UNLABELLED: The western honeybee (Apis mellifera) is the most important commercial insect pollinator. However, bees are under pressure from habitat loss, environmental stress, and pathogens, including viruses that can cause lethal epidemics. Slow bee paralysis virus (SBPV) belongs to the Iflaviridae family of nonenveloped single-stranded RNA viruses. Here we present the structure of the SBPV virion determined from two crystal forms to resolutions of 3.4 Å and 2.6 Å. The overall structure of the virion resembles that of picornaviruses, with the three major capsid proteins VP1 to 3 organized into a pseudo-T3 icosahedral capsid. However, the SBPV capsid protein VP3 contains a C-terminal globular domain that has not been observed in other viruses from the order Picornavirales The protruding (P) domains form "crowns" on the virion surface around each 5-fold axis in one of the crystal forms. However, the P domains are shifted 36 Å toward the 3-fold axis in the other crystal form. Furthermore, the P domain contains the Ser-His-Asp triad within a surface patch of eight conserved residues that constitutes a putative catalytic or receptor-binding site. The movements of the domain might be required for efficient substrate cleavage or receptor binding during virus cell entry. In addition, capsid protein VP2 contains an RGD sequence that is exposed on the virion surface, indicating that integrins might be cellular receptors of SBPV. IMPORTANCE: Pollination by honeybees is needed to sustain agricultural productivity as well as the biodiversity of wild flora. However, honeybee populations in Europe and North America have been declining since the 1950s. Honeybee viruses from the Iflaviridae family are among the major causes of honeybee colony mortality. We determined the virion structure of an Iflavirus, slow bee paralysis virus (SBPV). SBPV exhibits unique structural features not observed in other picorna-like viruses. The SBPV capsid protein VP3 has a large C-terminal domain, five of which form highly prominent protruding "crowns" on the virion surface. However, the domains can change their positions depending on the conditions of the environment. The domain includes a putative catalytic or receptor binding site that might be important for SBPV cell entry.

Department of Ecology Swedish University of Agricultural Sciences Uppsala Uppsala Sweden

Department of Zoology Fishery Hydrobiology and Apidology Faculty of Agronomy Mendel University in Brno Brno Czech Republic

Structural Virology Central European Institute of Technology Masaryk University Brno Czech Republic

Zobrazit více v PubMed

Allsopp MH, de Lange WJ, Veldtman R. 2008. Valuing insect pollination services with cost of replacement. PLoS One 3:e3128. doi:10.1371/journal.pone.0003128. PubMed DOI PMC

Biesmeijer JC, Roberts SP, Reemer M, Ohlemuller R, Edwards M, Peeters T, Schaffers AP, Potts SG, Kleukers R, Thomas CD, Settele J, Kunin WE. 2006. Parallel declines in pollinators and insect-pollinated plants in Britain and the Netherlands. Science 313:351–354. doi:10.1126/science.1127863. PubMed DOI

Vanengelsdorp D, Meixner MD. 2010. A historical review of managed honey bee populations in Europe and the United States and the factors that may affect them. J Invertebr Pathol 103(Suppl 1):S80–S95. PubMed

Dainat B, Vanengelsdorp D, Neumann P. 2012. Colony collapse disorder in Europe. Environ Microbiol Rep 4:123–125. doi:10.1111/j.1758-2229.2011.00312.x. PubMed DOI

van Engelsdorp D, Hayes J Jr, Underwood RM, Pettis J. 2008. A survey of honey bee colony losses in the U.S., fall 2007 to spring 2008. PLoS One 3:e4071. doi:10.1371/journal.pone.0004071. PubMed DOI PMC

Smith KM, Loh EH, Rostal MK, Zambrana-Torrelio CM, Mendiola L, Daszak P. 2013. Pathogens, pests, and economics: drivers of honey bee colony declines and losses. Ecohealth 10:434–445. doi:10.1007/s10393-013-0870-2. PubMed DOI

Chen YP, Siede R. 2007. Honey bee viruses. Adv Virus Res 70:33–80. doi:10.1016/S0065-3527(07)70002-7. PubMed DOI

Vanengelsdorp D, Evans JD, Saegerman C, Mullin C, Haubruge E, Nguyen BK, Frazier M, Frazier J, Cox-Foster D, Chen Y, Underwood R, Tarpy DR, Pettis JS. 2009. Colony collapse disorder: a descriptive study. PLoS One 4:e6481. doi:10.1371/journal.pone.0006481. PubMed DOI PMC

Williams GR, Tarpy DR, vanEngelsdorp D, Chauzat MP, Cox-Foster DL, Delaplane KS, Neumann P, Pettis JS, Rogers RE, Shutler D. 2010. Colony collapse disorder in context. Bioessays 32:845–846. doi:10.1002/bies.201000075. PubMed DOI PMC

Berthound HIA, Haueter M, Radloff S, Neumann P. 2010. Virus infections and winter losses of honey bee colonies (Apis mellifera). J Apic Res 49:60–65. doi:10.3896/IBRA.1.49.1.08. DOI

Genersh EvdO W, Kaatz H, Schroeder A, Otten C, Buchler R, Berg S, Ritter W, Muhlen W, Gisder S, Meixner M, Leibig G, Rosenkranz P. 2010. The German bee monitoring project: a long term study to understand periodically high winter losses of honey bee colonies. Adipologie 41:332–352.

Bailey L, Woods RD. 1974. Three previously undescribed viruses from the honey bee. J Gen Virol 25:175–186. doi:10.1099/0022-1317-25-2-175. PubMed DOI

Carreck NL, B B, Wilson JK, Allen MF. 2005. The epodemiology of slow paralysis virus in honey bee colonies infested by Varroa destructor in the UK, p 32–33. Proceedings of XXXIXth International Apicultural Congress, Dublin, Ireland.

Santillan-Galicia MT, Ball BV, Clark SJ, Alderson PG. 2010. Transmission of deformed wing virus and slow paralysis virus to adult bees (Apis mellifera L.) by Varroa destructor. J Apic Res 49:141–148. doi:10.3896/IBRA.1.49.2.01. DOI

de Miranda JR, Dainat B, Locke B, Cordoni G, Berthoud H, Gauthier L, Neumann P, Budge GE, Ball BV, Stoltz DB. 2010. Genetic characterization of slow bee paralysis virus of the honeybee (Apis mellifera L.). J Gen Virol 91:2524–2530. doi:10.1099/vir.0.022434-0. PubMed DOI

Fürst MA, McMahon DP, Osborne JL, Paxton RJ, Brown MJ. 2014. Disease associations between honeybees and bumblebees as a threat to wild pollinators. Nature 506:364–366. doi:10.1038/nature12977. PubMed DOI PMC

McMahon DP, Furst MA, Caspar J, Theodorou P, Brown MJ, Paxton RJ. 3 March 2015. A sting in the spit: widespread cross-infection of multiple RNA viruses across wild and managed bees. J Anim Ecol doi:10.1111/1365-2656.12345. PubMed DOI PMC

Le Gall O, Christian P, Fauquet CM, King AM, Knowles NJ, Nakashima N, Stanway G, Gorbalenya AE. 2008. Picornavirales, a proposed order of positive-sense single-stranded RNA viruses with a pseudo-T =3 virion architecture. Arch Virol 153:715–727. doi:10.1007/s00705-008-0041-x. PubMed DOI

Zhang J, Feng J, Liang Y, Chen D, Zhou ZH, Zhang Q, Lu X. 2001. Three-dimensional structure of the Chinese Sacbrood bee virus. Sci China C Life Sci 44:443–448. doi:10.1007/BF02879612. PubMed DOI

Lanzi G, de Miranda JR, Boniotti MB, Cameron CE, Lavazza A, Capucci L, Camazine SM, Rossi C. 2006. Molecular and biological characterization of deformed wing virus of honeybees (Apis mellifera L.). J Virol 80:4998–5009. doi:10.1128/JVI.80.10.4998-5009.2006. PubMed DOI PMC

Geng P, Li W, Lin L, de Miranda JR, Emrich S, An L, Terenius O. 2014. Genetic characterization of a novel iflavirus associated with vomiting disease in the Chinese oak silkmoth Antheraea pernyi. PLoS One 9:e92107. doi:10.1371/journal.pone.0092107. PubMed DOI PMC

de Miranda JR, Bailey L, Ball BV, Blanchard P, Budge G, Chejanovsky N, Chen Y-P, Gauthier L, Genersch E, De Graaf D, Ribière M, Ryabov E, De Smet L, van der Steen JJM. 2013. Standard methods for Apis mellifera pest and pathogen research. In Dietemann V, Ellis JD, Neumann P (ed), The COLOSS BEEBOOK, vol II IBRA, Treforest, United Kingdom.

Newman J. 2006. A review of techniques for maximizing diffraction from a protein crystal in stilla. Acta Crystallogr D Biol Crystallogr 62:27–31. doi:10.1107/S0907444905032130. PubMed DOI

Tong L, Rossmann MG. 1997. Rotation function calculations with GLRF program. Methods Enzymol 276:594–611. doi:10.1016/S0076-6879(97)76080-4. PubMed DOI

Brunger AT. 2007. Version 1.2 of the Crystallography and NMR system. Nat Protoc 2:2728–2733. doi:10.1038/nprot.2007.406. PubMed DOI

Kleywegt GJ, Read RJ. 1997. Not your average density. Structure 5:1557–1569. doi:10.1016/S0969-2126(97)00305-5. PubMed DOI

Kleywegt GJ, Jones TA. 1999. Software for handling macromolecular envelopes. Acta Crystallogr D Biol Crystallogr 55:941–944. doi:10.1107/S0907444999001031. PubMed DOI

Kleywegt GJ. 1999. Experimental assessment of differences between related protein crystal structures. Acta Crystallogr D Biol Crystallogr 55:1878–1884. doi:10.1107/S0907444999010495. PubMed DOI

Cowtan K. 2006. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr D Biol Crystallogr 62:1002–1011. doi:10.1107/S0907444906022116. PubMed DOI

Cowtan K. 2008. Fitting molecular fragments into electron density. Acta Crystallogr D Biol Crystallogr 64:83–89. doi:10.1107/S0907444907033938. PubMed DOI PMC

Jones TA, Zou JY, Cowan SW, Kjeldgaard M. 1991. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A 47(Part 2):110–119. PubMed

Emsley P, Cowtan K. 2004. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60:2126–2132. doi:10.1107/S0907444904019158. PubMed DOI

Afonine PV, Grosse-Kunstleve RW, Echols N, Headd JJ, Moriarty NW, Mustyakimov M, Terwilliger TC, Urzhumtsev A, Zwart PH, Adams PD. 2012. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr D Biol Crystallogr 68:352–367. doi:10.1107/S0907444912001308. PubMed DOI PMC

McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ. 2007. Phaser crystallographic software. J Appl Crystallogr 40:658–674. doi:10.1107/S0021889807021206. PubMed DOI PMC

Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie AG, McCoy A, McNicholas SJ, Murshudov GN, Pannu NS, Potterton EA, Powell HR, Read RJ, Vagin A, Wilson KS. 2011. Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr 67:235–242. doi:10.1107/S0907444910045749. PubMed DOI PMC

Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL. 1998. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54:905–921. PubMed

Rossmann MG, Arnold E, Erickson JW, Frankenberger EA, Griffith JP, Hecht HJ, Johnson JE, Kamer G, Luo M, Mosser AG, Rueckert RR, Sherry B, Vriend G. 1985. Structure of a human common cold virus and functional relationship to other picornaviruses. Nature 317:145–153. doi:10.1038/317145a0. PubMed DOI

Katpally U, Voss NR, Cavazza T, Taube S, Rubin JR, Young VL, Stuckey J, Ward VK, Virgin HW IV, Wobus CE, Smith TJ. 2010. High-resolution cryo-electron microscopy structures of murine norovirus 1 and rabbit hemorrhagic disease virus reveal marked flexibility in the receptor binding domains. J Virol 84:5836–5841. doi:10.1128/JVI.00314-10. PubMed DOI PMC

Taube S, Rubin JR, Katpally U, Smith TJ, Kendall A, Stuckey JA, Wobus CE. 2010. High-resolution x-ray structure and functional analysis of the murine norovirus 1 capsid protein protruding domain. J Virol 84:5695–5705. doi:10.1128/JVI.00316-10. PubMed DOI PMC

Prasad BV, Hardy ME, Dokland T, Bella J, Rossmann MG, Estes MK. 1999. X-ray crystallographic structure of the Norwalk virus capsid. Science 286:287–290. doi:10.1126/science.286.5438.287. PubMed DOI

Bergelson JM, Coyne CB. 2013. Picornavirus entry. Adv Exp Med Biol 790:24–41. doi:10.1007/978-1-4614-7651-1_2. PubMed DOI

Fuchs R, Blaas D. 2012. Productive entry pathways of human rhinoviruses. Adv Virol 2012:826301. PubMed PMC

Dodson G, Wlodawer A. 1998. Catalytic triads and their relatives. Trends Biochem Sci 23:347–352. doi:10.1016/S0968-0004(98)01254-7. PubMed DOI

Canaan S, Roussel A, Verger R, Cambillau C. 1999. Gastric lipase: crystal structure and activity. Biochim Biophys Acta 1441:197–204. doi:10.1016/S1388-1981(99)00160-2. PubMed DOI

Vaquero ME, Barriuso J, Martinez MJ, Prieto A. 2016. Properties, structure, and applications of microbial sterol esterases. Appl Microbiol Biotechnol 100:2047–2061. doi:10.1007/s00253-015-7258-x. PubMed DOI

Ongus JR, Peters D, Bonmatin JM, Bengsch E, Vlak JM, van Oers MM. 2004. Complete sequence of a picorna-like virus of the genus Iflavirus replicating in the mite Varroa destructor. J Gen Virol 85:3747–3755. doi:10.1099/vir.0.80470-0. PubMed DOI

Fujiyuki T, Takeuchi H, Ono M, Ohka S, Sasaki T, Nomoto A, Kubo T. 2004. Novel insect picorna-like virus identified in the brains of aggressive worker honeybees. J Virol 78:1093–1100. doi:10.1128/JVI.78.3.1093-1100.2004. PubMed DOI PMC

Bailey L, Gibbs AJ, Woods RD. 1964. Sacbrood virus of the larval honey bee (Apis Mellifera Linnaeus). Virology 23:425–429. doi:10.1016/0042-6822(64)90266-1. PubMed DOI

Wu CY, Lo CF, Huang CJ, Yu HT, Wang CH. 2002. The complete genome sequence of Perina nuda picorna-like virus, an insect-infecting RNA virus with a genome organization similar to that of the mammalian picornaviruses. Virology 294:312–323. doi:10.1006/viro.2001.1344. PubMed DOI

Farr GA, Zhang LG, Tattersall P. 2005. Parvoviral virions deploy a capsid-tethered lipolytic enzyme to breach the endosomal membrane during cell entry. Proc Natl Acad Sci U S A 102:17148–17153. doi:10.1073/pnas.0508477102. PubMed DOI PMC

Cao S, Lou Z, Tan M, Chen Y, Liu Y, Zhang Z, Zhang XC, Jiang X, Li X, Rao Z. 2007. Structural basis for the recognition of blood group trisaccharides by norovirus. J Virol 81:5949–5957. doi:10.1128/JVI.00219-07. PubMed DOI PMC

Hansman GS, Shahzad-Ul-Hussan S, McLellan JS, Chuang GY, Georgiev I, Shimoike T, Katayama K, Bewley CA, Kwong PD. 2012. Structural basis for norovirus inhibition and fucose mimicry by citrate. J Virol 86:284–292. doi:10.1128/JVI.05909-11. PubMed DOI PMC

Holm L, Rosenstrom P. 2010. Dali server: conservation mapping in 3D. Nucleic Acids Res 38:W545–W549. doi:10.1093/nar/gkq366. PubMed DOI PMC

Morgunova E, Dauter Z, Fry E, Stuart DI, Stel'mashchuk V, Mikhailov AM, Wilson KS, Vainshtein BK. 1994. The atomic structure of carnation mottle virus capsid protein. FEBS Lett 338:267–271. doi:10.1016/0014-5793(94)80281-5. PubMed DOI

York RL, Yousefi PA, Bogdanoff W, Haile S, Tripathi S, DuBois RM. 2015. Structural, mechanistic, and antigenic characterization of the human astrovirus capsid. J Virol 90:2254–2263. PubMed PMC

Chen NC, Yoshimura M, Guan HH, Wang TY, Misumi Y, Lin CC, Chuankhayan P, Nakagawa A, Chan SI, Tsukihara T, Chen TY, Chen CJ. 2015. Crystal structures of a piscine betanodavirus: mechanisms of capsid assembly and viral infection. PLoS Pathog 11:e1005203. doi:10.1371/journal.ppat.1005203. PubMed DOI PMC

Guo YR, Hryc CF, Jakana J, Jiang H, Wang D, Chiu W, Zhong W, Tao YJ. 2014. Crystal structure of a nematode-infecting virus. Proc Natl Acad Sci U S A 111:12781–12786. doi:10.1073/pnas.1407122111. PubMed DOI PMC

Jackson T, Mould AP, Sheppard D, King AM. 2002. Integrin alphavbeta1 is a receptor for foot-and-mouth disease virus. J Virol 76:935–941. doi:10.1128/JVI.76.3.935-941.2002. PubMed DOI PMC

Boonyakiat Y, Hughes PJ, Ghazi F, Stanway G. 2001. Arginine-glycine-aspartic acid motif is critical for human parechovirus 1 entry. J Virol 75:10000–10004. doi:10.1128/JVI.75.20.10000-10004.2001. PubMed DOI PMC

Kalynych S, Palkova L, Plevka P. 2016. The structure of human parechovirus 1 reveals an association of the RNA genome with the capsid. J Virol 90:1377–1386. doi:10.1128/JVI.02346-15. PubMed DOI PMC

Weinstock GM, et al. 2006. Insights into social insects from the genome of the honeybee Apis mellifera. Nature 443:931–949. doi:10.1038/nature05260. PubMed DOI PMC

Groppelli E, Tuthill TJ, Rowlands DJ. 2010. Cell entry of the aphthovirus equine rhinitis A virus is dependent on endosome acidification. J Virol 84:6235–6240. doi:10.1128/JVI.02375-09. PubMed DOI PMC

Bakker SE, Groppelli E, Pearson AR, Stockley PG, Rowlands DJ, Ranson NA. 2014. Limits of structural plasticity in a picornavirus capsid revealed by a massively expanded equine rhinitis A virus particle. J Virol 88:6093–6099. doi:10.1128/JVI.01979-13. PubMed DOI PMC

Garriga D, Pickl-Herk A, Luque D, Wruss J, Caston JR, Blaas D, Verdaguer N. 2012. Insights into minor group rhinovirus uncoating: the X-ray structure of the HRV2 empty capsid. PLoS Pathog 8:e1002473. doi:10.1371/journal.ppat.1002473. PubMed DOI PMC

Levy HC, Bostina M, Filman DJ, Hogle JM. 2010. Catching a virus in the act of RNA release: a novel poliovirus uncoating intermediate characterized by cryo-electron microscopy. J Virol 84:4426–4441. doi:10.1128/JVI.02393-09. PubMed DOI PMC

Bostina M, Levy H, Filman DJ, Hogle JM. 2011. Poliovirus RNA is released from the capsid near a twofold symmetry axis. J Virol 85:776–783. doi:10.1128/JVI.00531-10. PubMed DOI PMC

Tate J, Liljas L, Scotti P, Christian P, Lin T, Johnson JE. 1999. The crystal structure of cricket paralysis virus: the first view of a new virus family. Nat Struct Biol 6:765–774. doi:10.1038/11543. PubMed DOI

Squires G, Pous J, Agirre J, Rozas-Dennis GS, Costabel MD, Marti GA, Navaza J, Bressanelli S, Guerin DM, Rey FA. 2013. Structure of the Triatoma virus capsid. Acta Crystallogr D Biol Crystallogr 69:1026–1037. doi:10.1107/S0907444913004617. PubMed DOI PMC

Agirre J, Aloria K, Arizmendi JM, Iloro I, Elortza F, Sanchez-Eugenia R, Marti GA, Neumann E, Rey FA, Guerin DM. 2011. Capsid protein identification and analysis of mature Triatoma virus (TrV) virions and naturally occurring empty particles. Virology 409:91–101. doi:10.1016/j.virol.2010.09.034. PubMed DOI

Smyth M, Pettitt T, Symonds A, Martin J. 2003. Identification of the pocket factors in a picornavirus. Arch Virol 148:1225–1233. doi:10.1007/s00705-002-0974-4. PubMed DOI

Hadfield AT, Lee W, Zhao R, Oliveira MA, Minor I, Rueckert RR, Rossmann MG. 1997. The refined structure of human rhinovirus 16 at 2.15 A resolution: implications for the viral life cycle. Structure 5:427–441. doi:10.1016/S0969-2126(97)00199-8. PubMed DOI

Smith TJ, Kremer MJ, Luo M, Vriend G, Arnold E, Kamer G, Rossmann MG, McKinlay MA, Diana GD, Otto MJ. 1986. The site of attachment in human rhinovirus 14 for antiviral agents that inhibit uncoating. Science 233:1286–1293. doi:10.1126/science.3018924. PubMed DOI

Hadfield AT, Diana GD, Rossmann MG. 1999. Analysis of three structurally related antiviral compounds in complex with human rhinovirus 16. Proc Natl Acad Sci U S A 96:14730–14735. doi:10.1073/pnas.96.26.14730. PubMed DOI PMC

Grant RA, Hiremath CN, Filman DJ, Syed R, Andries K, Hogle JM. 1994. Structures of poliovirus complexes with anti-viral drugs: implications for viral stability and drug design. Curr Biol 4:784–797. PubMed

Hiremath CN, Grant RA, Filman DJ, Hogle JM. 1995. Binding of the antiviral drug WIN51711 to the sabin strain of type 3 poliovirus: structural comparison with drug binding in rhinovirus 14. Acta Crystallogr D Biol Crystallogr 51:473–489. PubMed

Guu TS, Liu Z, Ye Q, Mata DA, Li K, Yin C, Zhang J, Tao YJ. 2009. Structure of the hepatitis E virus-like particle suggests mechanisms for virus assembly and receptor binding. Proc Natl Acad Sci U S A 106:12992–12997. doi:10.1073/pnas.0904848106. PubMed DOI PMC

Chen R, Neill JD, Estes MK, Prasad BV. 2006. X-ray structure of a native calicivirus: structural insights into antigenic diversity and host specificity. Proc Natl Acad Sci U S A 103:8048–8053. doi:10.1073/pnas.0600421103. PubMed DOI PMC

Hopper P, Harrison SC, Sauer RT. 1984. Structure of tomato bushy stunt virus. V. Coat protein sequence determination and its structural implications. J Mol Biol 177:701–713. PubMed

Kleywegt GJ, Brunger AT. 1996. Checking your imagination: applications of the free R value. Structure 4:897–904. doi:10.1016/S0969-2126(96)00097-4. PubMed DOI

O'Flaherty S, Coffey A, Edwards R, Meaney W, Fitzgerald GF, Ross RP. 2004. Genome of staphylococcal phage K: a new lineage of Myoviridae infecting gram-positive bacteria with a low G+C content. J Bacteriol 186:2862–2871. doi:10.1128/JB.186.9.2862-2871.2004. PubMed DOI PMC

Honeybee Iflaviruses Pack Specific tRNA Fragments from Host Cells in Their Virions

Capsid opening enables genome release of iflaviruses

Virion structure and genome delivery mechanism of sacbrood honeybee virus

Structure of deformed wing virus, a major honey bee pathogen

Cryo-EM study of slow bee paralysis virus at low pH reveals iflavirus genome release mechanism