The genus Orthobunyavirus within the family Bunyaviridae constitutes an expanding group of emerging viruses, which threaten human and animal health. Despite the medical importance, little is known about orthobunyavirus structure, a prerequisite for understanding virus assembly and entry. Here, using electron cryo-tomography, we report the ultrastructure of Bunyamwera virus, the prototypic member of this genus. Whilst Bunyamwera virions are pleomorphic in shape, they display a locally ordered lattice of glycoprotein spikes. Each spike protrudes 18 nm from the viral membrane and becomes disordered upon introduction to an acidic environment. Using sub-tomogram averaging, we derived a three-dimensional model of the trimeric pre-fusion glycoprotein spike to 3-nm resolution. The glycoprotein spike consists mainly of the putative class-II fusion glycoprotein and exhibits a unique tripod-like arrangement. Protein–protein contacts between neighbouring spikes occur at membrane-proximal regions and intra-spike contacts at membrane-distal regions. This trimeric assembly deviates from previously observed fusion glycoprotein arrangements, suggesting a greater than anticipated repertoire of viral fusion glycoprotein oligomerization. Our study provides evidence of a pH-dependent conformational change that occurs during orthobunyaviral entry into host cells and a blueprint for the structure of this group of emerging pathogens.
References
[1]
Elliott RM (2008) Bunyaviruses: General Features. In: Mahy BWJ, M. Van Regenmortel, editor. Encyclopedia of Virology. 3rd ed. Oxford: Elsevier Academic Press. pp. 390–399.
[2]
Elliott RM, Blakqori G (2011) Molecular Biology of Orthobunyaviruses. In: Elliott RM, Plyusnin A, editors. Bunyaviridae Molecular and Cellular Biology. First ed. Great Britain: Caister Academic Press. pp. 1–40.
[3]
Nichol ST, Beaty BJ, Elliott RM, Goldbach R, Plyusnin A, et al.. (2005) Eighth Report of the International Committee on Taxonomy of Viruses. In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA, editors. Virus Taxonomy: Academic Press. pp. 695–716.
[4]
Vasconcelos HB, Nunes MR, Casseb LM, Carvalho VL, Pinto da Silva EV, et al. (2011) Molecular epidemiology of Oropouche virus, Brazil. Emerg Infect Dis 17: 800–806. doi: 10.3201/eid1705.101333
[5]
Tesh RB (1994) The emerging epidemiology of Venezuelan hemorrhagic fever and Oropouche fever in tropical South America. Ann N Y Acad Sci 740: 129–137. doi: 10.1111/j.1749-6632.1994.tb19863.x
[6]
Anderson CR, Spence L, Downs WG, Aitken TH (1961) Oropouche virus: a new human disease agent from Trinidad, West Indies. Am J Trop Med Hyg 10: 574–578.
[7]
McJunkin JE, de los Reyes EC, Irazuzta JE, Caceres MJ, Khan RR, et al. (2001) La Crosse encephalitis in children. N Engl J Med 344: 801–807. doi: 10.1056/nejm200103153441103
[8]
Haddow AD, Odoi A (2009) The incidence risk, clustering, and clinical presentation of La Crosse virus infections in the eastern United States, 2003–2007. PLoS One 4: e6145. doi: 10.1371/journal.pone.0006145
[9]
Gerhardt RR, Gottfried KL, Apperson CS, Davis BS, Erwin PC, et al. (2001) First isolation of La Crosse virus from naturally infected Aedes albopictus. Emerg Infect Dis 7: 807–811. doi: 10.3201/eid0705.017506
[10]
Lambert AJ, Blair CD, D'Anton M, Ewing W, Harborth M, et al. (2010) La Crosse virus in Aedes albopictus mosquitoes, Texas, USA, 2009. Emerg Infect Dis 16: 856–858. doi: 10.3201/eid1605.100170
[11]
Hoffmann B, Scheuch M, Hoper D, Jungblut R, Holsteg M, et al. (2012) Novel orthobunyavirus in Cattle, Europe, 2011. Emerg Infect Dis 18: 469–472. doi: 10.3201/eid1803.111905
[12]
Tarlinton R, Daly J, Dunham S, Kydd J (2012) The challenge of Schmallenberg virus emergence in Europe. Vet J 194: 10–18. doi: 10.1016/j.tvjl.2012.08.017
[13]
Garigliany MM, Bayrou C, Kleijnen D, Cassart D, Jolly S, et al. (2012) Schmallenberg virus: a new Shamonda/Sathuperi-like virus on the rise in Europe. Antiviral Res 95: 82–87. doi: 10.1016/j.antiviral.2012.05.014
[14]
Smithburn KC, Haddow AJ, Mahaffy AF (1946) A neurotropic virus isolated from Aedes mosquitoes caught in the Semliki forest. Am J Trop Med Hyg 26: 189–208.
[15]
Bridgen A, Elliott RM (1996) Rescue of a segmented negative-strand RNA virus entirely from cloned complementary DNAs. Proc Natl Acad Sci USA 93: 15400–15404. doi: 10.1073/pnas.93.26.15400
[16]
Shi X, Goli J, Clark G, Brauburger K, Elliott RM (2009) Functional analysis of the Bunyamwera orthobunyavirus Gc glycoprotein. J Gen Virol 90: 2483–2492. doi: 10.1099/vir.0.013540-0
[17]
Hollidge BS, Nedelsky NB, Salzano MV, Fraser JW, Gonzalez-Scarano F, et al. (2012) Orthobunyavirus entry into neurons and other mammalian cells occurs via clathrin-mediated endocytosis and requires trafficking into early endosomes. J Virol 86: 7988–8001. doi: 10.1128/jvi.00140-12
[18]
Ludwig GV, Israel BA, Christensen BM, Yuill TM, Schultz KT (1991) Monoclonal antibodies directed against the envelope glycoproteins of La Crosse virus. Microb Pathog 11: 411–421. doi: 10.1016/0882-4010(91)90037-b
[19]
Plassmeyer ML, Soldan SS, Stachelek KM, Roth SM, Martin-Garcia J, et al. (2007) Mutagenesis of the La Crosse Virus glycoprotein supports a role for Gc (1066–1087) as the fusion peptide. Virology 358: 273–282. doi: 10.1016/j.virol.2006.08.050
[20]
Garry CE, Garry RF (2004) Proteomics computational analyses suggest that the carboxyl terminal glycoproteins of Bunyaviruses are class II viral fusion protein (beta-penetrenes). Theor Biol Med Model 1: 10.
[21]
Pettersson RF, Melin L (1996) Synthesis, assembly, and intracellular transport of Bunyaviridae membrane proteins. In: Elliott RM, editor. The Bunyaviridae. New York, NY: Plenum Press. pp. 159–188.
[22]
Chothia C, Lesk AM (1986) The relation between the divergence of sequence and structure in proteins. EMBO J 5: 823–826.
[23]
Novoa RR, Calderita G, Cabezas P, Elliott RM, Risco C (2005) Key Golgi factors for structural and functional maturation of bunyamwera virus. J Virol 79: 10852–10863. doi: 10.1128/jvi.79.17.10852-10863.2005
[24]
Huiskonen JT, Overby AK, Weber F, Grunewald K (2009) Electron cryo-microscopy and single-particle averaging of Rift Valley fever virus: evidence for GN-GC glycoprotein heterodimers. J Virol 83: 3762–3769. doi: 10.1128/jvi.02483-08
[25]
Sherman MB, Freiberg AN, Holbrook MR, Watowich SJ (2009) Single-particle cryo-electron microscopy of Rift Valley fever virus. Virology 387: 11–15. doi: 10.1016/j.virol.2009.02.038
[26]
Overby AK, Pettersson RF, Grunewald K, Huiskonen JT (2008) Insights into bunyavirus architecture from electron cryotomography of Uukuniemi virus. Proc Natl Acad Sci USA 105: 2375–2379. doi: 10.1073/pnas.0708738105
[27]
Talmon Y, Prasad BV, Clerx JP, Wang GJ, Chiu W, et al. (1987) Electron microscopy of vitrified-hydrated La Crosse virus. J Virol 61: 2319–2321.
[28]
Martin ML, Lindsey-Regnery H, Sasso DR, McCormick JB, Palmer E (1985) Distinction between Bunyaviridae genera by surface structure and comparison with Hantaan virus using negative stain electron microscopy. Arch Virol 86: 17–28. doi: 10.1007/bf01314110
[29]
Huiskonen JT, Hepojoki J, Laurinmaki P, Vaheri A, Lankinen H, et al. (2010) Electron cryotomography of Tula hantavirus suggests a unique assembly paradigm for enveloped viruses. J Virol 84: 4889–4897. doi: 10.1128/jvi.00057-10
Baker TS, Olson NH, Fuller SD (1999) Adding the third dimension to virus life cycles: three-dimensional reconstruction of icosahedral viruses from cryo-electron micrographs. Microbiol Mol Biol Rev 63: 862–922. doi: 10.1128/mmbr.64.1.237-237.2000
[32]
Overby AK, Pettersson RF, Neve EP (2007) The glycoprotein cytoplasmic tail of Uukuniemi virus (Bunyaviridae) interacts with ribonucleoproteins and is critical for genome packaging. J Virol 81: 3198–3205. doi: 10.1128/jvi.02655-06
[33]
Obijeski JF, Bishop DH, Murphy FA, Palmer EL (1976) Structural proteins of La Crosse virus. J Virol 19: 985–997.
[34]
Dessau M, Modis Y (2013) Crystal structure of glycoprotein C from Rift Valley fever virus. Proc Natl Acad Sci U S A 110: 1696–1701. doi: 10.1073/pnas.1217780110
[35]
Vaney MC, Rey FA (2011) Class II enveloped viruses. Cell Microbiol 13: 1451–1459. doi: 10.1111/j.1462-5822.2011.01653.x
[36]
Kielian M (2006) Class II virus membrane fusion proteins. Virology 344: 38–47. doi: 10.1016/j.virol.2005.09.036
[37]
Freiberg AN, Sherman MB, Morais MC, Holbrook MR, Watowich SJ (2008) Three-dimensional organization of Rift Valley fever virus revealed by cryoelectron tomography. J Virol 82: 10341–13048. doi: 10.1128/jvi.01191-08
[38]
Bowden TA, Jones EY, Stuart DI (2011) Cells under siege: viral glycoprotein interactions at the cell surface. J Struct Biol 175: 120–126. doi: 10.1016/j.jsb.2011.03.016
[39]
Bowden TA, Crispin M, Jones EY, Stuart DI (2010) Shared paramyxoviral glycoprotein architecture is adapted for diverse attachment strategies. Biochem Soc Trans 38: 1349–1355. doi: 10.1042/bst0381349
[40]
Backovic M, Jardetzky TS (2011) Class III viral membrane fusion proteins. Adv Exp Med Biol 714: 91–101. doi: 10.1007/978-94-007-0782-5_3
[41]
Modis Y, Ogata S, Clements D, Harrison SC (2003) A ligand-binding pocket in the dengue virus envelope glycoprotein. Proc Natl Acad Sci USA 100: 6986–6991. doi: 10.1073/pnas.0832193100
[42]
Rey FA, Heinz FX, Mandl C, Kunz C, Harrison SC (1995) The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution. Nature 375: 291–298. doi: 10.1038/375291a0
[43]
Lamb RA, Jardetzky TS (2007) Structural basis of viral invasion: lessons from paramyxovirus F. Curr Opin Struct Biol 17: 427–436. doi: 10.1016/j.sbi.2007.08.016
[44]
Backovic M, Jardetzky TS (2009) Class III viral membrane fusion proteins. Curr Opin Struct Biol 19: 189–196. doi: 10.1016/j.sbi.2009.02.012
[45]
Tivol WF, Briegel A, Jensen GJ (2008) An improved cryogen for plunge freezing. Microsc Microanal 14: 375–379. doi: 10.1017/s1431927608080781
[46]
Sorzano CO, Marabini R, Velazquez-Muriel J, Bilbao-Castro JR, Scheres SH, et al. (2004) XMIPP: a new generation of an open-source image processing package for electron microscopy. J Struct Biol 148: 194–204. doi: 10.1016/j.jsb.2004.06.006
[47]
Heymann JB, Belnap DM (2007) Bsoft: image processing and molecular modeling for electron microscopy. J Struct Biol 157: 3–18. doi: 10.1016/j.jsb.2006.06.006
[48]
Heymann JB, Cardone G, Winkler DC, Steven AC (2008) Computational resources for cryo-electron tomography in Bsoft. J Struct Biol 161: 232–242. doi: 10.1016/j.jsb.2007.08.002
[49]
van Heel M, Harauz G, Orlova EV, Schmidt R, Schatz M (1996) A new generation of the IMAGIC image processing system. J Struct Biol 116: 17–24. doi: 10.1006/jsbi.1996.0004
[50]
Mastronarde DN (2005) Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152: 36–51. doi: 10.1016/j.jsb.2005.07.007
[51]
Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116: 71–76. doi: 10.1006/jsbi.1996.0013
[52]
Liljeroos L, Huiskonen JT, Ora A, Susi P, Butcher SJ (2011) Electron cryotomography of measles virus reveals how matrix protein coats the ribonucleocapsid within intact virions. Proc Natl Acad Sci USA 108: 18085–18090. doi: 10.1073/pnas.1105770108
[53]
Karotki L, Huiskonen JT, Stefan CJ, Ziolkowska NE, Roth R, et al. (2011) Eisosome proteins assemble into a membrane scaffold. J Cell Biol 195: 889–902. doi: 10.1083/jcb.201104040
[54]
Scheres SH, Chen S (2012) Prevention of overfitting in cryo-EM structure determination. Nat Methods 9: 853–854. doi: 10.1038/nmeth.2115
[55]
Hrabe T, Chen Y, Pfeffer S, Cuellar LK, Mangold AV, et al. (2012) PyTom: a python-based toolbox for localization of macromolecules in cryo-electron tomograms and subtomogram analysis. J Struct Biol 178: 177–188. doi: 10.1016/j.jsb.2011.12.003
[56]
Rosenthal PB, Henderson R (2003) Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J Mol Biol 333: 721–745. doi: 10.1016/j.jmb.2003.07.013
[57]
Castano-Diez D, Kudryashev M, Arheit M, Stahlberg H (2012) Dynamo: a flexible, user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments. J Struct Biol 178: 139–151. doi: 10.1016/j.jsb.2011.12.017
[58]
Scheres SH (2012) RELION: implementation of a Bayesian approach to cryo-EM structure determination. J Struct Biol 180: 519–530. doi: 10.1016/j.jsb.2012.09.006
[59]
Mindell JA, Grigorieff N (2003) Accurate determination of local defocus and specimen tilt in electron microscopy. J Struct Biol 142: 334–347. doi: 10.1016/s1047-8477(03)00069-8
