O-linked glycosylation is a ubiquitous protein modification in organisms belonging to several kingdoms. Both microbial and host protein glycans are used by many pathogens for host invasion and immune evasion, yet little is known about the roles of O-glycans in viral pathogenesis. Reportedly, there is no single function attributed to O-glycans for the significant paramyxovirus family. The paramyxovirus family includes many important pathogens, such as measles, mumps, parainfluenza, metapneumo- and the deadly Henipaviruses Nipah (NiV) and Hendra (HeV) viruses. Paramyxoviral cell entry requires the coordinated actions of two viral membrane glycoproteins: the attachment (HN/H/G) and fusion (F) glycoproteins. O-glycan sites in HeV G were recently identified, facilitating use of the attachment protein of this deadly paramyxovirus as a model to study O-glycan functions. We mutated the identified HeV G O-glycosylation sites and found mutants with altered cell-cell fusion, G conformation, G/F association, viral entry in a pseudotyped viral system, and, quite unexpectedly, pseudotyped viral F protein incorporation and processing phenotypes. These are all important functions of viral glycoproteins. These phenotypes were broadly conserved for equivalent NiV mutants. Thus our results identify multiple novel and pathologically important functions of paramyxoviral O-glycans, paving the way to study O-glycan functions in other paramyxoviruses and enveloped viruses.
References
[1]
Vigerust DJ, Shepherd VL (2007) Virus glycosylation: role in virulence and immune interactions. Trends Microbiol 15: 211–218. pmid:17398101 doi: 10.1016/j.tim.2007.03.003
[2]
Gerken TA (2004) Kinetic modeling confirms the biosynthesis of mucin core 1 (beta-Gal(1–3) alpha-GalNAc-O-Ser/Thr) O-glycan structures are modulated by neighboring glycosylation effects. Biochemistry 43: 4137–4142. pmid:15065856 doi: 10.1021/bi036306a
[3]
Tian E, Ten Hagen KG (2009) Recent insights into the biological roles of mucin-type O-glycosylation. Glycoconj J 26: 325–334. doi: 10.1007/s10719-008-9162-4. pmid:18695988
[4]
Van den Steen P, Rudd PM, Dwek RA, Opdenakker G (1998) Concepts and principles of O-linked glycosylation. Crit Rev Biochem Mol Biol 33: 151–208. pmid:9673446 doi: 10.1080/10409239891204198
[5]
Ono M, Hakomori S (2004) Glycosylation defining cancer cell motility and invasiveness. Glycoconj J 20: 71–78. pmid:14993838 doi: 10.1023/b:glyc.0000018019.22070.7d
[6]
Kim YJ, Varki A (1997) Perspectives on the significance of altered glycosylation of glycoproteins in cancer. Glycoconj J 14: 569–576. pmid:9298689
[7]
Ju T, Cummings RD (2005) Protein glycosylation: chaperone mutation in Tn syndrome. Nature 437: 1252. pmid:16251947 doi: 10.1038/4371252a
[8]
Iwashkiw JA, Seper A, Weber BS, Scott NE, Vinogradov E, et al. (2012) Identification of a general O-linked protein glycosylation system in Acinetobacter baumannii and its role in virulence and biofilm formation. PLoS Pathog 8: e1002758. doi: 10.1371/journal.ppat.1002758. pmid:22685409
[9]
Iwashkiw JA, Vozza NF, Kinsella RL, Feldman MF (2013) Pour some sugar on it: the expanding world of bacterial protein O-linked glycosylation. Mol Microbiol 89: 14–28. doi: 10.1111/mmi.12265. pmid:23679002
[10]
Abu-Qarn M, Eichler J, Sharon N (2008) Not just for Eukarya anymore: protein glycosylation in bacteria and archaea. Curr Opin Struct Biol 18: 544–550. doi: 10.1016/j.sbi.2008.06.010
[11]
Hu A, Cathomen T, Cattaneo R, Norrby E (1995) Influence of N-linked oligosaccharide chains on the processing, cell surface expression and function of the measles virus fusion protein. J Gen Virol 76 (Pt 3): 705–710. pmid:7897359 doi: 10.1099/0022-1317-76-3-705
[12]
Moll M, Kaufmann A, Maisner A (2004) Influence of N-glycans on processing and biological activity of the Nipah virus fusion protein. J Virol 78: 7274–7278. pmid:15194804 doi: 10.1128/jvi.78.13.7274-7278.2004
[13]
Segawa H, Yamashita T, Kawakita M, Taira H (2000) Functional analysis of the individual oligosaccharide chains of sendai virus fusion protein. J Biochem 128: 65–72. pmid:10876159 doi: 10.1093/oxfordjournals.jbchem.a022731
[14]
Aguilar HC, Matreyek KA, Filone CM, Hashimi ST, Levroney EL, et al. (2006) N-glycans on Nipah virus fusion protein protect against neutralization but reduce membrane fusion and viral entry. Journal of virology 80: 4878–4889. pmid:16641279 doi: 10.1128/jvi.80.10.4878-4889.2006
[15]
Biering SB, Huang A, Vu AT, Robinson LR, Bradel-Tretheway B, et al. (2012) N-Glycans on the Nipah virus attachment glycoprotein modulate fusion and viral entry as they protect against antibody neutralization. J Virol 86: 11991–12002. doi: 10.1128/JVI.01304-12. pmid:22915812
[16]
Bradel-Tretheway BG, Liu Q, Stone JA, McInally S, Aguilar HC (2015) Novel Functions of Hendra Virus G N-Glycans and Comparisons to Nipah Virus. J Virol. doi: 10.1128/jvi.00773-15
[17]
Carter JR, Pager CT, Fowler SD, Dutch RE (2005) Role of N-linked glycosylation of the Hendra virus fusion protein. J Virol 79: 7922–7925. pmid:15919949 doi: 10.1128/jvi.79.12.7922-7925.2005
[18]
Levroney EL, Aguilar HC, Fulcher JA, Kohatsu L, Pace KE, et al. (2005) Novel innate immune functions for galectin-1: galectin-1 inhibits cell fusion by Nipah virus envelope glycoproteins and augments dendritic cell secretion of proinflammatory cytokines. J Immunol 175: 413–420. pmid:15972675 doi: 10.4049/jimmunol.175.1.413
[19]
Garner OB, Yun T, Pernet O, Aguilar HC, Park A, et al. (2015) Timing of galectin-1 exposure differentially modulates Nipah virus entry and syncytium formation in endothelial cells. J Virol 89: 2520–2529. doi: 10.1128/JVI.02435-14. pmid:25505064
[20]
Garner OB, Aguilar HC, Fulcher JA, Levroney EL, Harrison R, et al. (2010) Endothelial galectin-1 binds to specific glycans on nipah virus fusion protein and inhibits maturation, mobility, and function to block syncytia formation. PLoS Pathog 6: e1000993. doi: 10.1371/journal.ppat.1000993. pmid:20657665
[21]
Gill DJ, Clausen H, Bard F (2011) Location, location, location: new insights into O-GalNAc protein glycosylation. Trends Cell Biol 21: 149–158. doi: 10.1016/j.tcb.2010.11.004. pmid:21145746
[22]
Machiels B, Lete C, Guillaume A, Mast J, Stevenson PG, et al. (2011) Antibody evasion by a gammaherpesvirus O-glycan shield. PLoS Pathog 7: e1002387. doi: 10.1371/journal.ppat.1002387. pmid:22114560
[23]
Wang J, Fan Q, Satoh T, Arii J, Lanier LL, et al. (2009) Binding of herpes simplex virus glycoprotein B (gB) to paired immunoglobulin-like type 2 receptor alpha depends on specific sialylated O-linked glycans on gB. J Virol 83: 13042–13045. doi: 10.1128/JVI.00792-09. pmid:19812165
[24]
Arii J, Wang J, Morimoto T, Suenaga T, Akashi H, et al. (2010) A single-amino-acid substitution in herpes simplex virus 1 envelope glycoprotein B at a site required for binding to the paired immunoglobulin-like type 2 receptor alpha (PILRalpha) abrogates PILRalpha-dependent viral entry and reduces pathogenesis. J Virol 84: 10773–10783. doi: 10.1128/JVI.01166-10. pmid:20686018
[25]
Mardberg K, Nystrom K, Tarp MA, Trybala E, Clausen H, et al. (2004) Basic amino acids as modulators of an O-linked glycosylation signal of the herpes simplex virus type 1 glycoprotein gC: functional roles in viral infectivity. Glycobiology 14: 571–581. pmid:15044392 doi: 10.1093/glycob/cwh075
[26]
Iversen MB Reinert L, Thomsen MK, Bagdonaite I, Nandakumar R, Cheshenko N, Prabakaran T, Vakhrushev SY, Krzyzowska M, Kratholm SK, Ruiz-Perez F, Petersen HH, Frische S, Holm CK, Paludan SR (2015) An innate antiviral pathway acting before interferons at epithelial surfaces. Nat Immunology. doi: 10.1038/ni.3319
[27]
Simmons G, Wool-Lewis RJ, Baribaud F, Netter RC, Bates P (2002) Ebola virus glycoproteins induce global surface protein down-modulation and loss of cell adherence. J Virol 76: 2518–2528. pmid:11836430 doi: 10.1128/jvi.76.5.2518-2528.2002
[28]
Bernstein BM, Gill JC (1993) Natural history and therapy of hepatitis B and C in patients with HIV disease. AIDS Clin Rev: 129–143. pmid:8217896
[29]
Overbaugh J, Rudensey LM (1992) Alterations in potential sites for glycosylation predominate during evolution of the simian immunodeficiency virus envelope gene in macaques. J Virol 66: 5937–5948. pmid:1527847
[30]
Feldmann H, Will C, Schikore M, Slenczka W, Klenk HD (1991) Glycosylation and oligomerization of the spike protein of Marburg virus. Virology 182: 353–356. pmid:2024471 doi: 10.1016/0042-6822(91)90680-a
[31]
Niemann H, Geyer R, Klenk HD, Linder D, Stirm S, et al. (1984) The carbohydrates of mouse hepatitis virus (MHV) A59: structures of the O-glycosidically linked oligosaccharides of glycoprotein E1. EMBO J 3: 665–670. pmid:6325180
[32]
Shida H, Dales S (1981) Biogenesis of vaccinia: carbohydrate of the hemagglutinin molecules. Virology 111: 56–72. pmid:7233832 doi: 10.1016/0042-6822(81)90653-x
[33]
Collins PL, Mottet G (1992) Oligomerization and post-translational processing of glycoprotein G of human respiratory syncytial virus: altered O-glycosylation in the presence of brefeldin A. J Gen Virol 73 (Pt 4): 849–863. pmid:1634876 doi: 10.1099/0022-1317-73-4-849
[34]
Ferreira L, Villar E, Munoz-Barroso I (2004) Gangliosides and N-glycoproteins function as Newcastle disease virus receptors. Int J Biochem Cell Biol 36: 2344–2356. pmid:15313478 doi: 10.1016/j.biocel.2004.05.011
[35]
Lamb RA, Paterson RG, Jardetzky TS (2006) Paramyxovirus membrane fusion: lessons from the F and HN atomic structures. Virology 344: 30–37. pmid:16364733 doi: 10.1016/j.virol.2005.09.007
[36]
Luby SP, Hossain MJ, Gurley ES, Ahmed BN, Banu S, et al. (2009) Recurrent zoonotic transmission of Nipah virus into humans, Bangladesh, 2001–2007. Emerg Infect Dis 15: 1229–1235. doi: 10.3201/eid1508.081237. pmid:19751584
[37]
Chua KB, Bellini WJ, Rota PA, Harcourt BH, Tamin A, et al. (2000) Nipah virus: a recently emergent deadly paramyxovirus. Science 288: 1432–1435. pmid:10827955 doi: 10.1126/science.288.5470.1432
[38]
Chang A, Dutch RE (2012) Paramyxovirus fusion and entry: multiple paths to a common end. Viruses 4: 613–636. doi: 10.3390/v4040613. pmid:22590688
[39]
Negrete OA, Levroney EL, Aguilar HC, Bertolotti-Ciarlet A, Nazarian R, et al. (2005) EphrinB2 is the entry receptor for Nipah virus, an emergent deadly paramyxovirus. Nature 436: 401–405. pmid:16007075 doi: 10.1038/nature03838
[40]
Bonaparte MI, Dimitrov AS, Bossart KN, Crameri G, Mungall BA, et al. (2005) Ephrin-B2 ligand is a functional receptor for Hendra virus and Nipah virus. Proc Natl Acad Sci U S A 102: 10652–10657. pmid:15998730 doi: 10.1073/pnas.0504887102
[41]
Negrete OA, Wolf MC, Aguilar HC, Enterlein S, Wang W, et al. (2006) Two key residues in ephrinB3 are critical for its use as an alternative receptor for Nipah virus. PLoS Pathog 2: e7. pmid:16477309 doi: 10.1371/journal.ppat.0020007
[42]
Bowden TA, Aricescu AR, Gilbert RJ, Grimes JM, Jones EY, et al. (2008) Structural basis of Nipah and Hendra virus attachment to their cell-surface receptor ephrin-B2. Nat Struct Mol Biol 15: 567–572. doi: 10.1038/nsmb.1435. pmid:18488039
[43]
Zhu Q, Biering SB, Mirza AM, Grasseschi BA, Mahon PJ, et al. (2013) Individual N-glycans added at intervals along the stalk of the Nipah virus G protein prevent fusion but do not block the interaction with the homologous F protein. J Virol 87: 3119–3129. doi: 10.1128/JVI.03084-12. pmid:23283956
[44]
Melanson VR, Iorio RM (2006) Addition of N-glycans in the stalk of the Newcastle disease virus HN protein blocks its interaction with the F protein and prevents fusion. J Virol 80: 623–633. pmid:16378965 doi: 10.1128/jvi.80.2.623-633.2006
[45]
Maar D, Harmon B, Chu D, Schulz B, Aguilar HC, et al. (2012) Cysteines in the stalk of the nipah virus G glycoprotein are located in a distinct subdomain critical for fusion activation. J Virol 86: 6632–6642. doi: 10.1128/JVI.00076-12. pmid:22496210
[46]
Bose S, Song AS, Jardetzky TS, Lamb RA (2014) Fusion activation through attachment protein stalk domains indicates a conserved core mechanism of paramyxovirus entry into cells. J Virol 88: 3925–3941. doi: 10.1128/JVI.03741-13. pmid:24453369
[47]
Bose S, Zokarkar A, Welch BD, Leser GP, Jardetzky TS, et al. (2012) Fusion activation by a headless parainfluenza virus 5 hemagglutinin-neuraminidase stalk suggests a modular mechanism for triggering. Proc Natl Acad Sci U S A 109: E2625–2634. pmid:22949640 doi: 10.1073/pnas.1213813109
[48]
Brindley MA, Suter R, Schestak I, Kiss G, Wright ER, et al. (2013) A stabilized headless measles virus attachment protein stalk efficiently triggers membrane fusion. J Virol 87: 11693–11703. doi: 10.1128/JVI.01945-13. pmid:23966411
[49]
Liu Q, Stone JA, Bradel-Tretheway B, Dabundo J, Benavides Montano JA, et al. (2013) Unraveling a three-step spatiotemporal mechanism of triggering of receptor-induced Nipah virus fusion and cell entry. PLoS Pathog 9: e1003770. doi: 10.1371/journal.ppat.1003770. pmid:24278018
[50]
Colgrave ML, Snelling HJ, Shiell BJ, Feng YR, Chan YP, et al. (2012) Site occupancy and glycan compositional analysis of two soluble recombinant forms of the attachment glycoprotein of Hendra virus. Glycobiology 22: 572–584. doi: 10.1093/glycob/cwr180. pmid:22171062
[51]
Negrete OA, Chu D, Aguilar HC, Lee B (2007) Single amino acid changes in the Nipah and Hendra virus attachment glycoproteins distinguish ephrinB2 from ephrinB3 usage. J Virol 81: 10804–10814. pmid:17652392 doi: 10.1128/jvi.00999-07
Aguilar HC, Aspericueta V, Robinson LR, Aanensen KE, Lee B (2010) A quantitative and kinetic fusion protein-triggering assay can discern distinct steps in the nipah virus membrane fusion cascade. J Virol 84: 8033–8041. doi: 10.1128/JVI.00469-10. pmid:20519383
[54]
Pager CT, Craft WW Jr., Patch J, Dutch RE (2006) A mature and fusogenic form of the Nipah virus fusion protein requires proteolytic processing by cathepsin L. Virology 346: 251–257. pmid:16460775 doi: 10.1016/j.virol.2006.01.007
[55]
Pager CT, Dutch RE (2005) Cathepsin L is involved in proteolytic processing of the Hendra virus fusion protein. J Virol 79: 12714–12720. pmid:16188974 doi: 10.1128/jvi.79.20.12714-12720.2005
[56]
Aguilar HC, Ataman ZA, Aspericueta V, Fang AQ, Stroud M, et al. (2009) A Novel Receptor-induced Activation Site in the Nipah Virus Attachment Glycoprotein (G) Involved in Triggering the Fusion Glycoprotein (F). J Biol Chem 284: 1628–1635. doi: 10.1074/jbc.M807469200. pmid:19019819
[57]
Apte-Sengupta S, Navaratnarajah CK, Cattaneo R (2013) Hydrophobic and charged residues in the central segment of the measles virus hemagglutinin stalk mediate transmission of the fusion-triggering signal. J Virol 87: 10401–10404. doi: 10.1128/JVI.01547-13. pmid:23864629
[58]
Bose S, Welch BD, Kors CA, Yuan P, Jardetzky TS, et al. (2011) Structure and mutagenesis of the parainfluenza virus 5 hemagglutinin-neuraminidase stalk domain reveals a four-helix bundle and the role of the stalk in fusion promotion. J Virol 85: 12855–12866. doi: 10.1128/JVI.06350-11. pmid:21994464
[59]
Navaratnarajah CK, Oezguen N, Rupp L, Kay L, Leonard VH, et al. (2011) The heads of the measles virus attachment protein move to transmit the fusion-triggering signal. Nat Struct Mol Biol 18: 128–134. doi: 10.1038/nsmb.1967. pmid:21217701
[60]
Paal T, Brindley MA, St Clair C, Prussia A, Gaus D, et al. (2009) Probing the spatial organization of measles virus fusion complexes. J Virol 83: 10480–10493. doi: 10.1128/JVI.01195-09. pmid:19656895
[61]
Aguilar HC, Matreyek KA, Choi DY, Filone CM, Young S, et al. (2007) Polybasic KKR motif in the cytoplasmic tail of Nipah virus fusion protein modulates membrane fusion by inside-out signaling. J Virol 81: 4520–4532. pmid:17301148 doi: 10.1128/jvi.02205-06
[62]
Patch JR, Crameri G, Wang LF, Eaton BT, Broder CC (2007) Quantitative analysis of Nipah virus proteins released as virus-like particles reveals central role for the matrix protein. Virol J 4: 1. pmid:17204159
[63]
Whitman SD, Smith EC, Dutch RE (2009) Differential rates of protein folding and cellular trafficking for the Hendra virus F and G proteins: implications for F-G complex formation. J Virol 83: 8998–9001. doi: 10.1128/JVI.00414-09. pmid:19553334
[64]
Liu Q, Bradel-Tretheway B, Monreal AI, Saludes JP, Lu X, et al. (2015) Nipah virus attachment glycoprotein stalk C-terminal region links receptor binding to fusion triggering. J Virol 89: 1838–1850. doi: 10.1128/JVI.02277-14. pmid:25428863
[65]
Landowski M, Dabundo J, Liu Q, Nicola AV, Aguilar HC (2014) Nipah virion entry kinetics, composition, and conformational changes determined by enzymatic virus-like particles and new flow virometry tools. J Virol 88: 14197–14206. doi: 10.1128/JVI.01632-14. pmid:25275126
[66]
Monaghan P, Green D, Pallister J, Klein R, White J, et al. (2014) Detailed morphological characterisation of Hendra virus infection of different cell types using super-resolution and conventional imaging. Virol J 11: 200. doi: 10.1186/s12985-014-0200-5. pmid:25428656
[67]
Lodish H, Berk A, Zipursky SL (2000) Protein Glycosylation in the ER and Golgi Complex. Molecular Cell Biology. 4 ed. New York: W.H. Freeman.
