VP3 is crucial for the stability of Nora virus virions

Vi us Resea ch 223 (2016) 20–27 Con en s lis s a ailable a ScienceDi ec Vi us Resea ch jou nalhomepage: www.else ie .com/loca e/ i us es VP3 is c ucial o he s abili y o No a i us i ions Sajna Anand Sadanandana, Jens-Ola Eks öma,c, Venka eswa a Rao Jonnab, Ande s Ho e b, Dan Hul ma ka,c,∗ aDepa men o Molecula Biology, Umeå Uni e si y, SE-901 87 Umeå, Sweden bDepa men o Medical Biochemis y and Biophysics, Umeå Uni e si y, SE-901 87 Umeå, Sweden cIns i u e o Biomedical Technology, Uni e si y o Tampe e, FI-33520 Tampe e, Finland a i c l e i n o A icle his o y: Recei ed 15 Ma ch 2016 Recei ed in e ised o m 15 June 2016 Accep ed 17 June 2016 A ailable online 18 June 2016 Keywo ds: RNA i uses No a i us Capsid s abili y Vi us biology a b s a c No a i usisanen e ic i us ha causes pe sis en ,non-pa hologicalin ec ion inD osophila melanogas e . I eplica es in he fly gu and is ansmi ed ia he ecal-o al ou e. No a i us has a single-s anded posi i e-senseRNAgenome,whichis ansla edin ou open eading ames.Reading ame h eeencodes he VP3 p o ein, he s uc u e and unc ion o which we ha e in es iga ed in his wo k. We ha e shown ha VP3 is a ime ha has an ␣-helical seconda y s uc u e, wi h a unc ionally impo an coiled-coil domain. In o de o iden i y he ole o VP3 in he No a i us li e cycle, we cons uc ed VP3-mu an s using he cDNA clone o he i us. Ou esul s show ha VP3 does no ha e a ole in he ac ual assembly o he i us pa icles, bu i ions ha lack VP3 o ha bo VP3 wi h a dis up ed coiled coil domain a e incapable o ansmission ia he ecal-o al ou e. Remo ing he egion downs eam o he pu a i e coiled coil appea s o ha e an e ec on he fi ness o he i us bu does no hampe i s eplica ion o ansmission. We also ound ha he VP3 p o ein and pa icula ly he coiled coil domain a e c ucial o he s abili y o No a i us i ions when exposed o hea o p o eases. Hence, we p opose ha VP3 is impe a i e o No a i us i ions as i con e s s abili y o he i al capsid. © 2016 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/). 1. In oduc ion No a i us is a small RNA i us ha causes pe sis en , non- pa hological in ec ion in D osophila melanogas e (Habayeb e al., 2006). I is an en e ic i us ha is ansmi ed ia he ecal-o al ou e and high i us i e s ha e been obse ed in he eces o in ec ed animals (Habayeb e al., 2009). I possibly defines a new amily o pico na-like i uses. Based on sequence compa isons o he eplica i e enzymes, he No a i us is mos closely ela ed o i uses o he amilies Pico na i idae, which in ec e eb a es, and Ifla i idae, which in ec insec s (Habayeb e al., 2006; Koonin e al., 2008). Ye , he No a i us has a unique size and o gani- za ion o i s genome. Pico na i idae includes impo an human en e o i uses such as he polio i us, while honeybee pa hogens like he de o med wing and sacb ood i uses belong o Ifla i idae. Some membe s o hese amilies a e able o es ablish pe sis en in ec ions in hei hos s, p obably con ibu ing o p oblems such as he pos -polio synd ome in humans and he colony collapse dis- ∗Co esponding au ho a : Depa men o Molecula Biology, Umeå Uni e si y, SE-901 87 Umeå, Sweden. E-mail add ess: [email p o ec ed] (D. Hul ma k). o de in honeybees (P isco e al., 2011; Baj e al., 2015). The No a i us may be a use ul model o s udy he phenomenon o RNA i us pe sis ence. Fo ha pu pose, we ound i impo an o cha ac e ize he i us in mo e de ail. No a i us has a 12333 nucleo ides long (Eks öm e al., 2011) single-s anded RNA genome o posi i e pola i y (Habayeb e al., 2006). Expe imen al wo k and sequence analysis ha e indica ed ha , ou o he ou open eading ames in he No a i us genome, ORF1 encodes an inhibi o o he RNAi de ense mechanism ( an Mie lo e al., 2012), while ORF2 encodes a long polyp o ein wi h he helicase, p o ease and polyme ase egions ha a e closely ela ed o he co esponding p o eins ound in he eplica i e casse es o pico na-like i uses (Habayeb e al., 2006; Koonin e al., 2008). No sequence simila i y was de ec ed be ween ORF4 and o he known i us p oduc s, bu expe imen al e idence has shown ha i encodes a polyp o ein ha is clea ed in o h ee capsid p o eins, VP4A, B and C (Eks öm e al., 2011). Howe e , he ORF3-encoded p o ein, VP3, emains la gely uncha ac e ized. Small amoun s o an ORF3-encoded p o ein, wi h an es ima ed molecula mass o 35kDa, we e de ec ed in he i ions du ing mass spec ome y analysis o he VP4 capsid p o eins (Eks öm e al., 2011). This sugges ed ha he VP3 p o ein was associa ed wi h he s uc u al p o eins. The fi s 71 nucleo ides o ORF3 o e lap wi h h p://dx.doi.o g/10.1016/j. i us es.2016.06.011 0168-1702/© 2016 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/). S.A. Sadanandan e al. / Vi us Resea ch 223 (2016) 20–27 21 he C- e minus o ORF2 and he e o e, ansla ion o ORF3 is likely o occu wi h a ame shi mechanism. The esul ing VP3 p o ein is 281–304 amino acids long, depending on he exac posi ion o he ame shi , and he p edic ed molecula mass app oxima ely 31–34 kDa. Based on he sequence, Eks öm e al. (2011) p edic ed an ␣-helical seconda y s uc u e o he N- e minal 200 amino acids o VP3, and ha his pa o he p o ein is likely o o m a coiled-coil domain. Howe e , expe imen al e idence is s ill lack- ing o his model. Thus a , he ole o VP3 in he li e cycle o No a i us has no been in es iga ed. We ha e now used he cDNA clone o No a i us (Eks öm e al., 2011) o s udy he impo ance o VP3 in i ion assembly and s uc- u al s abili y, by gene a ing VP3 mu an s. Du ing he cou se o his s udy, we obse ed ha VP3 was impo an o he ansmission o No a i us ia he ecal-o al ou e. Consequen ly, we hypo hesized ha he p esumed coiled-coil domain in his p o ein was impo an o he s abili y o he i us pa icles. To add ess his hypo hesis, we compa ed he s abili y o wild- ype i ions o ha o VP3 mu an i ions. We ound ha mu an i ions lacking VP3 o ha bo ing a dis up ion in he p edic ed coiled coil domain we e labile unde hea and p o ease ea men s, while he wild ype i ions su i ed hese condi ions. 2. Ma e ials and me hods 2.1. Cloning, exp ession and pu ifica ion o No a i us VP3 No a i us ORF3 was cloned in o he mul iple cloning si e o he pETM-MBP1a exp ession ec o (P yo and Lei ing, 1997) using o wa d (GCTTCCATGGCATTAAAAGAGGAGATTTTTGATCAA) and e e se (GCTTGGTACCTTACATAGAGTCATAAATTACTGATGT- GCT) p ime s. The pETM-MBP1a ec o con ains a hexa-His idine ag ollowed by he mal ose binding p o ein (MBP) and a Tobacco e ch i us (TEV) clea age si e, a e which he ORF3 sequence was inse ed. This cons uc was ans o med in o Rose a (DE3) cells (Fu e al., 2007), which we e hen g own o e nigh in an au o- induc ion LB medium (Edwin e al., 2014) a 20◦C. Since i is agged wi h he mal ose binding p o ein, he exp essed VP3 p o- ein could be isola ed using an amylose esin a fini y ma ix (NEB), acco ding o he ins uc ions p o ided by he manu ac u e . The isola ed p o ein was clea ed om i s MBP ag using he TEV p o- ease and he ea e pu ified by NiNTA aga ose ch oma og aphy (Qiagen) in o de o emo e bo h MBP and TEV p o ease. The now- un agged VP3 p o ein was subjec ed Q-Sepha ose (GE heal hca e) ch oma og aphy in o de o ob ain pu e VP3 (Fig. 1A, lane 2). 2.2. Analysis o VP3 oligome s by GEMMA Pu ified No a i us VP3 p o ein was dissol ed in 20 mM ammo- nium ace a e pH 7.8 o a final concen a ion o 0.05 mg/ml. GEMMA analysis was pe o med as desc ibed in Ro ouga an e al. (2008). 2.3. Analysis o VP3 oligome s by c oss-linking Fo c oss-linking, pu ified VP3 (0.25 mg/ml, final concen a ion) was ea ed wi h dime hyl pimelimida e (Sigma, 0.75 mg/ml, final concen a ion), bo h dissol ed in phospha e bu e ed saline, pH 8.0. The eac ion was incuba ed a oom empe a u e o 30 min, and he ea e s opped by addi ion o T is-HCL bu e , pH 8.0, o a final concen a ion o 20 mM. Bo h na i e and c oss-linked VP3 we e ea ed wi h 2× Laemmli p o ein sample bu e (100 mM T is pH 6.8, 2% SDS, 0.001% b omophenol blue, 20% Glyce ol and 2% ␤-me cap oe hanol) a a 1:1 a io, hea ed a 95◦C o 10 min, and hen un on a 13.6% SDS-polyac ylamide gel (29:1 ac ylamide/bis- ac ylamide) as desc ibed (Laemmli, 1970). Fig. 1. S uc u al Cha ac e iza ion o No a i us VP3 (A) SDS-polyac ylamide gel elec opho esis o VP3 monome and c oss-linked VP3 ime . Ma ke : PageRule p es ained p o ein ladde (The mo Scien ific). (B) Nano-Elec osp ay GEMMA anal- ysis o No a i us VP3 (0.05 mg/ml) a 2 psi capilla y p essu e. (C) Fa -UV Ci cula dich oism analysis o No a i us VP3, p esen ed as an a e age o 5 scans aken a 50 nm/min, showing a spec um ha is ypical o ␣-helical p o eins. 22 S.A. Sadanandan e al. / Vi us Resea ch 223 (2016) 20–27 2.4. Ci cula dich oism CD spec a o No a i us VP3 we e ob ained on a Jasco Spec- opola ime e model J-715 (Jasco Co po a ion). The spec a we e collec ed a 25◦C in a s anda d phospha e bu e ed saline (pH 7.4) using a qua z cu e e o 0.1 cm pa h leng h. Shown spec a ep e- sen he a e age o fi e scans a a speed o 50 nm/min. Only he a UV egion om 190 nm o 260 nm was analyzed. 2.5. Sequence da a and seconda y s uc u e p edic ion Seconda y s uc u es we e p edic ed using he Ne Su P 1.1 se e (Pe e sen e al., 2009) and he Ne Tu nP 1.0 se e (Pe e sen e al., 2010), accessed ia he CBS P edic ion Se e s (h p://www. cbs.d u.dk/se ices). P edic ion o coiled coil was pe o med using he Ma coil se e (Delo enzi and Speed, 2002) accessed ia he Bioin o ma ics Toolki a he Max-Planck Ins i u e o De elopmen- al Biology (h p:// oolki . uebingen.mpg.de/ma coil), he Pai coil2 se e (McDonnell e al., 2006) and he Mul iCoil se e (Wol e al., 1997), bo h accessed a he MIT Compu e Science and A ificial In elligence Labo a o y (h p://g oups.csail.mi .edu/pai coil2.h ml and h p://g oups.csail.mi .edu/cb/mul icoil/cgi-bin/mul icoil.cgi) 2.6. Es ablishing wild ype and mu an No a i us in ec ion in flies The in ec ious cDNA clone o No a i us (Eks öm e al., 2011) was used o cons uc VP3 mu an s using s anda d molecula cloning echniques. Lis o p ime s used o cons uc he di e - en mu an s is gi en in Table S1 o Supplemen a y ma e ials. All expe imen s we e pe o med using RelishE23 (Hedeng en e al., 1999) flies, ea ed a 25◦C on s anda d mashed-po a o fly ood (Yang e al., 2015). The cons uc ed clones we e mic oinjec ed in o decho iona ed syncy ial-s age emb yos (Sp adling, 1986). To induce exp ession he la ae we e subsequen ly ea ed on fly ood con aining 0.5 mM CuSO4, un il hey eached he adul s age. 2.7. Vi us pa icle pu ifica ion Vi us pa icles we e pu ified om whole fly ex ac and om he eces o in ec ed flies. To ex ac i us pa icles om in ec ed flies, 10–15 flies we e homogenized wi h a homogenizing pes le in NT- bu e (100 mM NaCl and 10 mM T is-HCl, pH 7.4). To ex ac i us pa icles om eces, eces was collec ed om 10 o 15 flies by keep- ing hem in 15-ml cen i uge ubes o 2–4 h and washing he ube walls wi h NT-bu e . T i on X-100 was added o a final concen a- ion o 0.5% o he whole fly ex ac and 0.01% o he eces. Vi us pu ifica ion was pe o med as desc ibed p e iously by Ande son e al. (1966). B iefly, he i us pa icles we e pelle ed by cen i u- ga ion a 38000 pm o 70 min using he SW41Ti o o (Beckman Coul e ). Pelle ed pa icles we e dissol ed in 1 ml NT-T bu e (NT bu e , 0.01% T i on X-100) and loaded on op o he suc ose g a- dien s. Ra e-zonal sepa a ion was pe o med on a 35–12% (w/ ) linea suc ose g adien in NT-T bu e a 38000 pm, o 90 min in 4◦C. G adien ac ions we e collec ed by punc u ing he bo om o he ubes and collec ing 0.5 ml/ ac ion. Quan i a i e RT-PCR was used o de e mine i us peak ac ions. 2.8. Quan i a i e RT-PCR To al RNA was ex ac ed om he samples using Au um To al RNA Mini Ki (BioRad). Quan i a i e RT-PCR was pe o med as desc ibed p e iously (Habayeb e al., 2009; Eks öm e al., 2011) using o wa d p ime : 5-TTTCACTTTACTGTTGGTCTCC-3, e e se P ime : 5-ATTCCATTTGTGACTGATTTTATTTC-3and Taq- man p obe: 5-FAM-AGAGTTAGTGGACAAGTTAGAGACTGGCAT- TAMRA-3. 2.9. P o ease clea age analysis Wild- ype and mu an i us pa icles pu ified by a e-zonal sepa a ion, we e subjec ed o p o ease clea age. They we e ea ed wi h, ei he 2000 U/ml ypsin (#T8003, Sigma-Ald ich) o 0.05 U/ml panc ea ic p o ease (#P4630, Sigma-Ald ich), o 2 h a 37◦C. Bo h ypsin and p o ease we e dissol ed in phospha e saline bu e , pH 7.4. Following his, he ea ed pa icles we e incuba ed wi h a p o ease inhibi o cock ail (#1697498, Sigma-Ald ich) and RNase A (The mo Scien ific), o 30 min a 37◦C. To al RNA was ex ac ed and i us i e was de e mined by quan i a i e RT-PCR (as desc ibed in Sec ion 2.6). 3. Resul s 3.1. VP3 o ms ime s In o de o cha ac e ize VP3 biochemically, we cloned and exp essed he p o ein in a bac e ial exp ession ec o . The pu ified VP3 p o ein uns like a 29–31 kDa p o ein on an SDS gel (Fig. 1A). This is in ag eemen wi h he molecula mass p edic ed om he sequence, 31–34 kDa, depending on he posi ion o he amino e - minus. I is also consis en wi h he p e iously es ima ed 35 kDa o VP3 om in ac i ions (Eks öm e al., 2011). Nano-Elec osp ay GEMMA de ec s he elec opho e ic mobil- i y o pa icles in ai , which is used o calcula e molecula diame e and consequen ly, he molecula mass o he pa icles. We ha e used his me hod o find he na i e oligome ic na u e o pu i- fied VP3 p o ein. GEMMA shows a s ong peak a 97 kDa (Fig. 1B), which co esponds wi h he ime ic o m o VP3. Mino peaks a e obse ed a 33, 68, and 190 kDa, sugges ing he p esence o minimal amoun s o in e media y monome ic, dime ic and hex- ame ic o ms o he p o ein. As an independen es o he esul s ob ained h ough GEMMA, we c oss-linked VP3 using dime hyl pimelimida e and analyzed he c oss-linked p o ein on an SDS- polyac ylamide gel (Fig. 1A). The c oss-linked p o ein appea s o be a ound 97 kDa, which would co espond o a VP3 ime . Taken oge he , ou esul s show ha No a i us VP3 has a p opensi y o o m ime s. 3.2. VP3 is p edominan ly ˛-helical Seconda y s uc u e p edic ion o VP3 had indica ed ha he N- e minal egion is ␣-helical and ha he egion downs eam o his showed g ea e likelihood o o ming ␤-s ands (Eks öm e al., 2011). Ci cula dich oism was pe o med on pu ified VP3 o expe imen ally alida e he p edic ed seconda y s uc u e. The CD spec um o VP3 (Fig. 1C) shows a posi i e band a 195 nm and wo nega i e bands a 209 nm and 220 nm. Such a spec um is cha ac- e is ic o ␣-helical p o eins (G eenfield, 2006). On calcula ing he pe cen ages o di e en s uc u al elemen s in he p o ein, using he CDNN CD decon olu ion so wa e (Böhm e al., 1992), i was ound o be app oxima ely 77% ␣-helical, 10% ␤- u n, 8% andom coil and he emaining an ipa allel/pa allel shee s. These esul s pa allel he seconda y s uc u e p edic ion o No a i us VP3 pe - o med p e iously by Eks öm e al. (2011) 3.3. A ime ic coiled-coil s uc u e is p edic ed o VP3 homologs in No a-like i uses The simila i y o No a i us o p e iously desc ibed i uses is la gely es ic ed o he eplica i e casse e encoded by ORF2, which shows homology o pico na-like i uses (Habayeb e al., S.A. Sadanandan e al. / Vi us Resea ch 223 (2016) 20–27 23 Fig. 2. Sequence analysis and seconda y s uc u e p edic ion o VP3. (A) P obabili ies o ␣-helix ( ed), ␤-s and (blue) and coiled coil acco ding o Ma coil (solid black) o Pai coil2 (dashed black), and ela i e p obabili ies o coiled coil dime s (pu ple) o ime s (g een) a e shown as sliding a e ages o 13 amino acids. (B) Schema ic ep esen a ion o p edic ed s uc u al elemen s in VP3. The p obabili y o each amino acid o eside in any o he se en possible posi ions, a-g, o a coiled coil is indica ed by di e en shades o ed. P edic ed ␤- u ns a e shown in g ey and helix-b eaking p oline esidues a e black. The p edic ed coiled coil segmen s, CC1-CC3, a e delinea ed by blue lines. (C) Sequence conse a ion be ween h ee D osophila No a i uses, D. melanogas e , D. immig ans and D. subobscu a; 3/3 indica es ull iden i y. The egion ha o e laps wi h ORF2 is shown g ey. The pe cen o esidues ha a e ully conse ed is indica ed o he di e en sub- egions below he g aph. Also shown is he egion ha is conse ed in i uses om he eph i id flies. 2006; Koonin e al., 2008). TBLASTN sea ch in he EST da abase had ea lie iden ified ORF3- and ORF4- ela ed EST sequences om he pa asi oid wasp, Nasonia i ipennis (Oli ei a e al., 2010; Eks öm e al., 2011), showing ha his wasp, o possibly i s hos , ha bo ed a i us ha is ela ed o he D osophila No a i us. Seconda y s uc- u e es ima ion showed ha he VP3 homolog in his Nasonia No a i us also had a pu a i e coiled coil domain in he N- e minal pa o he p o ein (Eks öm e al., 2011). Addi ional No a-like sequences a e now a ailable, allowing a mo e de ailed compa a i e s udy o VP3- ela ed sequences. Th ee no el No a-like i uses ha e ecen ly been desc ibed, wo om he D osophila species D. immig ans and D. subobscu a and one om he mo h Spodop e a exigua (Jakubowska e al., 2014; an Mie lo e al., 2014). Fu he mo e, h ough TBLASTN sea ches in he T ansc ip ome Sho gun Assembly da abase, we could e ie e addi ional No a-like i us sequence assemblies om h ee eph i id flies, wo addi ional mo hs, one an and one pa a- si oid wasp (Table S2), as well as sho e sequence agmen s om se e al o he insec species. These sequences we e defined as No a- like by wo c i e ia: a) hey encode ORF4 capsid p o eins o he unique No a i us ype and b) hei eplica ion casse es a e mo e closely ela ed o he D. melanogas e No a i us ORF2 p o eins han o o he pico na-like homologs. We will he e e e o all No a i us homologs as No a i uses. No iceably, he ela ionship be ween he di e en No a i uses closely mi o s ha o hei insec hos s, sug- ges ing a long his o y o co-e olu ion. ORF1 and ORF3 sequences we e poo ly conse ed be ween he di e en No a i uses, bu an ORF3 homolog could always be iden ified as a sepa a e open ead- ing ame be ween he be e conse ed ORF2 and ORF4- ela ed sequences. Fig. 2 illus a es p edic ed seconda y s uc u e elemen s in he VP3 p o ein o D. melanogas e No a i us. A egion spanning he fi s app oxima ely 200 amino acids displays a high p obabili y o an ␣-helical con o ma ion ( ed cu e in Fig. 2A), wi h a high p ob- abili y o coiled-coil o ma ion be ween esidues 36–200 (black cu e), while he egion downs eam is p edic ed o ha e s e ches o ␤-s ands (blue cu e) in e up ed by ␤- u ns (g ey-shaded in Fig. 2B). This p edic ion o a seconda y s uc u e is ema kably con- se ed among he No a i uses, in pa icula o he dip e an and lepidop e an i uses (Fig. S1). Howe e , he p ima y amino acid sequences a e poo ly conse ed, allowing a consis en alignmen only o he i uses om he h ee D osophila species (Fig. S2). Only when we used an algo i hm ha akes seconda y s uc u es in o conside a ion was i possible o align VP3 sequences om all No a- like i uses (Fig. S3). A bes , we see a modes ex en o sequence conse a ion o amino acids 1–200, while he egion downs eam is e y poo ly conse ed, ba ing a ew amino acid posi ions. A sequence simila i y wi h a consis ency sco e o 5 o abo e was seen only a 19 amino acid posi ions, all o which all wi hin he ␣-helical egion. Hence, seconda y s uc u e p edic ion is conse ed, despi e he modes sequence simila i y, sugges ing a i al unc ion o he ␣-helical s uc u al domain. A close inspec ion o he p edic ed coiled coil egion shows u - he in e es ing ea u es in VP3 o he D. melanogas e No a i us. Unlike he Ma coil p edic ion (solid black line in Fig. 2A), he Pai - coil algo i hm p edic s ha he coiled coil is spli in o h ee sepa a e segmen s, labeled CC1-3 in Fig. 2A, wi h a ela i ely low coiled coil p obabili y o he middle segmen (dashed line in Fig. 2). The epea uni o a coiled coil is se en amino acids, o abou wo u ns o he ␣-helix. Fig. 2B illus a es he colo -coded p obabili y o each amino acid in VP3 o be in any o hese se en posi ions, labeled a-g. Fo he p edic ed coiled coil segmen CC3 his gene a es a eg- ula pa e n whe e each 7-amino acid epea is ep esen ed as a ed diagonal. The phase o his pa e n shi s by one posi ion a he bo de be ween CC2 and CC3, sugges ing a dis up ion o he coiled coil s uc u e a his poin . A second se o diagonals in CC1 and CC2, shi ed by h ee posi ions om he majo diagonals, indica es wo possible o ien a ions o hese segmen s. In he D. immig ans No a i us he e a e p edic ed phase shi s be ween CC1 and CC2, as well as be ween CC2 and CC3 (Fig. S1), sugges ing discon inu- i ies be ween all h ee segmen s. Finally, while no phase shi s a e p edic ed in he co esponding coiled coil egion o he No a i us om D. subobscu a, a helix-b eaking p oline esidue a he bo de be ween CC2 and CC3 (black line in Fig. 2C) indica es ha he egu- la ␣-helical con o ma ion is in e up ed in his place. We conclude ha he mos likely con o ma ion o VP3 in all h ee D osophila i uses include wo o h ee sepa a e coiled coil segmen s. O hese, 24 S.A. Sadanandan e al. / Vi us Resea ch 223 (2016) 20–27 a Mul icoil p edic ion sugges s ha CC1 is mos likely engaged in coiled coil ime s, while he p edic ion o CC2 is mo e unce ain (g een cu es in Fig. 2 and Fig. S1). The p edic ion o a coiled coil ime is consis en wi h ou obse a ion wi h GEMMA ha VP3 has a endency o o m ime s (Fig. 1A). Howe e , CC3 is p edic ed o o m a dime in he D. immig ans and D. subobscu a i uses (pu - ple cu es) and he e is no p edic ion o i s highe o de s uc u e in he D. melanogas e No a i us. A sequence compa ison be ween he h ee D osophila No a i uses (Fig. 2C, Fig. S2) shows ha he CC1 segmen is highly con- se ed; 76% o he posi ions a e iden ical in all h ee species. CC2 is less well conse ed (23% iden ical) and CC3 is e en mo e a iable (12% iden ical) and he la e segmen has long indels a he N- e minus (Fig. S2). Ou side he coiled coil domains, he N- e minal pa is highly conse ed (78%), bu ha egion pa ially o e laps wi h he polyme ase o ORF2 (g ey egion in Fig. 2C and Fig. S2) and i is unce ain how much o ha sequence is included in he ma u e VP3. Finally, he C- e minal 1/3 o VP3, which is domina ed by p edic ed ␤-s ands and u ns, is poo ly conse ed. Only 16% o he esidues a e iden ical in he h ee D osophila i uses, mos o hem in he C- e minal 50 amino acids. The CC1 segmen is also well conse ed in he No a i uses om he eph i id flies, B. la i ons, B. do salis and C. capi a a. A egion o 68 esidues, ex ending 20 esidues ups eam o CC1 and downs eam in o he bo de egion o CC2, is 38–40% iden ical o he co esponding D. melanogas e sequence (Fig. 2C). Like D. sub- obscu a, he eph i id i uses ha e a phase shi in he p edic ed coiled coil a he CC1-CC2 bo de (Fig. S1). Remaining pa s o he eph i id i uses and he en i e sequences o he lepidop e an and hymenop e an i uses a e poo ly conse ed (Fig. S2) and hey can- no be unambiguously aligned wi h he D osophila i us sequences, bu he p edic ed seconda y s uc u es a e simila . All No a i uses ha e a egion wi h po en ial o o m coiled coil ime s. 3.4. VP3 is c i ical o he ecal-o al ansmission o No a i us In o de o cha ac e ize he unc ion o VP3 and he p edic ed coiled-coil mo i , we cons uc ed h ee mu an s (Fig. 3A) using he cDNA clone o No a i us. In he fi s mu an (NV-VP3), h ee consecu i e s op codons we e in oduced 97 nucleo ides down- s eam o he 5end o ORF3, o 24 nucleo ides downs eam o he o e lap wi h ORF2. The second mu an (NV-VP3CC) is a dele ion mu an , whe ein 54 nucleo ides co esponding o fi e u ns o he ␣-helix, we e dele ed om ORF3, dele ing pa s o CC1 and CC2. In he hi d mu an (NV- uncVP3), h ee consecu i e s op codons we e in oduced downs eam o he posi ed coiled-coil. The gene - a ed clones we e mic oinjec ed in o ui fly emb yos o es ablish i al in ec ion. To al RNA was ex ac ed om injec ed animals and hei o sp ing o quan i a i e RT-PCR analysis, in o de o e alu- a e i us i e s (Fig. 3B, no e he loga i hmic alues). The wild- ype cDNA clone o No a i us (NV-W ) was used as a con ol in all expe imen s. In p e ious expe imen s we injec ed fly emb yos wi h No a i us cDNA clones wi h non- eplica ing No a i us genome sequences. A e he injec ed emb yos g ew in o adul we nei he de ec ed emains o he injec ed cDNA no ansc ibed i al RNA. In con as , we de ec ed i us i e s in all h ee i us mu an s es ed in his wo k, indica ing ha he genomes we e eplica ing. The i us i e is educed in he NV-VP3 and NV-VP3CC mu an s, bu i is nea he wild- ype le el in NV- uncVP3. In addi ion, NV-W and he NV- uncVP3 mu an s a e also de ec ed in he o sp ing o he injec ed animals. Howe e , NV-VP3 and NV-VP3CC mu an s a e no de ec able in he o sp ing, abo e he le el whe e non-specific PCR p oduc s can be ound (dashed line in Fig. 3B). This indica es ha , al hough he NV-VP3 and NV-VP3CC mu an s ha e epli- ca ing RNA genomes, hey a e no ansmi ed o he o sp ing. I is in e es ing o no e ha dis up ion o he coiled coil egion leads o almos he same pheno ype as emo ing he en i e VP3 p o ein. Dele ing he egion downs eam o he coiled coil appea s o ha e a sligh e ec on he fi ness o he i us, bu no i s eplica ion o ansmission, since he NV- uncVP3 mu an is p esen bo h in he injec ed animals and hei o sp ing. This signifies ha he coiled coil egion is he impe a i e domain o VP3 and ha i is essen- ial o he ansmission o No a i us ia he ecal-o al ou e. This opens up se e al possibili ies, one o which is a ole o his egion in he assembly o i us pa icles. We p oceeded o examine whe he hese mu an s we e capa- ble o assembling i ions. To explo e his possibili y, we pu ified i us pa icles om whole fly ex ac and eces, collec ed om flies in ec ed wi h NV-W , NV-VP3, NV-VP3CC o NV- uncVP3. Ra e-zonal sepa a ion was used o pu i y he i us pa icles and quan i a i e RT-PCR was pe o med on o al RNA isola ed om he suc ose-g adien ac ions (Fig. 3C). In his scena io, any sig- nal ob ained o he i us will ha e o igina ed om assembled, in ac i ions. Also, unassembled capsid p o eins will sedimen e y di e en ly as compa ed o assembled i us pa icles. Hence, his me hod would gi e us an indica ion as o whe he i ions ha e been assembled o he di e en mu an s. Assembled i us pa icles we e de ec ed in in ec ed flies o all h ee mu an s, sig- ni ying ha mu a ion o VP3 did no a ec he ac ual assembly o he i ions. Pa icles we e no de ec ed in eces o he NV-VP3 and NV-VP3CC mu an s in con as o NV-W and NV- uncVP3 mu an pa icles, which we e p esen in he eces also. This implied ha , al hough i ions a e assembled o all he mu an s, only he NV- uncVP3 pa icles can be de ec ed in he eces. The ac ha he NV-VP3 and NV-VP3CC pa icles a e no de ec ed in he eces explains why hey a e no ho izon ally ansmi ed o he o sp ing. Taken oge he , hese esul s indica e ha he pu a i e coiled- coil mo i o VP3 is no in ol ed in he eplica ion o he i al genome o in he assembly o he i ions. This domain is ins ead c i ical o he success ul anspo o he assembled pa icles o he eces o o he s abili y o he pa icles du ing o a e ans- po a ion o he eces. 3.5. VP3-coiled coil domain aids ansmission by s abilizing he i us pa icles The e a e se e al p o eases and enzymes wi hin in ec ed cells ha can cause ha m o i ions. Addi ionally, since No a i us epli- ca es in he ui fly midgu (Habayeb e al., 2009), i a els h ough he hos ile en i onmen o he fly gu , wi h a ying pH condi ions, diges i e enzymes and p o eases (Lemai e and Miguel-Aliaga, 2013), be o e i eaches he eces. Upon a i al in he eces, he i ions may once again ace enzymes and p o eases, sec e ed by de eca ed gu mic obes, be o e success ully in ec ing a new hos . We pos ula ed ha he conse ed coiled-coil domain was c ucial o he s abili y o he i ions agains ad e se condi ions wi hin in ec ed cells, ei he while hey unde ook he pe ilous passage h ough he fly gu , o upon de eca ion along wi h he eces. Fi s , we compa ed he s abili y o hea ed mu an pa icles o ha o wild ype pa icles. Fo his pu pose, NV-W and mu an i i- ons we e pu ified om in ec ed flies by a e-zonal sepa a ion and hei con en o i al RNA was quan ified as shown in Fig. 3C. Only in ac assembled i us pa icles will sedimen a ac ions close o he sedimen a ion a e o wild- ype pa icles. Hence, RNA de ec ed by quan i a i e RT-PCR would ha e o igina ed om assembled i i- ons o i ion-like s uc u es. The 2.5 ml and 3.0 ml sedimen a ion ac ions we e pooled and aliquo ed in o wo equal olumes. One ba ch was subjec ed o hea ea men a 55◦C o 5 min, ollowed by RNase A ea men o 30 min a 37◦C. The second ba ch was subjec ed only o RNase A ea men . To al RNA was ex ac ed om bo h se s o pa icles o each mu an and analyzed by quan- i a i e RT-PCR. Any signal ob ained would ha e o igina ed om S.A. Sadanandan e al. / Vi us Resea ch 223 (2016) 20–27 25 0 10000 20000 1.0 1.5 2. 0 2.5 3.0 3.5 4.0 4.5 Vi us i e, a bi a y uni s Suc ose g adien ac ions, ml 0 10000 20000 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 Suc ose g adien ac ions, ml ORF 1 ORF 2 ORF 3 ORF 4 No a i us genome Helicase P o ease Polyme ase Capsid p o eins ORF 3 S op NV- VP3 ORF 3 P edic ed coiled coil ORF 3 ORF 3 Dele ion S op P edic ed coiled coil P edic ed coiled coil NV-VP3 CC NV- uncVP3 NV-w ...CGCCGTTT AATGAAGA......GTTTAGAA ...GAATTTAC NV-W NV- VP3 NV-VP 3 CC NV- uncVP3 A 0 2 4 6 8 10 Log i us i e, a bi a y uni s Injec ed animals O sp ing nd B C Whole lies Feces Vi us i e, a bi a y uni s NV-ΔVP3 NV-W NV- uncVP3 NV-VP3ΔCC Fig. 3. VP3 is equi ed o he ansmission o No a i us ia he ecal-o al ou e. (A) Schema ic ep esen a ion o No a i us VP3 mu an s gene a ed. (B) No a i us was quan ified by quan i a i e RT-PCR on o al RNA om RelishE23 flies and hei o sp ing, in ec ed wi h ei he unmodified No a i us (NV-W ) o any one o he VP3 mu an s (NV-VP3, NV-VP3CC and NV- uncVP3). The do ed line ep esen s he poin below which PCR a i ac s appea , a e 35 cycles o amplifica ion. Such signals a e also obse ed in nega i e con ols wi hou any empla e RNA. (C) Vi us pa icles we e pu ified om whole RelishE23 flies o om eces o such flies, in ec ed wi h ei he NV-W o any one o he VP3 mu an s, by a e-zonal sepa a ion on a suc ose g adien . Quan i a i e RT-PCR was pe o med on o al RNA ex ac ed om each collec ed ac ion o he suc ose g adien . Fo (B) and (C), he esul s shown a e an a e age o a leas 3 independen expe imen s and e o ba s indica e s anda d de ia ion. Be ween 5–30 flies we e used o each expe imen , and he same numbe o flies we e used o he con ol and he mu an being es ed in ha pa icula expe imen . in ac i us pa icles, as ee RNA eleased due o capsid disin e- g a ion would be deg aded du ing RNase A ea men . This would hence gi e us an es ima e o he pa icles ha su i ed hea ea - men wi hou deg ada ion. As shown in Fig. 4A, he NV-W and NV- uncVP3 pa icles esis hea ea men and can be de ec ed a i e s close o he unhea ed con ols. On he o he hand, he NV-VP3 and NV-VP3CC pa icles a e almos en i ely decima ed (a leas 1000- old dec ease) and can ba ely be de ec ed a e hea ea men . This clea ly shows ha he abili y o No a i us pa icles o wi hs and hea ea men is dependen on he coiled-coil mo i bu no he C- e minal hi d o he VP3 p o ein. We nex p oceeded in a simila way o examine whe he VP3 was impo an o he s abili y o he i ions when ea ed wi h p o eases. The combined 2.5 ml and 3.0 ml ac ions o a e-zonally pu ified NV-W and mu an i us pa icles we e di ided in o h ee equal olumes. The fi s se was ea ed wi h ypsin and he second se was ea ed wi h panc ea ic p o ease, bo h ollowed by ea - men wi h p o ease inhibi o cock ail and RNase A. The hi d se used as con ol was ea ed only wi h p o ease inhibi o and RNase A. Quan i a i e RT-PCR was pe o med on o al RNA om he pa - icles. Like in he hea - ea men expe imen , he NV-W pa icles su i ed bo h ypsin and p o ease ea men wi hou any signi - ican loss o i ions (Fig. 4B). The NV- uncVP3 i ions appea o be only pa ially esis an o ypsin and p o ease ea men as a en- old loss o i ions was obse ed. On he o he hand, NV-VP3 and NV-VP3CC mu an pa icles we e almos comple ely disin- eg a ed as e y li le i al RNA was de ec ed om hese pa icles. This clea ly signifies he impo ance o VP3 in he s abili y o he capsid s uc u e unde p o ease ea men . Based on he esul s om he hea - ea men expe imen and he p o ease clea age analysis, i is appa en ha VP3 is c i ical o he s abili y o No a i us i ions. Specifically, he coiled-coil mo i appea s o be a unc- ionally impo an domain in ol ed in con e ing s abili y o he capsid s uc u e. The egion downs eam o he coiled-coil also 26 S.A. Sadanandan e al. / Vi us Resea ch 223 (2016) 20–27 0 1 2 3 4 5 Log i us i e, a bi a y uni s RNase ea men Hea ➔ RNase ea men Log i us i e, a bi a y uni s 0 1 2 3 4 5 P o ease inhibi o ➔ RNase ea men P o ease ➔ P o ease inhibi o ➔ RNase ea men T ypsin ➔ P o ease inhibi o ➔ RNase ea men A B NV-ΔVP3 NV-W NV- uncVP3 NV-VP3ΔCC NV-ΔVP3 NV-W NV- uncVP3 NV-VP3ΔCC Fig. 4. VP3 is c ucial o he s abili y o No a i us Vi ions. (A) Pu ified i us pa icles we e hea - ea ed a 55◦C o 5 min, ollowed by RNase A ea men . Con ol samples we e ea ed only wi h RNase. qRT-PCR was pe o med on RNA om bo h hea - ea ed and unhea ed pa icles o es ima e i us i e . This g aph shows he loss o NV-VP3 and NV-VP3CC i ions upon hea - ea men . (B) Pu ified i us pa icles we e 1) ea ed wi h ypsin, ollowed by ea men wi h a p o ease inhibi o cock ail and RNase, 2) ea ed wi h p o ease, ollowed by ea men wi h a p o ease inhibi o cock ail and RNase, 3) ea ed wi h only he p o ease inhibi o cock ail and RNase. Quan i a i e RT-PCR was pe o med on RNA om all pa icles o calcula e i us i e . This g aph shows he se e e disin eg a ion o NV-VP3 and NV-VP3CC i ions and a pa ial loss o NV- uncVP3 i ions upon ea men wi h ypsin and p o ease. Fo (A) and (B), he esul s shown a e an a e age o 3 expe imen s, whe ein he e o ba s indica e s anda d de ia ion. Be ween 10–20 flies we e used o each expe imen , bu he same numbe o flies we e used o he con ol and he mu an being es ed in ha pa icula expe imen . The do ed line ep esen s he poin below which all alues a e conside ed o be PCR a i ac s since hese a e ob ained om quan i a i e RT-PCR signals ha appea a e 35 cycles o amplifica ion. Such signals a e also obse ed in nega i e con ols wi hou any empla e RNA. seems o ha e a mode a e e ec on he s abili y o capsid p o eins, bu i is appa en ly no c ucial. 4. Discussion Ou esul s show ha No a i us VP3 is a p ima ily ␣-helical p o ein ha o ms ime s in i s na i e con o ma ion. The ␣-helical egion is p edic ed o o m a coiled-coil domain, spli in o h ee pa s, and compa isons wi h sequences om o he No a i uses ha e shown ha his a angemen is well conse ed. To he bes o ou knowledge, no known s uc u al p o eins o he pico na- like i uses ha e been p edic ed o shown o ha e his mo i . The appea ance o VP3, along wi h VP4 p o eins in i us pa icles (Eks öm e al., 2011) sugges ed ha i should be associa ed wi h he capsid p o eins. I was shown ha he s uc u al p o eins o No a i us a e dis inc ly di e en om ha o known pico na-like i uses (Eks öm e al., 2011), bu he ole o VP3 o i s associa ion wi h he capsid has no been s udied be o e. In Pico na i idae, each capsid p o ein, VP1, VP2, VP3 and VP4 (un ela ed o he No a i us p o eins wi h simila names), has a ole in he di e en s ages o capsid assembly, ma u a ion and s a- bili y (A nold e al., 1987; Hellen and Wimme , 1992a; Hellen and Wimme , 1992b; Cu y e al., 1997; Lin e al., 2009; Jiang e al., 2014). Hence, mu a ions in any o hese p o eins can ad e sely a ec one o mo e s ages o he i us li e cycle. Fo example, mu a- ions in he Polio i us VP2 p o ein can dis up he comple ion o mo phogenesis and he e en ual o ma ion o ma u e i al pa i- cles (Comp on e al., 1990). Fu he , single amino acid subs i u ions in he Foo and Mou h disease i us VP1 p o ein can ende he i us acid-s able, while amino acid subs i u ions in he VP3 p o- ein ende he i us acid-labile (Ca idi e al., 2015). Simila e ec s ha e been obse ed in o he i us amilies such as he Calici i idae and Toga i idae also. In he No walk i us, he VP2 p o ein asso- cia ed wi h he shell domain o he i al capsid (Vongpunsawad e al., 2013) is known o be c ucial o he exp ession and s abili y o he capsid p o eins (Be olo i-Cia le e al., 2003). An Alpha i us nucleocapsid p o ein con ains a p edic ed ␣-helical coiled coil domain ha is essen ial o he assembly o he nucleocapsid co e (Pe e a e al., 2001). Simila ly, he coiled coil domain o adeno i us p o ein pIX is s a ed o ha e an impo an ole in sel -associa ion and ime o ma ion o pIX molecules (Rosa-Cala a a e al., 2001). The co ec s uc u al associa ion o pIX is c i ical o he icosahe- d al capsid s uc u e o adeno i uses ( an Oos um and Bu ne , 1985; Fu cini i e al., 1989; S ewa e al., 1993). Thus, he ole o ce ain capsid-associa ed p o eins in he assembly, s abili y and ma u a ion o i ions ha e been p o en in se e al i uses. Ou wo k on No a i us VP3 has e ealed ha his capsid-associa ed p o ein is indispensable o he s abili y o he i ion s uc u e. Dis up ing he coiled-coil domain o No a i us VP3 has se ious implica ions on he s abili y o he i ions, such ha i a ec s he ho izon al ansmission o he i us. This u he ein o ces ou p emise ha No a i us VP3 is absolu ely essen ial o he s uc u al s abili y o he capsid p o eins. To unde s and he molecula mechanisms in ol ed in capsid s abili y is impo an no only o gain knowledge o an elemen a y aspec o No a i us biology, bu also o cha ac e ize he hos - pa hogen ela ionship be ween a small RNA i us and i s hos . This ela ionship be ween No a i us and he ui fly also happens o be an excellen model o s udy no el pa hways in ol ed in immune esponses agains small RNA i uses. Unde s anding he biology o i uses also helps us unde s and be e he di e en echniques hey use o modi y cellula beha io and o supp ess an i- i al esponses. I would indeed be e y in e es ing o u he dissec he mechanism behind how VP3 p o ides s abili y o he No a i us i ions. Conflic o in e es s The au ho s decla e ha he e is no conflic o in e es s. S.A. Sadanandan e al. / Vi us Resea ch 223 (2016) 20–27 27 Acknowledgemen s The pu ifica ion o VP3 was planned and pe o med by he Umeå P o ein Expe ise Pla o m. We hank Jö gen Åden (Depa men o Chemis y, Umeå Uni e si y) o helping us pe o m Ci cula Dich oism on VP3. This esea ch was suppo ed by g an s om he Swedish Resea ch Council, he Academy o Finland and he Sig id Juselius Founda ion. Appendix A. Supplemen a y da a Supplemen a y da a associa ed wi h his a icle can be ound, in he online e sion, a h p://dx.doi.o g/10.1016/j. i us es.2016.06. 011. Re e ences Ande son, N.G., Ha is, W.W., Ba be , A.A., Rankin J ., C.T., Candle , E.L., 1966. Sepa a ion o subcellula componen s and i uses by combined a e- and isopycnic-zonal cen i uga ion. J. Na l. Cance Ins . Monog . 21, 253–283. A nold, E., Luo, M., V iend, G., Rossmann, M.G., Palmenbe g, A.C., Pa ks, G., Nicklin, M.J.H., Wimme , E., 1987. Implica ions o he pico na i us capsid s uc u e o polyp o ein p ocessing. P oc. Na l. Acad. Sci. 84, 21–25. Baj, A., Colombo, M., Headley, J.L., McFa lane, J.R., Lie ho , M.A., Toniolo, A., 2015. Pos -poliomyeli is synd ome as a possible i al disease. In . J. In ec . Dis. 35, 107–116. Böhm, G., Muh , R., Jaenicke, R., 1992. Quan i a i e analysis o p o ein a UV ci cula dich oism spec a by neu al ne wo ks. P o ein Eng. 5, 191–195. Be olo i-Cia le , A., C aw o d, S.E., Hu son, A.M., Es es, M.K., 2003. The 3’ end o No walk i us mRNA con ains de e minan s ha egula e he exp ession and s abili y o he i al capsid p o ein VP1: a no el unc ion o he VP2 p o ein. J. Vi ol. 77, 11603–11615. Ca idi, F., Vázquez-Cal o, A., Sob ino, F., Ma ín-Acebes, M.A., 2015. The pH s abili y o oo -and-mou h disease i us pa icles is modula ed by esidues loca ed a he pen ame ic in e ace and in he N e minus o VP1. J. Vi ol. 89, 5633–5642. Comp on, S.R., Nelsen, B., Ki kegaa d, K., 1990. Tempe a u e-sensi i e polio i us mu an ails o clea e VP0 and accumula es p o i ions. J. Vi ol. 64, 4067–4075. Cu y, S., F y, E., Blakemo e, W., Abu-Gazaleh, R., Jackson, T., King, A., Lea, S., Newman, J., S ua , D., 1997. Dissec ing he oles o VP0 clea age and RNA packaging in pico na i us capsid s abiliza ion: he s uc u e o emp y capsids o oo -and-mou h disease i us. J. Vi ol. 17, 9743–9752. Delo enzi, M., Speed, T., 2002. An HMM model o coiled-coil domains and a compa ison wi h PSSM-based p edic ions. Bioin o ma ics 18, 617–625. Edwin, A., G unds öm, C., Wai, S.N., Öhman, A., S ie , G., Saue -E iksson, A.E., 2014. Domain isola ion, exp ession, pu ifica ion and p o eoly ic ac i i y o he me allop o ease P V om Vib io chole ae. P o ein Exp . Pu i . 96, 39–47. Eks öm, J.O., Habayeb, M.S., S i as a a, V., Kieselbach, T., Wingsle, G., Hul ma k, D., 2011. D osophila No a i us capsid p o eins di e om hose o o he pico na-like i uses. Vi us Res. 160, 51–58. Fu, W., Lin, J., Cen, P., 2007. 5-Aminole ulina e p oduc ion wi h ecombinan Esche ichia coli using a a e codon op imize hos s ain. Appl. Mic obiol. Bio echnol. 75, 777–782. Fu cini i, P.S., an Oos um, J., Bu ne , R.M., 1989. Adeno i us polypep ide IX e ealed as capsid cemen by di e ence images om elec on mic oscopy and c ys allog aphy. EMBO J. 8, 3563–3570. G eenfield, N.J., 2006. Using ci cula dich oism spec a o es ima e p o ein seconda y s uc u e. Na . P o oc. 1, 2876–2890. Habayeb, M.S., Ekeng en, S.K., Hul ma k, D., 2006. No a i us, a pe sis en i us in D osophila, defines a new pico na-like i us amily. J. Gen. Vi ol. 87, 3045–3051. Habayeb, M.S., Can e a, R., Casano a, G., Eks öm, J.O., Alb igh , S., Hul ma k, D., 2009. The D osophila No a i us is an en e ic i us, ansmi ed ia eces. J. In e eb . Pa hol. 101, 29–33. Hedeng en, M., Åsling, B., Dushay, M.S., Ando, I., Ekeng en, S., Wihlbo g, M., Hul ma k, D., 1999. Relish, a cen al ac o in he con ol o humo al bu no cellula immuni y in D osophila. Mol. Cell 4, 827–837. Hellen, C.U.T., Wimme , E., 1992a. Ma u a ion o polio i us capsid p o eins. Vi ology 187, 391–397. Hellen, C.U.T., Wimme , E., 1992b. The ole o p o eoly ic p ocessing in he mo phogenesis o i us pa icles. Expe ien ia 48, 201–215. Jakubowska, A.K., D’Angiolo, M., González-Ma ínez, R.M., Millán-Lei a, A., Ca ballo, A., Mu illo, R., Caballe o, P., He e o, S., 2014. Simul aneous occu ence o co e in ec ions wi h small RNA i uses in he lepidop e an Spodop e a exigua. J. In e eb . Pa hol. 121, 56–63. Jiang, P., Liu, Y., Ma, H.C., Paul, A.V., Wimme , E., 2014. Pico na i us mo phogenesis. Mic obiol. Mol. Biol. Re . 78, 418–437. Koonin, E.V., Wol , Y.I., Nagasaki, K., Dolja, V.V., 2008. The Big Bang o pico na-like i us e olu ion an eda es he adia ion o euka yo ic supe g oups. Na . Re . Mic obiol. 6, 925–939. Laemmli, U.K., 1970. Clea age o s uc u al p o eins du ing he assembly o he head o he bac e iophage T4. Na u e 227, 680–685. Lemai e, B., Miguel-Aliaga, I., 2013. The diges i e ac o D osophila melanogas e . Annu. Re . Gene . 47, 377–404. Lin, J.Y., Chen, T.C., Weng, K.F., Chang, S.C., Chen, L.L., Shih, S.R., 2009. Vi al and hos p o eins in ol ed in pico na i us li e cycle. J. Biomed. Sci. 16, 103. McDonnell, A.V., Jiang, T., Kea ing, A.E., Be ge , B., 2006. Pai coil2: imp o ed p edic ion o coiled coils om sequence. Bioin o ma ics 22, 356–358. Oli ei a, D.C.S.G., Hun e , W.B., Ng, J., Desja dins, C.A., Dang, P.M., We en, J.H., 2010. Da a mining cDNAs e eals h ee new single s anded RNA i uses in Nasonia (Hymenop e a: P e omalidae). Insec Mol. Biol. 19 (Suppl. 1), 99–107. Pe e a, R., Owen, K.E., Tellinghuisen, T.L., Go balenya, A.E., Kuhn, R.J., 2001. Alpha i us nucleocapsid p o ein con ains a pu a i e coiled coil ␣-helix impo an o co e assembly. J. Vi ol. 75, 1–10. Pe e sen, B., No dahl Pe e sen, T., Ande sen, P., Nielsen, M., Lundegaa d, C., 2009. A gene ic me hod o assignmen o eliabili y sco es applied o sol en accessibili y p edic ions. BMC S uc . Biol. 9, 51. Pe e sen, B., Lundegaa d, C., No dahl Pe e sen, T., 2010. Ne Tu nP − neu al ne wo k p edic ion o be a- u ns by use o e olu iona y in o ma ion and p edic ed p o ein sequence ea u es. PLoS One 5, e15079. P isco, G.D., Zhang, X., Pennacchio, F., Cap io, E., Li, J., E ans, J.D., DeG andi-Ho man, G., Hamil on, M., Chen, Y.P., 2011. Dynamics o pe sis en and acu e de o med wing i us in ec ions in honey bees, Apis melli e a. Vi uses 3, 2425–2441. P yo , K.D., Lei ing, B., 1997. High-le el exp ession o soluble p o ein in Esche ichia coli using a His6- ag and mal ose-binding-p o ein double-a fini y usion sys em. P o ein Exp . Pu i . 10, 309–319. Ro ouga an, R., C ona, M., Vodnala, M., Sjöbe g, B.M., Ho e , A., 2008. Oligome iza ion s a us di ec s o e all ac i i y egula ion o he Esche ichia coli class Ia ibonucleo ide educ ase. J. Biol. Chem. 283, 35310–35318. Rosa-Cala a a, M., G a e, L., Pu ion-Du illeul, F., Cha on, B., Kedinge , C., 2001. Func ional analysis o adeno i us p o ein IX iden ifies domains in ol ed in capsid s abili y, ansc ip ional ac i i y, and nuclea eo ganiza ion. J. Vi ol. 75, 7131–7141. Sp adling, A.C., 1986. P elemen -media ed ans o ma ion. In: Robe s, D.B. (Ed.), D osophila: A P ac ical App oach. IRL P ess, Ox o d, pp. 175–197. S ewa , P.L., Fulle , S.D., Bu ne , R.M., 1993. Di e ence imaging o adeno i us: b idging he esolu ion gap be ween X- ay c ys allog aphy and elec on mic oscopy. EMBO J. 12, 2589–2599. an Mie lo, J.T., B onkho s , A.W., O e heul, G.J., Sadanandan, S.A., Eks öm, J.-O., Hees e mans, M., Hul ma k, D., An oniewski, C., an Rij, R.P., 2012. Con e gen e olu ion o a gonau e-2 slice an agonism in wo dis inc insec RNA i uses. PLoS Pa hog. 8, e1002872. an Mie lo, J.T., O e heul, G.J., Obadia, B., an Clee , K.W.R., Webs e , C.L., Saleh, M.-C., Obba d, D.J., an Rij, R.P., 2014. No el D osophila i uses encode hos -specific supp esso s o RNAi. PLoS Pa hog. 10, e1004256. an Oos um, J., Bu ne , R.M., 1985. Molecula composi ion o he adeno i us ype 2 i ion. J. Vi ol. 56, 439–448. Vongpunsawad, S., Venka a am P asad, B.V., Es es, M.K., 2013. No walk i us mino capsid p o ein VP2 associa es wi hin he VP1 shell domain. J. Vi ol. 87, 4818–4825. Wol , E., Kim, P.S., Be ge , B., 1997. Mul iCoil: a p og am o p edic ing wo- and h ee-s anded coiled coils. P o ein Sci. 6, 1179–1189. Yang, H., K onhamn, J., Eks öm, J.O., Ko ku , G.G., Hul ma k, D., 2015. JAK/STAT signaling in D osophila muscles con ols he cellula immune esponse agains pa asi oid in ec ion. EMBO Rep. 16, 1664–1672.