UNLABELLED: Viruses of the family Dicistroviridae can cause substantial economic damage by infecting agriculturally important insects. Israeli acute bee paralysis virus (IAPV) causes honeybee colony collapse disorder in the United States. High-resolution molecular details of the genome delivery mechanism of dicistroviruses are unknown. Here we present a cryo-electron microscopy analysis of IAPV virions induced to release their genomes in vitro We determined structures of full IAPV virions primed to release their genomes to a resolution of 3.3 Å and of empty capsids to a resolution of 3.9 Å. We show that IAPV does not form expanded A particles before genome release as in the case of related enteroviruses of the family Picornaviridae The structural changes observed in the empty IAPV particles include detachment of the VP4 minor capsid proteins from the inner face of the capsid and partial loss of the structure of the N-terminal arms of the VP2 capsid proteins. Unlike the case for many picornaviruses, the empty particles of IAPV are not expanded relative to the native virions and do not contain pores in their capsids that might serve as channels for genome release. Therefore, rearrangement of a unique region of the capsid is probably required for IAPV genome release. IMPORTANCE: Honeybee populations in Europe and North America are declining due to pressure from pathogens, including viruses. Israeli acute bee paralysis virus (IAPV), a member of the family Dicistroviridae, causes honeybee colony collapse disorder in the United States. The delivery of virus genomes into host cells is necessary for the initiation of infection. Here we present a structural cryo-electron microscopy analysis of IAPV particles induced to release their genomes. We show that genome release is not preceded by an expansion of IAPV virions as in the case of related picornaviruses that infect vertebrates. Furthermore, minor capsid proteins detach from the capsid upon genome release. The genome leaves behind empty particles that have compact protein shells.

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

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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

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

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

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

Genersh E, von der Ohe 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. Apidologie 41:332–352. doi:10.1051/apido/2010014. DOI

Govan VA, Leat N, Allsopp M, Davison S. 2000. Analysis of the complete genome sequence of acute bee paralysis virus shows that it belongs to the novel group of insect-infecting RNA viruses. Virology 277:457–463. doi:10.1006/viro.2000.0616. PubMed DOI

Maori E, Lavi S, Mozes-Koch R, Gantman Y, Peretz Y, Edelbaum O, Tanne E, Sela I. 2007. Isolation and characterization of Israeli acute paralysis virus, a dicistrovirus affecting honeybees in Israel: evidence for diversity due to intra- and inter-species recombination. J Gen Virol 88:3428–3438. doi:10.1099/vir.0.83284-0. PubMed DOI

Chen YP, Pettis JS, Corona M, Chen WP, Li CJ, Spivak M, Visscher PK, DeGrandi-Hoffman G, Boncristiani H, Zhao Y, vanEngelsdorp D, Delaplane K, Solter L, Drummond F, Kramer M, Lipkin WI, Palacios G, Hamilton MC, Smith B, Huang SK, Zheng HQ, Li JL, Zhang X, Zhou AF, Wu LY, Zhou JZ, Lee ML, Teixeira EW, Li ZG, Evans JD. 2014. Israeli acute paralysis virus: epidemiology, pathogenesis and implications for honey bee health. PLoS Pathog 10:e1004261. doi:10.1371/journal.ppat.1004261. PubMed DOI PMC

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. doi:10.1016/j.jip.2009.06.014. PubMed DOI

de Miranda JR, Genersch E. 2010. Deformed wing virus. J Invertebr Pathol 103(Suppl 1):S48–S61. doi:10.1016/j.jip.2009.06.012. PubMed DOI

Di Prisco G, Pennacchio F, Caprio E, Boncristiani HF Jr, Evans JD, Chen Y. 2011. Varroa destructor is an effective vector of Israeli acute paralysis virus in the honeybee, Apis mellifera. J Gen Virol 92:151–155. doi:10.1099/vir.0.023853-0. PubMed DOI

Maori E, Paldi N, Shafir S, Kalev H, Tsur E, Glick E, Sela I. 2009. IAPV, a bee-affecting virus associated with colony collapse disorder can be silenced by dsRNA ingestion. Insect Mol Biol 18:55–60. doi:10.1111/j.1365-2583.2009.00847.x. 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

Cox-Foster DL, Conlan S, Holmes EC, Palacios G, Evans JD, Moran NA, Quan PL, Briese T, Hornig M, Geiser DM, Martinson V, vanEngelsdorp D, Kalkstein AL, Drysdale A, Hui J, Zhai J, Cui L, Hutchison SK, Simons JF, Egholm M, Pettis JS, Lipkin WI. 2007. A metagenomic survey of microbes in honey bee colony collapse disorder. Science 318:283–287. doi:10.1126/science.1146498. PubMed DOI

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

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

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

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

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

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. doi:10.1099/vir.0.048553-0. PubMed DOI

Tuthill TJ, Groppelli E, Hogle JM, Rowlands DJ. 2010. Picornaviruses. Curr Top Microbiol Immunol 343:43–89. doi:10.1007/82_2010_37. 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

Lyu K, Ding J, Han JF, Zhang Y, Wu XY, He YL, Qin CF, Chen R. 2014. Human enterovirus 71 uncoating captured at atomic resolution. J Virol 88:3114–3126. doi:10.1128/JVI.03029-13. 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. doi:10.1038/nsmb.2255. 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. doi: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. doi:10.1128/JVI.00531-10. PubMed DOI PMC

Fricks CE, Hogle JM. 1990. Cell-induced conformational change in poliovirus: externalization of the amino terminus of VP1 is responsible for liposome binding. J Virol 64:1934–1945. PubMed PMC

Greve JM, Forte CP, Marlor CW, Meyer AM, Hoover-Litty H, Wunderlich D, McClelland A. 1991. Mechanisms of receptor-mediated rhinovirus neutralization defined by two soluble forms of ICAM-1. J Virol 65:6015–6023. PubMed PMC

Prchla E, Kuechler E, Blaas D, Fuchs R. 1994. Uncoating of human rhinovirus serotype 2 from late endosomes. J Virol 68:3713–3723. PubMed 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. doi:10.1038/ncomms2889. 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. doi:10.1128/JVI.06425-11. 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

Tuthill TJ, Harlos K, Walter TS, Knowles NJ, Groppelli E, Rowlands DJ, Stuart DI, Fry EE. 2009. Equine rhinitis A virus and its low pH empty particle: clues towards an aphthovirus entry mechanism? PLoS Pathog 5:e1000620. doi:10.1371/journal.ppat.1000620. PubMed DOI PMC

Chalcoff VR, Aizen MA, Galetto L. 2006. Nectar concentration and composition of 26 species from the temperate forest of South America. Ann Bot 97:413–421. PubMed 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. doi:10.1073/pnas.1312128110. 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

Shakeel S, Westerhuis BM, Ora A, Koen G, Bakker AQ, Claassen Y, Wagner K, Beaumont T, Wolthers KC, Butcher SJ. 2015. Structural basis of human parechovirus neutralization by human monoclonal antibodies. J Virol 89:9571–9580. doi:10.1128/JVI.01429-15. PubMed DOI PMC

Zhu L, Wang X, Ren J, Porta C, Wenham H, Ekstrom JO, Panjwani A, Knowles NJ, Kotecha A, Siebert CA, Lindberg AM, Fry EE, Rao Z, Tuthill TJ, Stuart DI. 2015. Structure of Ljungan virus provides insight into genome packaging of this picornavirus. Nat Commun 6:8316. doi:10.1038/ncomms9316. PubMed DOI PMC

Hewat EA, Neumann E, Blaas D. 2002. The concerted conformational changes during human rhinovirus 2 uncoating. Mol Cell 10:317–326. doi:10.1016/S1097-2765(02)00603-2. PubMed DOI

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. doi:10.1128/JVI.74.3.1342-1354.2000. PubMed DOI PMC

Belnap DM, McDermott BM Jr, Filman DJ, Cheng N, Trus BL, Zuccola HJ, Racaniello VR, Hogle JM, Steven AC. 2000. Three-dimensional structure of poliovirus receptor bound to poliovirus. Proc Natl Acad Sci U S A 97:73–78. doi:10.1073/pnas.97.1.73. PubMed DOI PMC

Wien MW, Curry S, Filman DJ, Hogle JM. 1997. Structural studies of poliovirus mutants that overcome receptor defects. Nat Struct Biol 4:666–674. doi:10.1038/nsb0897-666. PubMed DOI

Liu Y, Sheng J, Fokine A, Meng G, Shin WH, Long F, Kuhn RJ, Kihara D, Rossmann MG. 2015. Structure and inhibition of EV-D68, a virus that causes respiratory illness in children. Science 347:71–74. doi:10.1126/science.1261962. PubMed DOI PMC

Lee H, Shingler KL, Organtini LJ, Ashley RE, Makhov AM, Conway JF, Hafenstein S. 2016. The novel asymmetric entry intermediate of a picornavirus captured with nanodiscs. Sci Adv 2:e1501929. doi:10.1126/sciadv.1501929. PubMed DOI PMC

Shukla A, Padhi AK, Gomes J, Banerjee M. 2014. The VP4 peptide of hepatitis A virus ruptures membranes through formation of discrete pores. J Virol 88:12409–12421. doi:10.1128/JVI.01896-14. PubMed DOI PMC

Panjwani A, Strauss M, Gold S, Wenham H, Jackson T, Chou JJ, Rowlands DJ, Stonehouse NJ, Hogle JM, Tuthill TJ. 2014. Capsid protein VP4 of human rhinovirus induces membrane permeability by the formation of a size-selective multimeric pore. PLoS Pathog 10:e1004294. doi:10.1371/journal.ppat.1004294. PubMed DOI PMC

Shaikh TR, Gao H, Baxter WT, Asturias FJ, Boisset N, Leith A, Frank J. 2008. SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs. Nat Protoc 3:1941–1974. doi:10.1038/nprot.2008.156. PubMed DOI PMC

Tang G, Peng L, Baldwin PR, Mann DS, Jiang W, Rees I, Ludtke SJ. 2007. EMAN2: an extensible image processing suite for electron microscopy. J Struct Biol 157:38–46. doi:10.1016/j.jsb.2006.05.009. PubMed DOI

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

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. PubMed DOI

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. PubMed DOI PMC

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. PubMed DOI

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 47:110–119. doi:10.1107/S0108767390010224. PubMed DOI

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

Vagin AA, Steiner RA, Lebedev AA, Potterton L, McNicholas S, Long F, Murshudov GN. 2004. REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use. Acta Crystallogr D Biol Crystallogr 60:2184–2195. doi:10.1107/S0907444904023510. PubMed DOI

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

Davis IW, Leaver-Fay A, Chen VB, Block JN, Kapral GJ, Wang X, Murray LW, Arendall WB III, Snoeyink J, Richardson JS, Richardson DC. 2007. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res 35:W375–W383. doi:10.1093/nar/gkm216. PubMed DOI PMC

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