J. Mol. Biol. (1995) 252, 379–385

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The Avian Adenovirus Penton: Two Fibres andOne Base

Michael Hess 1, Alain Cuzange 2, Rob W. H. Ruigrok 3

Jadwiga Chroboczek 2 and Bernard Jacrot 2*

1Institute of Poultry Diseases The penton capsomer of mammalian adenoviruses consists of a trimeric, longFree University of Berlin and thin fibre inserted into a pentameric base. The avian adenoviruses

possess a penton which presents another symmetry mismatch: eachKoserstrasse 21, 1000 Berlinpentameric base is associated with two fibres. Here we have studied the33, Germanymorphology of the penton of CELO virus, an avian adenovirus, and we have2Institut de Biologie determined the sequence of both fibres, one long and one short. The short fibre

Structural, 41 Avenue des is probably associated with the base in the same way as the mammalian viralMartyrs, 38027 Grenoble fibres and we will discuss how the long fibre could be attached. The shaftscedex 1, France of all known adenovirus fibres consist of a series of 15-residue repeats. The

avian virus fibres show a more complicated and less regular shaft repeat3EMBL Grenoble Outstationstructure with single, double and triple repeats. The sequences of the receptorc/o ILL, BP 156, 38042binding (head) domains of both fibres are very different from all other knownGrenoble cedex 9, Francefibre head domains and very different from each other, suggesting that thetwo fibres might bind to different receptors. The genome organization of thesequenced region is rather different from that in human adenoviruses. Inparticular, a region homologous to the human virus E3 region was not foundat the position where it normally occurs in the human virus genome.

7 1995 Academic Press Limited

*Corresponding author Keywords: avian adenovirus; penton; fibre; CELO; receptor binding

Mammalian adenoviruses are icosahedral virusescontaining 240 hexamer and 12 pentamer capsomers.These major capsomer building blocks are cementedtogether by a number of minor capsid proteins(Stewart et al., 1991, 1993). The pentameric capsomeris composed of two types of protein: a penton basewhich is anchored in the capsid and a thin fibre thatextends outward. The fibre consists of an amino-ter-minal tail that is inserted into the base, a thin shaftof variable length depending on the serotype, and acarboxy-terminal knob or head domain containingthe receptor binding site (see review by Chroboczeket al., 1995). The penton presents an interestingstructural mismatch. The penton base is a homo-pen-tamer and the fibre is a homo-trimer (van Oostrum& Burnett, 1985; Ruigrok et al., 1990). In the case ofthe mammalian adenoviruses, it has been suggestedthat this mismatch could be solved by the flexibilityof the tail of the fibre and, on the side of the pentonbase, by the synthesis of polypeptides with different

lengths from two closely spaced initiation codons(van Oostrum & Burnett, 1985; van Oostrum et al.,1987).

The second group of adenoviruses, the avianviruses, has a general architecture like themammalian viruses, but their pentons show adifferent mismatch; every penton base is complexedwith two fibres (Laver et al., 1971; Gelderblom &Maichle-Lauppe, 1982). In order to study thisproblem further, we determined the sequence ofboth fibres of CELO virus (or FAV1) and we re-examined the morphology of the pentons of CELOvirus.

Isolated CELO pentons were observed in anegatively stained preparation of virus. As forhuman and bovine adenovirus pentons (Ruigroket al., 1990, 1994; Kidd et al., 1993), we coulddistinguish two orientations of the penton base(Figure 1), one in which the base is lying on its side(side-view) and one in which the base is lying withits top on the carbon support film (end-on). In theend-on orientation the two fibres must bend in orderto lie flat on the carbon support film. The images ofpentons in end-on view are very similar to thosepublished before (Laver et al., 1971; Gelderblom &

Abbreviations used: CELO virus, chick embryo lethalorphan virus; EM, electron microscopy; FAV, fowladenovirus; HAd, human adenovirus; MAd, mouseadenovirus; ORF, open reading frame.

0022–2836/95/390379–07 $12.00/0 7 1995 Academic Press Limited

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A B

Figure 1. A, Electron micrographs of negatively stained pentons of CELO virus (FAV1). Two views of the penton canbe seen, one with the penton base lying on its side (side view), another with the base lying end-on, showing a rounderor pentagonal outline. In the end-on orientation both fibres need to bend at a 90° angle in order to lie in the plane of thecarbon support film. In the images shown the fibres can be seen to connect to the base, which means that the base isadsorbed onto the film with its upper surface, since the stain that we used (SST, see below) is known to outline mainly(or only) the sides of the proteins that are touching the carbon support film. Virus was grown in primary chicken kidneycells and purified through CsCl gradients as described (Laver et al., 1971; Bauer et al., 1990). Pentons were observed ina sample of purified virus which was applied directly from the CsCl stock onto the carbon film. Sample preparation,negative staining with 1% sodium silicotungstate and low-dose electron microscopy were as described (Ruigrok et al.,1990). The magnification was calibrated using negatively stained crystals of catalase and the magnification bar represents500 A. B, Schematical drawings of the CELO penton in side-view and end-on view (not to scale). The dimensions are givenin A as the mean 2 standard deviation and correspond to the values given in Table 1 where also the number ofmeasurements are given. The question mark in the side-view indicates that we do not know how the long fibre is attachedto the base (see the text).

Maichle-Lauppe, 1982). Two fibres per penton, a longand a short one, can easily be recognised and in mostcases the fibres seem to come out of the centre of thebase. However, in the side-view, it can clearly beseen that the short fibre comes out of the base in astraight manner, like the fibres of the human andbovine viruses (Ruigrok et al., 1990, 1994), but thatthe long fibre comes out at a 90° angle. This long fibreshows two places where it can bend, leading to arather curved bend or to a zig-zag. We found one casewhere the short fibre did not come out straight(Figure 1, top right) but here it also seemed to beattached to the middle of the base whereas the longfibre seemed attached at the side. The observedmorphology suggests that the two fibres on theCELO virus penton are not associated with the basein an equivalent manner.

CELO pentons were measured from positiveprints and dimensions of total penton length, theheight and width of the penton bases and fibre headsare given in Figure 1B and Table 1. The dimensionsof the short fibre plus penton base (which we willname the short penton) were measured both inend-on and side-view (see Figure 1B). The length of

the long CELO penton was only measured on theend-on view pentons (see Figure 1B) since it was notclear where to start the measurement of the long fibrecoming out of the base at a 90° angle. In some casesthe fibre seemed attached to the top of the base, inother cases to the shaft of the short fibre but often anunambiguous decision could not be made on whereto start the measurement.

We sequenced the region of the CELO genomeexpected to contain the two fibres. Since theamino-terminal tails of the fibres associate with thebase, the sequence of these tails, in comparison withthe known sequences of the mammalian virus fibres,may help to understand the structure of the penton.In addition, it might be possible to correlate themorphological features of the fibres with theirsequences. We determined the sequence of 7355 nt,between co-ordinates 63 and 80% (EMBL data baseaccession number: X84724). This region contains tenORFs coding for polypeptides larger than 100 aminoacid residues (Figure 2). The three largest ORFs canbe identified through homologies with correspond-ing proteins in human serotypes. Going from left toright on the viral DNA they code for protein pVIII,

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Table 1. Dimensions of the CELO virus pentonPenton length Derived Fibre head dimensions Penton base

Side-view End-on view shaft-lengtha Height Width Height Width

CELOLong N.D. 583 2 16(57) N.D. 47 2 4(34) 66 2 4(34) 113 2 4(34) 95 2 3(28)Short 268 2 6(34) 232 2 9(91) 128 45 2 4(26) 67 2 3(26)HAd2b 431 2 6 400 2 9 290 49 2 5 56 2 4 100 2 6 94 2 6

Dimensions in A of the penton of CELO virus measured from electron micrographs from negatively stained samples. The values aregiven as the mean 2 standard deviation, with the number of measurements between brackets. The measurements were performed asschematically indicated in Figure 1A.

a The length of the shaft of the fibre can be derived from the dimensions of the intact penton in comparison with the data from theHAd2 penton and fibre and assuming similar dimensions for the CELO penton components. For the HAd2 penton it could be calculatedthat the length of the fibre was 58 A shorter than the length of the penton in side-view and 27 A shorter than the length of the pentonin end-on view. For the short fibre this would lead to two independent estimates of the fibre length of 210 and 205 A, respectively (207 Aaverage). The length of the shaft can be calculated from the fibre length by subtracting the contributions of the height of the head andthe length of the N-terminal tail (for the CELO short fibre 45 and 34 A, respectively).

b The dimension of the penton and fibre head of human adenovirus type 2 are given for comparison (Ruigrok et al., 1990).

a long fibre and a short fibre. The proteinhomologous to pVIII in the human viruses has 246amino acid residues and a molecular mass of26,878 Da. The overall amino acid identity withhuman pVIII (HAd40 or HAd2) is about 25%. Thisgene has a TATA-box 15 nt before the putativeinitiation codon. The nucleotide context of this

initiation codon is close to the consensus sequencefor the start of translation (Kozak, 1989). A secondATG is present 9 nt after the first but is in anunfavourable context for translation initiation. Aputative polyadenylation signal AATAAA is present34 nt after the stop codon. A large ORF coding for aprotein of 792 amino acid residues (Mr 82,918 Da)

Figure 2. Comparison of the genome organisation of CELO and HAd40. The bottom part shows the positions of theCELO genes which have been localised so far, in comparison with their counterparts in HAd40. The position of the genefor the penton base is that of FAV10 (Sheppard & Trist, 1992). The upper part shows the ORFs found in the sequencereported in this article. CELO virus (FAV1, phelps strain) was three times plaque purified and propagated in primarychicken kidney cells from 14-day old specific-pathogen-free chicken. Purification of virus and viral DNA was as describedearlier (Laver et al., 1971; Bauer et al., 1990). The HindIII fragments B (06.9 kbp) and D (04.9 kbp) located between 63%to 80% of the CELO genome according to the physical map (Shimada et al., 1983), were cloned separately into pBluescriptII SK+ vector (Stratagene). DNA was purified with the Qiagen Kit (Helden, Germany) and double-stranded DNAsequencing was performed by the dideoxy chain-termination method with the SequenaseTM version 2.0 DNA Sequencingkit (USB, Cleveland, Ohio, USA) and deoxyadenosine 5'-[a-35S]thiotriphosphate (>1000 Ci/mmol; Amersham). To resolvesome band compressions, dlTP was used as described in the kit. With the exception of the 100 nucleotides on the rightside of the fragment, the sequence was determined on both strands at least twice. The contigs were created and sequenceanalysis was performed using the DNASTAR program.

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Figure 3. Amino acid sequences of the long and short fibres of CELO virus. (a) Tail domains of the long and the shortfibres are shown together with that of the human adenovirus HAd2 (Herisse & Galiber, 1981) and mouse adenovirusMAd1 (Raviprakash et al., 1989) fibres. In bold are shown the homologies between the sequences. The VYPY/F andPPFV/F sequences discussed in the text are boxed. (b) Shaft domains of the CELO long and short fibres and the MAd1fibre (Raviprakash et al., 1989). The sequences in the shaft domain are shown in their repeat form (see the text) alignedby hand. In bold are the conserved residues and boxed are two very homologous triple repeats (see the text). In italicsin the short fibre sequence is a repeat of which we cannot say whether it is an actual shaft repeat or whether it belongsto the head domain. (c) Head domains. In bold and boxed are homologies between the two head domains. The sequencein italics represents the same residues as shown in the shaft domain, i.e. although they are shown twice, they exist onlyonce.

(a)

(b)

(c)

starts 226 nt after the stop codon of pVIII. Thededuced amino acid sequence suggests that thisprotein is the long fibre (see Figure 3). The third largeORF starts 47 nt after the end of the gene for the longfibre. The gap between these two ORFs contains 12successive GCA repeats. The third ORF also containstwo closely spaced ATGs at the beginning of thegene. In view of their nucleotide context it seemslikely that translation starts at the second ATG, 57 ntdownstream from the first, leading to a protein with

a molecular mass of 41,134 Da. The stop codon is partof a polyadenylation signal, AATAAA, a situationidentical to that found for the fibre gene in HAd2.The amino acid sequence of this third ORF alsocorresponds to that of an adenovirus fibre (Figure 3).The molecular weight of the fibres may be comparedwith the data by Li et al. (1984) who showed SDS/gels of the CELO penton consisting of three proteinswith apparent molecular masses 44.5, 65 and 92 kDa.The polypeptide of the penton base of FAV10 has a

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molecular mass of 57.4 kDa and it is likely that thepenton base of CELO has a similar size (Sheppard &Trist, 1992). This would mean that the 44.5 and92 kDa bands correspond to the fibres, in reasonableagreement with the molecular masses suggested here.

The genome of the avian adenoviruses seems tocontain between 43 to 47 kb (Sheppard & Trist, 1992,1993; Cai & Weber, 1993), some 7 to 12 kb largerthan that of the mammalian viruses (Roberts et al.,1984; Chroboczek et al., 1992; Davison et al., 1993;Sprengel et al., 1994). We have tried to compare theorganisation of the genome of CELO virus with thatof HAd40, the human serotype which also containstwo fibre genes (Figure 2). In human adenovirusesthe genes for pVIII and for the fibre are separated byseveral thousands of nucleotides. The E3 transcrip-tion unit is essentially located in this interval,although it does overlap with these two late genes.We found no homologies with human adenovirus E3products, neither in the ORFs of the fragmentsequenced here, nor in the sequences between 80and 100% (Akopian et al., 1990; Akopian et al.,unpublished sequence, accession number Z172160).The CELO equivalent of E3, if it exists, does not seemto be in the same localisation as that in humanadenoviruses. This may be important for those whowant to replace the hypothetical E3 region ofavian adenovirus for gene transfer purposes. It isclear from Figure 2 that the layout of the genes for thestructural proteins of CELO and HAd40 is quitesimilar. However, in the CELO genome, there seemsto be an extra 8 kb downstream of the fibre genes andwe do not yet know what they code for.

The sequences for the long and the short fibres areshown in Figure 3(a) to (c). The sequences can bedevided into tail, shaft and head domains and inthe following we will discuss the specific CELOsequence features and try to relate the observedCELO penton morphology to the sequence. Theamino terminus of the short fibre shows stronghomology with that of the mammalian virus fibres,as shown in Figure 3(a) for the HAd2 fibre tail. Thefirst shaft repeat of HAd2 follows directly after theend of the tail sequence shown. However, the first 30residues of the N-terminal sequence of the long fibreare very different from that of the other fibres. Thenfollows a region of strong homology with 19 residuesof the tail domains of the short CELO fibre and thehuman virus fibres and then a poly(G) stretch. InFigure 3(a) we have included this poly(G) in the taildomain but it may also form a connection betweenthe tail and the shaft without belonging to either. TheKRAR sequence in the HAd2 fibre (residues 2 to 5)is probably a nuclear localization signal (Hong &Engler, 1991). This sequence is missing in the CELOfibres, although the KKPR in the long fibre (residues20 to 23) could have the same function.

The shaft of human and bovine adenovirusfibres contains a series of 15 amino acid repeatswith the following consensus sequence: 1-HyXHyX-HyXXP(G)LXHyXXXX-15, where Hy stands for ahydrophobic residue and P(G) indicates proline orglycine at position 8, giving PL or GL repreats (Green

et al., 1983; Kidd et al., 1993; Chroboczek et al., 1995).The first three residues (Hy-X-Hy) and residuesL-X-Hy are thought to form short b-strands and theintervening sequences sharp turns (Green et al.,1983). A model for the three-dimensional structure ofthe shaft, based on the features of these repeats(motifs), consists of a triple helical coil in which theb-strands form inter-chain hydrogen bonds, result-ing in three long and thin b-sheets running along thelength of the shaft, with all hydrophobic residuesburied in the interior (Stouten et al., 1992). However,the shafts of the canine and murine adenovirus fibres(Dragulev et al., 1991; Raviprakash et al., 1989) showa slightly more complicated organisation (see MAd1in Figure 3(b) and Chroboczek et al., 1995). Largesections of the shafts of the dog and mouse virusfibres are made up of double repeats consisting ofalternating, irregular 20-residue PL repeats and veryregular 15-residue GL repeats.

The shafts of the long and short CELO fibres showa similar but more complicated pattern (Figure 3(b)).The short fibre shaft consists of four long repeats of32 to 37 residues, each comprising a more irregularfirst half (left part of Figure 3(b)) characterized byhydrophobic residues at positions 1 and 3, aconserved Asp at position 6, but no PL or GL,followed by a more regular 15-residue second half(right part of Figure 3(b)). The end of the short fibreshaft is not quite clear. We have shown the samerepeat in italics both at the end of the shaft domainand at the beginning of the head (Figure 3(b) and (c),respectively) to indicate that it could belong to either.Therefore, the shaft of the short fibre consists eitherof four double repeats (equivalent of eight ‘‘single’’repeats) plus possibly a ninth single repeat. Theamino-terminal part of the CELO short fibre hasprobably the same length as that of the HAd2 fibre,34 A (Ruigrok et al., 1990), which means that the shaftlength of the short fibre is about 128 A long (Table 1).For human and bovine adenoviruses we have foundthat the length contribution per shaft repeat wasbetween 13 and 14 A (Ruigrok et al., 1994). Thus, alength of 128 A could be made up of about nine singlerepeats, in agreement with the sequence analysis.

The shaft of the long CELO fibre consists of 11regular 15-residue PL and GL repeats (right part ofFigure 3(b)), six double repeats, much like those ofthe short fibre, and two very homologous triplerepeats. There is one more irregular repeat startingat residue 522, leading to the equivalent of 30 singlerepeats in total. The shaft of the long fibre ends withanother poly(G) stretch. It is very difficult to make acalculation for the shaft length of the long fibrebecause we do not know the length contribution ofthe tail nor how the fibre is attached to the base.However, a rough estimate of the shaft length wouldlead to between 30 and 32 repeats, in agreement withthe sequence. The long fibre is bent in two placesclose to each other. The sites are situated at297(213) A (n = 18) and 333(211) A (n = 22) fromthe top of the head, i.e. between 18 and 20 repeatsfrom the C-terminal end of the shaft and, thus, couldcorrespond to the triple repeats.

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The sequences for the head domains are shown inFigure 3(c). The C-terminal knob (or head) domainshave no homology with the mammalian fibre heads.Classical secondary structure analysis predictsalternating b-strands and turns, which would agreewith the three-dimensional structure of the HAd5head (Xia et al., 1994), but the absence of the wellconserved residues in the mammalian fibres makesit impossible to align the CELO fibre heads with theother known sequences. In mammalian subtypes,there is a conserved TLWT sequence in the firstb-strand of the head (Xia et al., 1994). This sequenceis absent in CELO fibres which makes it impossibleto define where the head domain actually starts. Wehave assumed that the head of the long fibre startsafter the second poly(G) sequence and that the headof the short fibre starts with the sequence in italicsFigure 3(c)). This would lead to head domains ofidentical sizes, 212 residues for both CELO fibres,which is in the range of the sizes of the heads of themammalian fibres, i.e. from 183 amino acids for thehuman virus fibres to 227 for the MAd1 head(Chroboczek et al., 1995). Finally, although there existsome short stretches of sequence homology betweenthe heads of the short and long CELO fibres (in boldand boxed), it is not possible to align the entiresequences without introducing unacceptablylong gaps. The absence of homology between theCELO fibre head domains and those of mammalianfibres could be reflected in the different dimensionsobserved in EM (Table 1), i.e. the CELO headsare considerably wider and less high than thehuman and bovine fibre heads (Ruigrok et al., 1990,1994).

A question which we have not yet addressed, iswhy CELO virus has two fibres. The mammalianviruses have only one fibre which interacts with theinitial receptor on the cell surface, permitting virusattachment. The penton base then interacts throughits RGD motif with an integrin complex on the cellsurface, this being a prerequisite for internalisationof the virus (Wickham et al., 1993; Mathias et al.,1994). However, there are possibly some exceptionsto this way of virus internalisation: the penton baseof HAd40, which also has two different fibresalthough only one per penton, has no RGD motif(Davison et al., 1993) and neither has the FAV10penton base (Sheppard & Trist, 1992). It is possiblethat two fibres are needed for binding to twodifferent receptors; one for virus attachment and onefor internalization (although not to the sameintegrins since neither the HAd40 nor the CELO fibreheads contain an RGD sequence). Certainly, theabsence of homology between the two CELO headsmakes it very likely that they will bind to differentreceptors. Bending of the long CELO fibre willallow attachment of the short fibre without sterichindrance.

We still do not know how both CELO fibres areattached to the single penton base. The stoichiometryof CELO proteins (Li et al., 1984) is compatible withboth CELO fibres being trimers. This is confirmed bythe finding of the repeat structure in the CELO fibre

shafts, which implies a similar structure for allknown fibres, and by the similar overall morphologybetween CELO and HAd fibres as seen in EM. Thepenton base, as observed in EM, is clearly apentamer. The only known avian virus nucleotidesequence for a penton base is that of FAV10 whichcodes for a protein that is smaller than the HAd2 base(525 compared to 571 amino acids) but shows 42%homology with the HAd2 base. A central region of93 amino acids is even 71% homologous (Sheppard& Trist, 1992). It is probable that this highly con-served region is involved in the binding of the fibretail. Although the CELO penton base sequence is notknown, all FAV serotype penton bases seem to berather similar as suggested by DNA hybridizationexperiments (Sheppard & Trist, 1992). The EMobservations on the CELO penton (Figure 1) suggestthat the short fibre is attached like the mammalianvirus fibres. This would agree with the fact that theshort fibre has an N-terminal sequence almostidentical to that found in mammalian adenoviruses.

It has been suggested that complementary linearsequences are involved in the interaction betweenfibre and penton base in HAd2 (Caillet-Boudin,1989). This would involve on the fibre the conservedVYPY/F sequence, present in the tail regions of bothCELO fibres. The central conserved part of the FAV10penton base sequence also contains the complemen-tary sequence proposed for HAd2. We can envisagetwo possibilities for the attachment of the long fibrebut experimental evidence is still lacking.

First possibility: Compared to the short fibre, thelong one could be attached completely differently,possibly to the top of the penton base through itsstrongly charged first 30 amino acids. Even thoughthe FAV10 base is smaller than the HAd2 base, thevalue for its height is larger, 113 versus 100 A. Theincrease in stain-excluding volume could be causedby extra mass of the long CELO fibre associated withthe base. This first possibility could agree with howthe long fibre comes out of the base as seen in EM.

Second possibility: The short and the long fibrescould both be attached to the base by the VYPY/Fsequence. This would make a mismatch of five basemonomers to six fibre monomers, not necessarilyworse than five to three as in mammalian pentons.An argument for this could come from thecomparison of the tails of the CELO fibres with theN-terminal sequence of the MAd1 fibre (Figure 3(a)).The MAd1 tail contains the VYPY/F sequence twicebut the other absolutely conserved PPFV/F sequenceonly once (see Figure 3(a)). A trimer of MAd1 wouldrepresent six VYPY/F and three PPFV/F sequences,just like the situation caused by a trimer of the longCELO fibre plus a trimer of the short fibre. If thissecond hypothesis is true then the need for sixVYPY/F and three PPFV/F could also explain whyevery penton has one long and one short fibre and nottwo long or two short. If the long CELO fibre isattached like the short fibre, the morphologicaldifference between the attachments as seen in EMcould be caused by the poly(G) sequence, whichexists only in the long fibre.

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AcknowledgementsWe thank Stephen Cusack (Grenoble) for discussions,

ideas and for help with Figure 3, and Sabine Schuller(Berlin) for growing virus for EM observations.

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Edited by M. F. Moody

(Received 31 March 1995; accepted 22 June 1995)

Note added in proof: After submission of our manuscript a partial sequence of an ovine adenovirus waspublished (Vrati, S., Boyle, D., Kochearhans, R. & Both, G. W. (1995). Sequence of ovine adenovirushomologues for 100 K hexon assembly, 33 K, pVIII and fiber genes: early region E3 is not in the expectedlocation. Virology, 209, 400–408) in which the E3 genes are also not present between pVIII and fibergenes.