: 12463-12472Crossref PubMed Scopus (199) Google Scholar) was synthesized based on the ZEBOV-May GP1,2 protein sequence (GenBank™ accession number NP_066246), using the same strategy. ORFs were ligated into a previously described pCDM8-derived expression vector (35Farzan M. Mirzabekov T. Kolchinsky P. Wyatt R. Cayabyab M. Gerard N.P. Gerard C. Sodroski J. Choe H. Cell. 1999; 96: 667-676Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar), encoding the CD5 signal sequence upstream of the ORF insert, and the Fc region of human immunoglobulin G1 downstream (MARV-Mus GP1-(17–432)-Fc and ZEBOV-May GP1-(33–308)-Fc). Vectors encoding N- and C-terminal truncation variants were generated by inverse PCR amplification using plasmids encoding MARV-Mus GP1-(17–432)-Fc or ZEBOV-May GP1-(33–308)-Fc as templates. An ORF encoding MARV-Mus GP1,2 residues 17–681 was cloned into a variant of the pCDM8 expression vector encoding the CD5 signal sequence and a C-terminal C9 tag (amino acid sequence GTETSQVAPA) derived from the rhodopsin C terminus (MARV-Mus GP1,2). Plasmid encoding a ZEBOV-May GP1,2 variant lacking its mucin-like domain, ZEBOV-May GP1,2-(Δ309–489) (4Wool-Lewis R.J. Bates P. J. Virol. 1998; 72: 3155-3160Crossref PubMed Google Scholar), was generously provided by Dr. James Cunningham. Plasmids encoding MARV-Ang GP1-Fc variants were generated by altering their equivalent MARV-Mus GP1-Fc variants at codon 74 (T74A), using the QuikChange method (Stratagene). Expression of Filovirus Envelope Glycoprotein Variants—For protein purification, 293T cells were transfected with plasmids encoding MARV-Mus GP1-(17–432)-Fc or ZEBOV-May GP1-(33–308)-Fc, their truncation variants, or control proteins (severe acute respiratory syndrome coronavirus strain Tor2 S(318–510)-Fc (SARS-CoV RBD-Fc) and human immunodeficiency virus type 1 (HIV-1) strain ADA gp120-Fc (36Choe H. Li W. Wright P.L. Vasilieva N. Venturi M. Huang C.C. Grundner C. Dorfman T. Zwick M.B. Wang L. Rosenberg E.S. Kwong P.D. Burton D.R. Robinson J.E. Sodroski J.G. Farzan M. Cell. 2003; 114: 161-170Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 37Wong S.K. Li W. Moore M.J. Choe H. Farzan M. J. Biol. Chem. 2004; 279: 3197-3201Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar)), using the calcium-phosphate method. Cells were washed in Dulbecco's phosphate-buffered saline (Invitrogen) 6 h post-transfection and grown at 37 °C in 293 SFM II medium (Invitrogen) supplemented with 100 IU/ml penicillin and 100 μg/ml streptomycin, 100 μm minimum Eagle's medium nonessential amino acids solution (Invitrogen), 2 mm sodium butyrate (Sigma), and 4 mm l-glutamine (Sigma). Medium was harvested after 48 h, and cell debris was removed by centrifugation and filtration through a 0.22-μm pore size filter (Corning Glass). Proteins were precipitated with protein A-Sepharose fast flow beads (Amersham Biosciences) at 4 °C for 16 h in the presence of Complete protease inhibitor (Roche Applied Science). Beads were washed once with 30-bed volumes of 0.5 m sodium chloride/phosphate-buffered saline, pH 7.4 (NaCl, Fisher; PBS, Invitrogen), and once with 10-bed volumes of PBS. Proteins were eluted with 50 mm sodium citrate, 50 mm glycine, pH 2 (sodium citrate, Fisher; glycine, Bio-Rad), neutralized with sodium hydroxide (Fisher), dialyzed in PBS, and concentrated with Centricon centrifugal filter units (Millipore). Purified proteins were assayed for size and concentration by comparison to bovine serum albumin standards (Sigma) by SDS-PAGE followed by Bio-Safe Coomassie (Bio-Rad) staining, and by using the Micro BCA protein assay kit (Pierce) according to the manufacturer's instructions. Cell Binding Assays—293T cells and Vero E6 cells were detached with PBS, 5 mm EDTA (Invitrogen) 48 h after plating, resuspended in an equal volume of PBS, 5 mm MgCl2 (Sigma), and washed twice in PBS, 2% goat serum (Sigma). Jurkat lymphocytes were harvested and washed twice in PBS, 2% goat serum. GP1-Fc constructs, truncation variants thereof, and control proteins were added to 5 × 105 cells to a final concentration of 100 nm and incubated on ice for 1.5 h. Cells were washed twice in PBS, 2% goat serum and incubated for 45 min on ice with a 1:40 dilution of goat Fc-specific fluorescein isothiocyanate (FITC)-conjugated anti-human IgG antibody (Sigma) in PBS, 2% goat serum. Cells were washed three times with PBS, 2% goat serum, once in PBS, and fixed with PBS, 2% formaldehyde (Sigma). Cell-surface binding of constructs was detected by flow cytometry with 10,000 events counted per sample. Base-line fluorescence was determined by measuring cells treated only with goat Fc-specific FITC-conjugated anti-human IgG antibody, which was then subtracted from binding values of the tested constructs and control proteins. Infection Assay with Filovirus Envelope Glycoprotein-pseudotyped Retroviruses—To generate retroviral pseudotypes, 293T cells were transfected by the calcium phosphate method with plasmid encoding MARV-Mus GP1,2, ZEBOV-May GP1,2-(Δ309–489), or vesicular stomatitis Indiana virus (VSV) G protein, together with the pQCXIX vector (BD Biosciences) expressing green fluorescent protein (GFP), and plasmid encoding the Moloney murine leukemia virus (MLV) gag and pol genes (38Moore M.J. Dorfman T. Li W. Wong S.K. Li Y. Kuhn J.H. Coderre J. Vasilieva N. Han Z. Greenough T.C. Farzan M. Choe H. J. Virol. 2004; 78: 10628-10635Crossref PubMed Scopus (177) Google Scholar) using equal concentrations of each plasmid. Cell supernatants were harvested 48 h post-transfection, cleared of cellular debris by centrifugation, filtered through a 0.45-μm pore size filter (Corning Glass), and stored at 4 °C. Supernatants containing pseudotyped viruses were added to 293T or Vero E6 cells in the presence or absence of the indicated concentrations of filovirus Fc truncation variants or control proteins. After 5 h, cells were washed once in PBS and replenished with fresh media. After 48 h, cells were imaged by fluorescent microscopy and detached with trypsin for analysis by flow cytometry. Infection Assay with Recombinant Green Fluorescent Protein-expressing Zaire Ebolavirus—All experiments with infectious filovirus were performed under biosafety level 4 conditions. Vero E6 cells were infected with a GFP-expressing ZEBOV-May created by reverse genetics (39Towner J.S. Paragas J. Dover J.E. Gupta M. Goldsmith C.S. Huggins J.W. Nichol S.T. Virology. 2005; 332: 20-27Crossref PubMed Scopus (148) Google Scholar). Virus was incubated with cells at a multiplicity of infection equal to 1 for 1 h in the presence or absence of 800 nm of filovirus truncation variants or control protein. Virus was removed, cells were washed in PBS, and media and protein were replenished. After 48 h, cells were fixed in 10% neutral buffered formalin. After 3 days of fixation, cells were removed from the biosafety level 4 suite, and the percentage of GFP-expressing cells was measured with a Discovery-1 automated microscope (Molecular Devices Corp.) by measuring nine individual spots per well. MARV-Mus GP1 Truncation Variant 38–188-Fc Efficiently Binds to Filovirus-permissive Cells—The envelope glycoproteins of a number of viruses include discrete, independently folded domains that bind cellular receptors as efficiently as their entire ectodomain regions. We sought to identify similar RBDs of MARV-Mus and ZEBOV-May. To determine the location of the MARV-Mus GP1 RBD, we synthesized a codon-optimized gene encoding the full-length mature MARV-Mus GP1 protein fused to the Fc region of human immunoglobulin G1 at the C terminus (17–432-Fc). Four sets of seven truncation variants were created, starting at N-terminal residues 17, 38, 61, or 87 and ending at C-terminal residues 432, 308, 265, 230, 188, 167, or 134 (Fig. 1A). All 28 constructs expressed efficiently in 293T cells as Fc fusion proteins (Fig. 1B). Equivalent concentrations of each variant were incubated with MARV-Mus-permissive African green monkey kidney Vero E6 and human embryonic kidney 293T cells and with nonpermissive Jurkat lymphocytes (5Chan S.Y. Speck R.F. Ma M.C. Goldsmith M.A. J. Virol. 2000; 74: 4933-4937Crossref PubMed Scopus (125) Google Scholar), and cell-surface association was determined by flow cytometry (Fig. 2, A–C). The RBDs of the severe acute respiratory syndrome coronavirus (SARS-CoV) S protein (residues 318–510) and HIV-1 gp120, expressed as Fc fusion proteins (SARS-CoV RBD-Fc, gp120-Fc), were used as controls (36Choe H. Li W. Wright P.L. Vasilieva N. Venturi M. Huang C.C. Grundner C. Dorfman T. Zwick M.B. Wang L. Rosenberg E.S. Kwong P.D. Burton D.R. Robinson J.E. Sodroski J.G. Farzan M. Cell. 2003; 114: 161-170Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 37Wong S.K. Li W. Moore M.J. Choe H. Farzan M. J. Biol. Chem. 2004; 279: 3197-3201Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). As reported previously, SARS-CoV RBD-Fc efficiently bound SARS-CoV-permissive Vero E6 cells but not 293T cells or Jurkat lymphocytes (40Li W. Moore M.J. Vasilieva N. Sui J. Wong S.K. Berne M.A. Somasundaran M. Sullivan J.L. Luzuriaga K. Greenough T.C. Choe H. Farzan M. Nature. 2003; 426: 450-454Crossref PubMed Scopus (4289) Google Scholar). Also as expected, gp120-Fc bound CD4-expressing Jurkat lymphocytes but not Vero E6 or 293T cells. All 28 MARV-Mus proteins bound to Vero E6 and 293T cells with varying efficiencies, whereas little or no association was observed with Jurkat lymphocytes in most cases. Successive truncation of the C termini of MARV-Mus GP1 variants initiated with residues 17, 38, 61, or 87 led to successively increased cell-surface binding to Vero E6 cells, up through the C-terminal truncation at residue 188 (Fig. 2A). Further truncation beyond residue 188 decreased cell association. A single exception to this trend was observed with the 87–432-Fc variant, which bound Vero E6 cells with higher affinity than 87–308-Fc and 87–265-Fc. Variants initiated with residues 38, 61, and 87 bound more efficiently than those initiated with residue 17, with MARV-Mus-(38–188)-Fc consistently binding most efficiently to Vero E6 and 293T cells (Fig. 2B). These data identify a cell-binding region of MARV-Mus, located between GP1 residues 38 and 188.FIGURE 2Binding of MARV-Mus and ZEBOV-May GP1-Fc truncation variants to the surface of nonhuman primate and human cells. The indicated MARV-Mus (A–C) and ZEBOV-May GP1-Fc constructs (D–F) and control proteins were incubated with filovirus-permissive African green monkey kidney (Vero E6) cells (A and D), filovirus-permissive 293T cells (B and E), and filovirus-nonpermissive Jurkat lymphocytes (C and F) and analyzed by flow cytometry using an Fc-specific FITC-conjugated secondary antibody. Bars indicate mean fluorescence intensity (M.F.I.) averages of two or more experiments. Error bars indicate standard deviations.View Large Image Figure ViewerDownload Hi-res image Download (PPT) ZEBOV-May GP1 Truncation Variant 54–201-Fc Efficiently Binds to Filovirus-permissive Cells—Deletion of the mucin-like domain has been demonstrated to markedly increase efficiency of ZEBOV GP1,2-mediated infection (13Jeffers S.A. Sanders D.A. Sanchez A. J. Virol. 2002; 76: 12463-12472Crossref PubMed Scopus (199) Google Scholar, 16Yang Z.-Y. Duckers H.J. Sullivan N.J. Sanchez A. Nabel E.G. Nabel G.J. Nat. Med. 2000; 6: 886-889Crossref PubMed Scopus (348) Google Scholar, 17Manicassamy B. Wang J. Jiang H. Rong L. J. Virol. 2005; 79: 4793-4805Crossref PubMed Scopus (135) Google Scholar, 18Chandran K. Sullivan N.J. Felbor U. Whelan S.P. Cunningham J.M. Science. 2005; 308: 1643-1645Crossref PubMed Scopus (684) Google Scholar). To determine the location of the ZEBOV-May GP1 RBD, we synthesized a codon-optimized gene encoding the mature ZEBOV GP1 protein, lacking its mucin-like domain, and fused to the IgG1 Fc region (33–308-Fc). Three sets of four truncation variants were created, starting at N-terminal residues 33, 54, or 76 and ending at C-terminal residues 308, 201, 172, or 156 (Fig. 1C). With the exception of variant 76–172-Fc, all variants expressed efficiently (Fig. 1D). As with the MARV-Mus variants, equivalent concentrations of each variant were incubated with ZEBOV-May-permissive Vero E6 and 293T cells and with nonpermissive Jurkat lymphocytes, and cell association was again assayed by flow cytometry. All 11 ZEBOV-May GP1 variants bound to Vero E6 and 293T cells, whereas binding to Jurkat lymphocytes was negligible in all cases (Fig. 2, D–F). ZEBOV-May GP1 truncation variants showed a pattern of association to Vero E6 and 293T cells similar to that observed with MARV-Mus variants. In particular, 54–201-Fc and 76–201-Fc bound more efficiently than all other ZEBOV-May GP1 variants assayed, with 54–201-Fc binding slightly but consistently better than 76–201-Fc to Vero E6 cells (Fig. 2, D–E). These data identify a cell-binding region of ZEBOV-May, located between GP1 residues 54 and 201, which corresponds to the cell-binding region of MARV-Mus. MARV Strains Angola and Musoke GP1 Truncation Variants Bind to Filovirus-permissive Cells with Comparable Efficiency—The largest and most severe marburgvirus disease outbreak to date occurred in Angola in early 2005 (41Hovette P. Méd. Trop. (Mars.). 2005; 65: 127-128PubMed Google Scholar, 42World Health OrganizationWkly. Epidemiol. Rec. 2005; 80: 298Google Scholar). The envelope glycoprotein amino acid sequence of the strain responsible for this outbreak, MARV Angola (MARV-Ang), is homologous to that of the MARV-Mus strain (43Towner J.S. Khristova M.L. Sealy T.K. Vincent M.J. Erickson B.R. Bawiec D.A. Hartman A.L. Comer J.A. Zaki S.R. Ströher U. Gomes da Silva F. del Castillo F. Rollin P. Ksiazek T.G. Nichol S.T. J. Virol. 2006; (in press)Google Scholar). In particular, a comparison between MARV-Mus GP1 amino acid residues 38–188 with the corresponding region of MARV-Ang yielded only one amino acid change, threonine 74 to alanine (T74A). This alteration was introduced into four MARV-Mus GP1 truncation variants (MARV-Ang GP1-(38–188)-Fc, -(38–167)-Fc, -(61–188)-Fc, and -(61–167)-Fc; see Fig. 3A). Cell association of each of these variants was compared with those of MARV-Mus. Each MARV-Ang variant bound Vero E6 cells slightly less efficiently than its MARV-Mus counterpart (Fig. 3B). These data largely exclude the possibility that more efficient cellular association of the MARV-Ang cell-binding region contributes to increased severity of disease. Both MARV-Mus and ZEBOV-May GP1 Cell-binding Regions Inhibit Entry of Retroviruses Pseudotyped with the GP1,2 of Either Filovirus—To determine whether the identified GP1 cell-binding regions associated with factors necessary for infection, we assayed the ability of MARV-Mus-(38–188)-Fc and ZEBOV-May-(54–201)-Fc to inhibit entry of pseudotyped retroviruses. A Moloney murine leukemia virus vector expressing GFP was pseudotyped with the GP1,2 of MARV-Mus (MARV/MLV), a mucin-like domain-deleted GP1,2 of ZEBOV-May (ZEBOV/MLV), or with the G protein of vesicular stomatitis Indiana virus (VSV/MLV). Vero E6 cells were incubated with these pseudotyped retroviruses and varying concentrations of MARV-Mus-(38–188)-Fc, ZEBOV-May-(54–201)-Fc, or SARS-CoV RBD-Fc (Fig. 4, A–C). No Fc fusion protein inhibited VSV/MLV, but both MARV-Mus-(38–188)-Fc and ZEBOV-May-(54–201)-Fc efficiently inhibited both MARV/MLV and ZEBOV/MLV. SARS-CoV RBD-Fc did not inhibit infection of either pseudotyped virus. MARV-Mus-(38–188) was the more potent of the two cellular bindin" @default.
• W2139749521 created "2016-06-24" @default.
• W2139749521 creator A5010775046 @default.
• W2139749521 creator A5014510012 @default.
• W2139749521 creator A5014946505 @default.
• W2139749521 creator A5016215059 @default.
• W2139749521 creator A5030091007 @default.
• W2139749521 creator A5054720102 @default.
• W2139749521 creator A5061775143 @default.
• W2139749521 creator A5068707910 @default.
• W2139749521 creator A5074436491 @default.
• W2139749521 creator A5085274916 @default.
• W2139749521 creator A5090719481 @default.
• W2139749521 creator A5091344677 @default.
• W2139749521 date "2006-06-01" @default.
• W2139749521 modified "2023-09-27" @default.
• W2139749521 title "Conserved Receptor-binding Domains of Lake Victoria Marburgvirus and Zaire Ebolavirus Bind a Common Receptor" @default.
• W2139749521 cites W1789264759 @default.
• W2139749521 cites W1965435260 @default.
• W2139749521 cites W1966238900 @default.
• W2139749521 cites W1974662746 @default.
• W2139749521 cites W1974974166 @default.
• W2139749521 cites W1979071280 @default.
• W2139749521 cites W1981692189 @default.
• W2139749521 cites W1982187685 @default.
• W2139749521 cites W1992292664 @default.
• W2139749521 cites W1993048073 @default.
• W2139749521 cites W1993347536 @default.
• W2139749521 cites W2001349146 @default.
• W2139749521 cites W2002944156 @default.
• W2139749521 cites W2022339063 @default.
• W2139749521 cites W2025592853 @default.
• W2139749521 cites W2025864818 @default.
• W2139749521 cites W2040453631 @default.
• W2139749521 cites W2046542318 @default.
• W2139749521 cites W2055027989 @default.
• W2139749521 cites W2057513374 @default.
• W2139749521 cites W2067510101 @default.
• W2139749521 cites W2075604706 @default.
• W2139749521 cites W2080374377 @default.
• W2139749521 cites W2086155380 @default.
• W2139749521 cites W2089715049 @default.
• W2139749521 cites W2092664889 @default.
• W2139749521 cites W2097231877 @default.
• W2139749521 cites W2098576769 @default.
• W2139749521 cites W2106702228 @default.
• W2139749521 cites W2107717338 @default.
• W2139749521 cites W2108609481 @default.
• W2139749521 cites W2109176931 @default.
• W2139749521 cites W2113132163 @default.
• W2139749521 cites W2121472371 @default.
• W2139749521 cites W2134189877 @default.
• W2139749521 cites W2136729029 @default.
• W2139749521 cites W2140823754 @default.
• W2139749521 cites W2143312620 @default.
• W2139749521 cites W2146321853 @default.
• W2139749521 cites W2146476111 @default.
• W2139749521 cites W2146540261 @default.
• W2139749521 cites W2146991250 @default.
• W2139749521 cites W2147679296 @default.
• W2139749521 cites W2149789513 @default.
• W2139749521 cites W2156643487 @default.
• W2139749521 cites W2156897762 @default.
• W2139749521 cites W2164195236 @default.
• W2139749521 cites W4231286510 @default.
• W2139749521 doi "https://doi.org/10.1074/jbc.m601796200" @default.
• W2139749521 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16595665" @default.
• W2139749521 hasPublicationYear "2006" @default.
• W2139749521 type Work @default.
• W2139749521 sameAs 2139749521 @default.
• W2139749521 citedByCount "115" @default.
• W2139749521 countsByYear W21397495212012 @default.
• W2139749521 countsByYear W21397495212013 @default.
• W2139749521 countsByYear W21397495212014 @default.
• W2139749521 countsByYear W21397495212015 @default.
• W2139749521 countsByYear W21397495212016 @default.
• W2139749521 countsByYear W21397495212017 @default.
• W2139749521 countsByYear W21397495212018 @default.
• W2139749521 countsByYear W21397495212019 @default.
• W2139749521 countsByYear W21397495212020 @default.
• W2139749521 countsByYear W21397495212021 @default.
• W2139749521 countsByYear W21397495212022 @default.
• W2139749521 countsByYear W21397495212023 @default.
• W2139749521 crossrefType "journal-article" @default.
• W2139749521 hasAuthorship W2139749521A5010775046 @default.
• W2139749521 hasAuthorship W2139749521A5014510012 @default.
• W2139749521 hasAuthorship W2139749521A5014946505 @default.
• W2139749521 hasAuthorship W2139749521A5016215059 @default.
• W2139749521 hasAuthorship W2139749521A5030091007 @default.
• W2139749521 hasAuthorship W2139749521A5054720102 @default.
• W2139749521 hasAuthorship W2139749521A5061775143 @default.
• W2139749521 hasAuthorship W2139749521A5068707910 @default.
• W2139749521 hasAuthorship W2139749521A5074436491 @default.
• W2139749521 hasAuthorship W2139749521A5085274916 @default.
• W2139749521 hasAuthorship W2139749521A5090719481 @default.
• W2139749521 hasAuthorship W2139749521A5091344677 @default.
• W2139749521 hasBestOaLocation W21397495211 @default.
• W2139749521 hasConcept C107824862 @default.

• next