Infections of Kashmir bee virus (KBV) are lethal for honeybees and have been associated with colony collapse disorder. KBV and closely related viruses contribute to the ongoing decline in the number of honeybee colonies in North America, Europe, Australia, and other parts of the world. Despite the economic and ecological impact of KBV, its structure and infection process remain unknown. Here we present the structure of the virion of KBV determined to a resolution of 2.8 Å. We show that the exposure of KBV to acidic pH induces a reduction in inter-pentamer contacts within capsids and the reorganization of its RNA genome from a uniform distribution to regions of high and low density. Capsids of KBV crack into pieces at acidic pH, resulting in the formation of open particles lacking pentamers of capsid proteins. The large openings of capsids enable the rapid release of genomes and thus limit the probability of their degradation by RNases. The opening of capsids may be a shared mechanism for the genome release of viruses from the family Dicistroviridae ImportanceThe western honeybee (Apis mellifera) is indispensable for maintaining agricultural productivity as well as the abundance and diversity of wild flowering plants. However, bees suffer from environmental pollution, parasites, and pathogens, including viruses. Outbreaks of virus infections cause the deaths of individual honeybees as well as collapses of whole colonies. Kashmir bee virus has been associated with colony collapse disorder in the US, and no cure of the disease is currently available. Here we report the structure of an infectious particle of Kashmir bee virus and show how its protein capsid opens to release the genome. Our structural characterization of the infection process determined that therapeutic compounds stabilizing contacts between pentamers of capsid proteins could prevent the genome release of the virus.

Centro de Estudios Parasitológicos y de Vectores Boulevard 120 s n e 60 y 64 1900 La Plata Argentina

Department of Biochemistry and Molecular Biology University of the Basque Country B° Sarriena S N 48940 Leioa Vizcaya Spain

Department of Zoology Fishery Hydrobiology and Apidology Faculty of Agronomy Mendel University in Brno Zemědělská 1 1665 613 00 Brno Czech Republic

Structural Virology Group Central European Institute of Technology Masaryk University Kamenice 753 5 62500 Brno Czech Republic

Zobrazit více v PubMed

de Miranda JR, Gauthier L, Ribiere M, Chen YP. 2012. Honey bee viruses and their effect on bee and colony health, p 71–102. In Sammataro DYJ (ed), Honey bee colony health: challenges and sustainable solutions. CRC Press, Boca Raton, FL.

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

Bailey L. 1976. Viruses attacking the honey bee. Adv Virus Res 20:271–304. 10.1016/s0065-3527(08)60507-2. PubMed DOI

de Miranda JR, Cordoni G, Budge G. 2010. The Acute bee paralysis virus-Kashmir bee virus-Israeli acute paralysis virus complex. J Invertebr Pathol 103(Suppl 1):S30–S47. 10.1016/j.jip.2009.06.014. PubMed DOI

Mullapudi E, Přidal A, Pálková L, de Miranda JR, Plevka P. 2016. Virion structure of Israeli acute bee paralysis virus. J Virol 90:8150–8159. 10.1128/JVI.00854-16. PubMed DOI PMC

Spurny R, Přidal A, Pálková L, Kiem HKT, de Miranda JR, Plevka P. 2017. Virion structure of black queen cell virus, a common honeybee pathogen. J Virol 91:e02100-16. 10.1128/JVI.02100-16. 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. 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. 10.1107/S0907444913004617. PubMed DOI PMC

Bonning BC, Miller WA. 2010. Dicistroviruses. Annu Rev Entomol 55:129–150. 10.1146/annurev-ento-112408-085457. PubMed DOI

Procházková M, Škubník K, Füzik T, Mukhamedova L, Přidal A, Plevka P. 2020. Virion structures and genome delivery of honeybee viruses. Curr Opin Virol 45:17–24. 10.1016/j.coviro.2020.06.007. PubMed DOI

Mullapudi E, Füzik T, Přidal A, Plevka P. 2017. Cryo-electron microscopy study of the genome release of the dicistrovirus Israeli acute bee paralysis virus. J Virol 91:e02060-16. 10.1128/JVI.02060-16. PubMed DOI PMC

Ren J, Wang X, Hu Z, Gao Q, Sun Y, Li X, Porta C, Walter TS, Gilbert RJ, Zhao Y, Axford D, Williams M, McAuley K, Rowlands DJ, Yin W, Wang J, Stuart DI, Rao Z, Fry EE. 2013. Picornavirus uncoating intermediate captured in atomic detail. Nat Commun 4:1929. 10.1038/ncomms2889. 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. 10.1371/journal.ppat.1002473. PubMed DOI PMC

Shingler KL, Yoder JL, Carnegie MS, Ashley RE, Makhov AM, Conway JF, Hafenstein S. 2013. The enterovirus 71 A-particle forms a gateway to allow genome release: a cryoEM study of picornavirus uncoating. PLoS Pathog 9:e1003240. 10.1371/journal.ppat.1003240. 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. 10.1128/JVI.00531-10. PubMed DOI PMC

Wang X, Peng W, Ren J, Hu Z, Xu J, Lou Z, Li X, Yin W, Shen X, Porta C, Walter TS, Evans G, Axford D, Owen R, Rowlands DJ, Wang J, Stuart DI, Fry EE, Rao Z. 2012. A sensor-adaptor mechanism for enterovirus uncoating from structures of EV71. Nat Struct Mol Biol 19:424–429. 10.1038/nsmb.2255. PubMed DOI PMC

Seitsonen JJ, Shakeel S, Susi P, Pandurangan AP, Sinkovits RS, Hyvonen H, Laurinmaki P, Yla-Pelto J, Topf M, Hyypia T, Butcher SJ. 2012. Structural analysis of coxsackievirus A7 reveals conformational changes associated with uncoating. J Virol 86:7207–7215. 10.1128/JVI.06425-11. PubMed DOI PMC

Yuan H, Li P, Ma X, Lu Z, Sun P, Bai X, Zhang J, Bao H, Cao Y, Li D, Fu Y, Chen Y, Bai Q, Zhang J, Liu Z. 2017. The pH stability of foot-and-mouth disease virus. Virol J 14:233. 10.1186/s12985-017-0897-z. PubMed DOI PMC

Buchta D, Fuzik T, Hrebik D, Levdansky Y, Sukenik L, Mukhamedova L, Moravcova J, Vacha R, Plevka P. 2019. Enterovirus particles expel capsid pentamers to enable genome release. Nat Commun 10:1138. 10.1038/s41467-019-09132-x. PubMed DOI PMC

Sanchez-Eugenia R, Durana A, Lopez-Marijuan I, Marti GA, Guerin DMA. 2016. X-ray structure of Triatoma virus empty capsid: insights into the mechanism of uncoating and RNA release in dicistroviruses. J Gen Virol 97:2769–2779. 10.1099/jgv.0.000580. PubMed DOI

Snijder J, Uetrecht C, Rose RJ, Sanchez-Eugenia R, Marti GA, Agirre J, Guerin DM, Wuite GJ, Heck AJ, Roos WH. 2013. Probing the biophysical interplay between a viral genome and its capsid. Nat Chem 5:502–509. 10.1038/nchem.1627. PubMed DOI

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

Harrison SC, Olson AJ, Schutt CE, Winkler FK, Bricogne G. 1978. Tomato bushy stunt virus at 2.9 A resolution. Nature 276:368–373. 10.1038/276368a0. PubMed DOI

Bennett MJ, Schlunegger MP, Eisenberg D. 1995. 3D domain swapping: a mechanism for oligomer assembly. Protein Sci 4:2455–2468. 10.1002/pro.5560041202. 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. 10.1007/s00705-008-0041-x. PubMed DOI

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. 10.1016/j.virol.2010.09.034. PubMed DOI

Fisher AJ, Johnson JE. 1993. Ordered duplex RNA controls capsid architecture in an icosahedral animal virus. Nature 361:176–179. 10.1038/361176a0. PubMed DOI

Munshi S, Liljas L, Cavarelli J, Bomu W, McKinney B, Reddy V, Johnson JE. 1996. The 2.8 A structure of a T = 4 animal virus and its implications for membrane translocation of RNA. J Mol Biol 261:1–10. 10.1006/jmbi.1996.0437. PubMed DOI

Zlotnick A, Reddy VS, Dasgupta R, Schneemann A, Ray WJ, Jr, Rueckert RR, Johnson JE. 1994. Capsid assembly in a family of animal viruses primes an autoproteolytic maturation that depends on a single aspartic acid residue. J Biol Chem 269:13680–13684. 10.1016/S0021-9258(17)36883-7. PubMed DOI

Gopal A, Zhou ZH, Knobler CM, Gelbart WM. 2012. Visualizing large RNA molecules in solution. RNA 18:284–299. 10.1261/rna.027557.111. PubMed DOI PMC

Jiang P, Liu Y, Ma HC, Paul AV, Wimmer E. 2014. Picornavirus morphogenesis. Microbiol Mol Biol Rev 78:418–437. 10.1128/MMBR.00012-14. PubMed DOI PMC

Fout GS, Medappa KC, Mapoles JE, Rueckert RR. 1984. Radiochemical determination of polyamines in poliovirus and human rhinovirus 14. J Biol Chem 259:3639–3643. 10.1016/S0021-9258(17)43142-5. PubMed DOI

Mounce BC, Olsen ME, Vignuzzi M, Connor JH. 2017. Polyamines and their role in virus infection. Microbiol Mol Biol Rev 81:e00029-17. 10.1128/MMBR.00029-17. PubMed DOI PMC

Harutyunyan S, Kumar M, Sedivy A, Subirats X, Kowalski H, Kohler G, Blaas D. 2013. Viral uncoating is directional: exit of the genomic RNA in a common cold virus starts with the poly-(A) tail at the 3'-end. PLoS Pathog 9:e1003270. 10.1371/journal.ppat.1003270. 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. 10.1128/JVI.02393-09. PubMed DOI PMC

Pickl-Herk A, Luque D, Vives-Adrian L, Querol-Audi J, Garriga D, Trus BL, Verdaguer N, Blaas D, Caston JR. 2013. Uncoating of common cold virus is preceded by RNA switching as determined by X-ray and cryo-EM analyses of the subviral A-particle. Proc Natl Acad Sci U S A 110:20063–20068. 10.1073/pnas.1312128110. PubMed DOI PMC

Kalynych S, Füzik T, Přidal A, de Miranda J, Plevka P. 2017. Cryo-EM study of slow bee paralysis virus at low pH reveals iflavirus genome release mechanism. Proc Natl Acad Sci U S A 114:598–603. 10.1073/pnas.1616562114. PubMed DOI PMC

Agirre J, Goret G, LeGoff M, Sanchez-Eugenia R, Marti GA, Navaza J, Guerin DM, Neumann E. 2013. Cryo-electron microscopy reconstructions of triatoma virus particles: a clue to unravel genome delivery and capsid disassembly. J Gen Virol 94:1058–1068. 10.1099/vir.0.048553-0. PubMed DOI

Groppelli E, Levy HC, Sun E, Strauss M, Nicol C, Gold S, Zhuang X, Tuthill TJ, Hogle JM, Rowlands DJ. 2017. Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes. PLoS Pathog 13:e1006197. 10.1371/journal.ppat.1006197. PubMed DOI PMC

Belnap DM, Filman DJ, Trus BL, Cheng N, Booy FP, Conway JF, Curry S, Hiremath CN, Tsang SK, Steven AC, Hogle JM. 2000. Molecular tectonic model of virus structural transitions: the putative cell entry states of poliovirus. J Virol 74:1342–1354. 10.1128/jvi.74.3.1342-1354.2000. PubMed DOI PMC

Sanchez-Eugenia R, Goikolea J, Gil-Carton D, Sanchez-Magraner L, Guerin DM. 2015. Triatoma virus recombinant VP4 protein induces membrane permeability through dynamic pores. J Virol 89:4645–4654. 10.1128/JVI.00011-15. PubMed DOI PMC

Zheng SQ, Palovcak E, Armache JP, Verba KA, Cheng Y, Agard DA. 2017. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat Methods 14:331–332. 10.1038/nmeth.4193. PubMed DOI PMC

Zhang K. 2016. Gctf: real-time CTF determination and correction. J Struct Biol 193:1–12. 10.1016/j.jsb.2015.11.003. PubMed DOI PMC

Wagner T, Merino F, Stabrin M, Moriya T, Antoni C, Apelbaum A, Hagel P, Sitsel O, Raisch T, Prumbaum D, Quentin D, Roderer D, Tacke S, Siebolds B, Schubert E, Shaikh TR, Lill P, Gatsogiannis C, Raunser S. 2019. SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM. Commun Biol 2:218. 10.1038/s42003-019-0437-z. PubMed DOI PMC

Scheres SH. 2012. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J Struct Biol 180:519–530. 10.1016/j.jsb.2012.09.006. PubMed DOI PMC

Scheres SH, Chen S. 2012. Prevention of overfitting in cryo-EM structure determination. Nat Methods 9:853–854. 10.1038/nmeth.2115. PubMed DOI PMC

Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zwart PH. 2010. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66:213–221. 10.1107/S0907444909052925. PubMed DOI PMC

Emsley P, Lohkamp B, Scott WG, Cowtan K. 2010. Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66:486–501. 10.1107/S0907444910007493. PubMed DOI PMC

Murshudov GN, Skubak P, Lebedev AA, Pannu NS, Steiner RA, Nicholls RA, Winn MD, Long F, Vagin AA. 2011. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr D Biol Crystallogr 67:355–367. 10.1107/S0907444911001314. PubMed DOI PMC

Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC. 2010. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr 66:12–21. 10.1107/S0907444909042073. PubMed DOI PMC

Krissinel E, Henrick K. 2007. Inference of macromolecular assemblies from crystalline state. J Mol Biol 372:774–797. 10.1016/j.jmb.2007.05.022. PubMed DOI

Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE. 2004. UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612. 10.1002/jcc.20084. PubMed DOI