J. gen. Virol. (1989), 70, 3203-3214.

Printed in Great Britain 3203

Key words: adenovirus type 40/nucleotide sequence/3D structure

The Adenovirus Type 40 Hexon: Sequence, Predicted Structure and

Relationship to Other Adenovirus Hexons
By C E L I A I. A. T O O G O O D , 1. R A M A C H A N D R A N M U R A L I , 2 R O G E R M.
B U R N E T T 2 AND R O N A L D T. H A Y 1
1Department of Biochemistry and Microbiology, University of St Andrews, Irvine Building,
St Andrews, Fife KY16 9AL, U.K. and 2The Wistar Institute, Philadelphia, Pennsylvania
19104-4268, U.S.A.

(Accepted 18 August 1989)

SUMMARY

The gene encoding the major capsid protein (hexon) of human adenovirus type 40
(Ad40) has been isolated and sequenced. Comparison of the predicted amino acid
sequence of the Ad40 hexon with the corresponding polypeptide of the human enteric
adenovirus, Ad41, reveals an overall identity of 88 ~. The majority of the changes in
sequence are located in two areas, amino acids 131 to 287 and 390 to 425. Regions in the
hexon protein that vary between Ad40 and Ad41 (subgroup F) were the same regions
that varied between Ad2 and Ad5 (subgroup C) suggesting that these areas of the
protein represent type-specific antigenic determinants. Other areas were conserved
within members of a subgroup but varied between subgroups. Fitting of the Ad40
hexon sequence to the known three-dimensional structure of the Ad2 hexon
demonstrates that the variable regions are located in the 11,12 and 14loops that form the
surface of the virion. Of major significance is the absence in Ad40 of the highly acidic
region present in both Ad2 and Ad5. In Ad2 this region stretches down into the D-
strand of the fl-barrel forming the P1 domain. Molecular modelling indicates that the
amino acids in Ad40 which correspond to the acidic region of Ad2 can also be
accommodated in the eight-stranded fl-barrel, thereby maintaining the integrity of the
barrel. Since the acidic region is also absent from the hexon of Ad41, the sequence of
amino acids that replaces the acidic residues may be responsible for some of the
distinctive biological properties of the subgroup F adenoviruses.

INTRODUCTION
To date 41 distinct serotypes of adenoviruses that infect humans have been recognized and
shown to cause a correspondingly wide range of disease symptoms. The enteric adenoviruses
types 40 (Ad40) and 41 were first identified in stool samples (Flewett et al., 1975) and evidence
has since accumulated to suggest their involvement in the aetiology of acute infantile
gastroenteritis (Whitelaw et al., 1977; Johansson et al., 1980; Hammond et al., 1987). Ad40
and Ad41 form adenovirus subgroup F and share many of the common characteristics of
adenoviruses but are particularly different with respect to their growth in tissue culture. They
have been termed fastidious for their inability to grow in conventional cell lines (de Jong et al.,
1983), but can be successfully propagated in 293 cells, a human embryonic kidney cell line
transformed by Ad5 (Graham et al., 1977).
Although the biological properties of this virus group are diverse, all human adenoviruses
share a common architecture and genome organization. A single copy of linear double-stranded
DNA of approximately 35000 bp is encapsidated in a non-enveloped icosahedral protein shell.
There are at least 11 virus-encoded structural polypeptides of which the hexon (polypeptide II) is
the most abundant (reviewed by Ginsberg, 1984). Hexons comprise 240 out of the 252
capsomeres, while pentons, which are located at the vertices of the icosahedron, form the
remaining 12. Hexon constitutes a large proportion of the surface of the virus and it has been

0000-8989 © 1989 SGM

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3204 c.I.A. TOOGOOD AND OTHERS
shown that this protein contains determinants for type- and group-specific neutralizing
antibodies (Haase & Pereira, 1972; Kjell6n & Pereira, 1968; Norrby, 1969; Willcox & Mautner,
1976).
To determine the basis for the antigenic variation among h u m a n adenoviruses, we have
examined the sequence of the hexons of the enteric adenoviruses. Sequence comparison of the
hexons of the subgroup C adenoviruses types 2 and 5 revealed large blocks of conserved residues
separated by regions containing amino acid residues that differed between the two viruses
(Kinloch et al., 1984). We recently determined the sequence of the Ad41 hexon gene (Toogood &
Hay, 1988) and, although comparison of the predicted amino acid sequence with that of Ad2
gave a similar pattern to the changes observed between Ad2 and Ad5, important differences
were also noted. A n extremely acidic region present in the subgroup C adenoviruses types 2 and
5 is completely absent in Ad41.
To extend this analysis, and to determine whether the pattern of variable amino acids and
absence of the extended acidic region present in Ad2 and Ad5 was a characteristic of enteric
adenoviruses, we have examined the hexon of the other m e m b e r of subgroup F, Ad40. We have
determined the nucleic acid sequence of the Ad40 hexon gene and have predicted the amino acid
sequence of the corresponding polypeptide. Utilizing the three-dimensional structure of the Ad2
hexon (Roberts et al., 1986), changes in amino acid sequence between Ad40 and Ad2 can be
localized on the hexon molecule. Whereas the base of the polypeptide is highly conserved, the
regions that are exposed on the surface show considerable type-specific variation.

METHODS
Cells and virus. Ad40 (strain Dugan) was propagated in 293 cells (Graham et al., 1977) which were grown in
Glasgow-modifiedEagle's medium (GMEM) containing 10~ newborn calf serum, and maintainedpost-infection
in GMEM containing 2~ newborn calf serum. Virus was purified (Mautner & Willcox, 1974),and viral DNA was
extracted by the method of Pettersson & Sambrook (1973).
Preparation o f probes. Recombinant M13 bacteriophage containing fragments of the Ad41 hexon gene were
generated as described previously (Toogood & Hay, 1988). mA41.1 and mA41.2 contained, respectively, the 5'
and 3' ends of the Ad41 gene. Single-strandedradioactive probes were synthesized by primer extension (Jeffreys et
al., 1985) using the M13 universal primer, dCTP, dTTP and dGTP in the presence of Klenow polymerase and
[~-32p]dATP. Following Bali digestion and denaturation, DNA was fractionated by electrophoresis in a 6%
polyacrylamide gel containing 50% (w/v) urea. Radioactive fragments were located by autoradiography, excised
and electroeluted, mA41.1 yielded a 129 nucleotide fragment spanning nucleotides 5 to 133 of the hexon gene.
mA41.2 yielded a 185 nucleotide fragment extending from nucleotide 2602 to eight nucleotides past the
translational termination site.
Identification o f the Ad40 hexon gene. Ad40 DNA was digested with PstI, fractionated by agarose gel
electrophoresis, and the DNA transferred to a nylon membrane (Southern, 1975). The blot was probed, using
conditions described previously (Hay et al., 1984), with the 129 nucleotide fragment isolated from mA41.1. After
removal of the mA41.1 probe, the membrane was reprobed with the 185 nucleotide fragment from mA41.2. In
both cases a single hybridizing species of 5-6 kb was identified. A PstI digest of Ad40 DNA was ligated into
pUC13 and used to transform Escherichia coil JM83 (Maniatis et al., 1982). Ampicillin-resistant colonies were
probed with the 129 nucleotide mA41.1 fragment described above and a positive colony, containing the plasmid
designated pCT40, was used to prepare DNA by CsCl-ethidium bromide centrifugation (Hay et al., 1984).
DNA sequencing. Plasmid DNA was sonicated and blunt ends were generated by treatment with T4 DNA
polymerase in the presence of dNTPs. DNA was size-fractionated on an agarose gel and fragments of 400 to 800
bp were ligated into Smal-cut dephosphorylated M 13mp8 replicative form DNA. The PstI fragment was isolated
from pCT40, nick-translated (Rigby et al., 1977)and used to identify recombinant phage containingAd40 hexon
DNA by plaque hybridization. Phage DNA was sequenced by the dideoxynucleotide procedure (Sanger et al.,
1977, 1980) using buffer gradient gels (Biggin et al., 1983). The sequence was assembled and analysed using the
Staden suite 9f computer programs (Staden, 1982).
Sequence alignment and molecular modelling. The amino acid sequence alignment was performed using the
program ALIGN in PRONUC (Bourne & Desai, 1987). The program uses the algorithm described by Needleman
& Wunsch (1970). Alignment of sequence was performed pairwise, with each against Ad2. The penalty for
breakpoints was varied to get the best alignment score, but in each case maximum alignment was obtained with a
value of 12. Models were displayed and manipulated using the molecular modelling program FRODO installed on
an Evans and Suthedand PS350 Graphics Display.

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 Characterization o f adenovirus type 40 hexon 3205
Materials. Media and bovine serum were from Gibco. Klenow polymerase was purified by the method of Joyce
& Grindley (1983). Restriction enzymes, T4 DNA ligase, E. coli DNA polymerase I and bacterial alkaline
phosphatase were obtained commercially and used as specified by the manufacturers. [c~-32P]dATP (3000
Ci/mmol) was obtained from Amersham.

RESULTS AND DISCUSSION

Sequence of the Ad40 hexon gene and comparison with the hexons of other human adenoviruses
The Ad40 hexon gene, identified by hybridization to homologous regions of the Ad41 hexon
gene (Toogood & Hay, 1988), was contained on a 5.6 kb PstI fragment (Fig. 1a) which was
cloned into pUC 13 prior to sequencing. The complete sequence of the adenovirus hexon gene
was determined by shotgun dideoxy sequencing (Fig. 2) and its location within the 5-6 kb PstI
fragment is represented in Fig. 1 (b). A coding region of 2772 nucleotides predicts a polypeptide
of 922 amino acids which is two amino acids shorter than its Ad41 counterpart and 45 amino
acids shorter than that of Ad2. Comparison of the amino acid sequences of the Ad40 and Ad41
hexons, inserting gaps for maximal alignment of the two polypeptides (Fig. 3) reveals an overall
identity of 88 % (each insertion is taken to be equivalent to one mismatch). However, the major
amino acid changes are concentrated in two regions, 131 to 287 and 390 to 425 (unless otherwise
stated amino acid numbering refers to the Ad40 hexon sequence) where the percentage of
identical amino acids drops to 56 and 27%, respectively. For the remaining 79% of the
polypeptide the identity is 98 %.

1 2 3
(a) (b)

PstI PstI

:: ~,: : ~ •

" • • r ' : :

I I
0 20 40 60 80 100

Fig. 1. (a) Identification of the Ad40 hexon gene by hybridization to homologous regions in Ad41 D NA.
Purified viral DNA was digested with restriction enzymes, fractionated by electrophoresis in a 0-7%
agarose gel and transferred to a nylon membrane. Lane 1, Ad2 D N A cleaved with HindIII and probed
with nick-translated Ad2 DNA to provide size markers; lane 2, Ad40 DNA cleaved with PstI and
probed with the 129 nucleotide fragment from mA41.1 representing the 5' end of the hexon gene; lane
3, hybridized radioactive DNA was removed from the membrane described in lane 2 and reprobed with
a 185 nucleotide fragment from mA41.2 representing the 3" end of the hexon gene. (b) Location of the
Ad40 hexon gene (shaded region) within the cloned PstI fragment and its position on the Ad40 genome
(100 map units represents approximately 36000 bp). Location of the hexon gene within the genome of
Ad40 was determined by aligning restriction sites in the hexon gene with published restriction maps
(Takiff et al., 1984).

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3206 C. I. A. T O O G O O D AND OTHERS

M A T P 3 M M P Q W S Y M H I A G Q D A S E Y L S P G L V Q F A R A T D T Y F S
KTGC~3CACCCCCTCGA~ A ~ CCGCAA~GTCT TACAII~CACATCGCCGGGCAGGACGCCTCGGAGTAC CTGAGCCCGGGCCTGGTGCAGTTCG CC~C CAC ~ A T A C ~ A ~ ~ A G C
10 20 30 ~0 50 60 70 80 90 100 110 120

L G N K F R N P T V A P T ~ D V T T D 2 S Q R t T L 2 F V P V D R E E T A Y S T
C ~.,C~GAACAAGTTCAC~ACCC CACCG TGGCTCC CACCCACGATGTAACCACAGACAGGTCGCAGCGACTGACGCTGCG CETCGTGCCCGTCGACCGCGAGGAAACCGCCTACTCTTAC
130 lqO 150 160 170 180 190 200 210 220 230 2/40

K V 8 F T L • V G D N 2 V L D M A S T Y F D I R G V L D E G P S F K P Y 2 G T •
EAAGTG C~CTTT•CGCIGGCCGTGGGCG•C•ACCGGGT TTTGGAC•TSGCCAG CAC CTACTTT~•C• TCOfiCGGCGTGCTG G•~GTGGTCCC•GCTTT~CC~•T~GGGC•CTGC•
250 260 270 280 290 300 310 320 330 3qO 350 360

Y N S L • P K G • P N P S Q W T N Q N K T 2 S F G Q • P ¥ I G Q K I T N Q G V Q
T•CA•CTCC C~GGCCCC CA•AGGTGCTCC CAATC CTAGC C•G TOG•CAAAC CAAA•CAAA •CAA•CTC CTT TGG•C•AG CTC C CT•T•T•GG•C•~•~ T C • C C ~ T C • 0 0 G ~
370 380 390 400 ~IO 1120 ~30 ~40 ~50 460 ~7o 480

V G L D S N N 2 D V F • D K T ¥ Q P E P Q V G Q T Q W 2 I N P M Q N • A G 2 I L
GTGGGCT TAGACTC C•ACAATCGCGATGTGTTTGCCGATKAAACGTACCAACCGGAGCCTCAAG TGGGGCAGACGCAATGGAACA TT•ATC CAATG CAAAACGCTG CGGGAAGAAT•CTA
1190 500 510 520 530 5~0 550 560 570 580 590 600

K Q T T 2 M Q P C Y G S Y ~ R P T N 2 K G G Q • K L V K N D D N Q T T T T N V G
AAACAAAC CACGCCCATGCAGCCA TGTTA TSGGTCATACGCT AOACCA•CAAACGAAAA•GGAGGTCAAGC CAAGCTGGTAAAAAA TGACGACAA TCAGACCACAA~•C~A~TAGGT
610 620 630 6~ 0 650 660 670 680 690 700 710 720

L N F F T T • T E T • N F S P K V V L ¥ S E D V N L E • P D T H L V F K P D V N
TTAAACTTTTTT•CCACTO CCACTO•G•CCGCT ~ATTTTIC•CC•A~OTGGT TCTGT•C•GCO•G GA TGTT••CT TAG••G CGCCCG•T•C CC•C CT T G ~ T T T ~ C ~ O • ~ T C A • C
730 7~0 750 760 770 780 790 800 810 820 830 8aO

G T S • E L L L G Q Q • • P N R P N Y I G F 2 D N F I G L M ¥ Y N S T G N M G V
GGC•CA•OTGCCG•GCTTTT•CTGGGTCAOC~GCCGCTCC CAATCG•C CTAATT•C•T TGGTTTTAGGG•C••CTTC•T I~GGTTT G • T G T • C T • C A • T T C C • C T G G ~ A ~ G • G ~
850 860 870 880 890 900 910 920 930 9~0 950 960

i • G Q A S Q t N • V V D L ~ D 2 N T EL S ¥ O L M L D • L G D 2 S H ¥ F S M W
CTGGCC~GfiCIAGCTTCTCAGCTCAACGCAGTGGTGGACT T•C•AG•T~G•AAC•CGG•GCT~TCTTACCAGTTAATGCT TG•CGCTTT~GGG•TCGG•GTCG•T•~ ~ T C C • ~ T G G
970 980 990 1000 I0 I0 1020 1030 I0~0 1050 1060 1070 1080

~ A Y D S • D P D ~ R I I E ~ ~ G ~ E D R L P N ~ C F P L N G ~ 0 I 8 ~ S Y
KACC ~n,GC•GT~ G•C•GCT• TG•CC C•G•CGTG•~AATT•TTC~TC•TGGC~TGGA•G•CGAGCTCC~CA•CT•TTGCTTT~CTCTTAATGGG~••GGAATAT~••~•GTT~CC~
1090 1100 1110 1120 7130 11~0 1150 1160 1170 1180 1190 1200

G V K T D N G T N W S O N N T D V S S N N E I S I G N V F A M E I N L • • N L W
C~C~TOAAGACTGACAA~3G•ACTAACTGGTCTC•GAATAATACAG•CGTCTCAAGCAACA ATGAAATTTCCATTGGCAATG TGTTTGCCATGGAGATTAATCTGG~CT~CT~G
1210 1220 1230 12~0 1250 1260 1270 1280 1290 1300 1310 1320

$ F L Y S B q • L Y L P D S Y K I T P D ~ I T L P 0 ~ K ~ T Y k ~ M N G 2 V A

AGAAGCT TCTT&TACTCJLAATGTAGC CTTGTACTTGC CTGACTCTTACJU~TAACCCC CGATAACATTACTTTACC C G A C A A C J ~ A T A C A T A ~ C ~ A C A ~ G T ~ G f i T ~ C C

1330 13110 1350 1360 1370 1380 1390 I~00 I~I0 11120 I~30 I~0

V P $ A L D T ¥ V N I G A 2 W S P D P M D N V N F F N H H R N A G L H Y E S M L
GTC 000AGCGCCCTGG•T•C•T•CGTKK•C•TCGGGGCGCGGTGfiTCTC CAG•C CCCATGGAC••CGTT•ATC 00~ TT••C CAC C•C OGCA•TGCTGGTCTGCGCT•C~TTCTA~ ~ C
11150 I~60 I~70 Iq 80 I~ 90 1500 1510 1520 1530 15~0 1550 1560

L G N G 2 Y V P F H I Q V P Q K F F A I K N L L L L P G S Y T Y E W N F 2 K D V
C ~ G T A A O 3 G C CG CT•CGTC~C CTTTTCACATC CAAGTGCCC CAGAAATTTTTCGC CATTAAAAATCTC CTG CTC CTGCC ~ G G T C ~ • C A C ~ • ~ A G ~ G A • ~ G A A G ~
1570 1580 1590 1600 1610 1620 1630 16~0 1650 1660 1670 1680

N M I L Q S S L 0 N D L R V D G A S V 2 F D S I N L Y A N F F P M A H N T A S T
~AC•T~ATTCTC C~CAGTCTCGGTAAC~ACCTCAGGGTCGATGGAGC CAGCGTCAGGTTTGACAGCA TTAAC CTGTATGCCAACTTTTTC CCCA%~GCTCACAAC•C O ~ ~CC
1690 1700 17 I0 1720 1730 17~0 1750 1760 1770 1780 1790 1800

i E A M L 2 N D T N D Q 8 F N D Z L C A A N M L Y P I P A N • T 8 V P I S I P S
TTGG~AGc~ATGCTTCGT~iTGATAC~•CGAT~AGTCTTTCAACG•CT•CCTCTG~GCCGCA~•CATG~TT•C~C~TACCCGC~ACG~A~•GCG~CC~T~ATT~T~
1810 1820 1830 18~0 1850 1860 1870 1880 1890 1900 1910 1920

E N W A ~ F 2 G W S F T 2 L K T K E T P S L G 3 G F D P ¥ F T Y S G S Y P ¥ h D
CG~AATTGGGCTG CT TTTCGGGGGTGGAGTTTTACT AGACT •AAAACT ~ A A A C CCCCTCT T I G ~ T C ~ T T ~ A ~ C•TATT~•C ~ A ~ C T G G ~ C ~ T C C ~ T • C T ~ G A T
1930 19110 1950 1960 1970 1980 1990 2000 2010 2020 2030 20110

G T ~ ~ L ~ ~ T ~ K K ~ ~ ~ M T D S 3 ~ 3 ~ ~ G ~ D ~ L L T P ~ E ~ E I K ~ T
OG~CCF/TTAC CTGAACC~TACTTTTAE~A~GTGTC CGTTA TGTTCGACTCCTCTGTGAGC~GC C T ~ G T A A C G A C C G A C T A C T T A C T C C C A A C G A G T T ~ T C ~ C A C ~
2050 2060 2070 2080 2090 2100 2110 2120 2130 21110 2150 2160
D 0 E G ¥ N V • Q C N M T K D W F L I Q M L S H ¥ N I G ¥ Q G F ~ V P E S ¥ K D
GAT~G~AAfiGATAC~ACGTGGCTCAATGTKACATGAC ~AGGACTGGTTC CTCATACA~ATGCTCAGTCACTACAATAT~GCTAC~GGGTT~ CACGTAC C A G A A ~ A C ~ G G • C
2170 2180 2190 2200 2210 2220 2230 22~0 2250 2260 2270 2280

R M ¥ 8 F F R ~l F ~ P M S R ~ %' %' 0 T T T ¥ T ~ Y ~ ~ V T L P F ~ ~ , ~ ~ 3 G F V
A~ATGTACTCCTTTTTCCCaAACr TCCAACCCATGAC~C~CCAGGT3GTAGACACT•CCAC CTAC•~GA~ATCA~ ~TAACTC~C ~T~CAG~T~T•A~TC~G~T~TA
8290 2300 2310 2320 2330 23~10 2350 2360 2370 2380 2390 2~100
G ~ M O p A 1 2 E G Q • I P A N Y P ~ P L I fi ~ T A V P S L T ~ K K F L C D 8 T
~ATACA~ACCT~CCATAC~0OAGG~ACAAGCTTAC CCC~CCAACTATC C A T A C C C C C T T • T ~ G T C • G A C G G C C ~ T A C ~ C ~ G A ~ C A G A A ~ T T ~ T ~ C G A ~ G T • C C
2~110 2~.20 21130 2~.~i0 21150 2~460 21170 2~80 2~190 2500 2510 2520

M W R I P ~ S S N ~ M ~ M G A L T D L G ~ N M L ¥ A N 8 A E A L D M T F E V D P
ATGT~3COCATTCC~/~TTCCAGCAACFfTA T~TCTA ~0GOGOCC CI~A CT~AC C~O~GCAAAACA ~ C ~ T A ~ C ~ A C T C C G 00~ ~ ~A~ ~A ~TT ~AGG~ACC CC
2530 25~0 2550 2560 2570 8580 2590 2600 2610 8820 2630 26~0
M D E P T L L ¥ V L F E V F D V V 2 I E Q P H 2 0 V I E A V ¥ L 2 T P F S A 0 I~
ATOOATOAOCCCACACTTCTCTA~OTTCTOTTC~AA~TTT~ACOTTGTOC~CATC CAC ~ G C ~ C ~ T C A ~ A G G C ~ T ~ A C ~TA~C~T~T~GC~GTA~

2650 2660 2670 2680 2690 2700 2710 2720 2730 27110 ~750 2760
A T T •
OCCaCC&C~T~A
2770

Fig. 2. Nucleic acid sequence of the Ad40 hexon gene and predicted amino acid sequence of the
corresponding polypeptide. Translation in the single-letter amino acid code is shown above the middle
base of the codon.

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2 ATP SMMPQWS YMH ]~GQDASEYLS PGLVQFARATETYF S I ~ K F R N P TVAP THDVTTDRSQRLT LRF IPVDREDT 1 75
5 ATP SMMPQWS YMH ~ G Q D A S EYLS PGLVQFAKATETYFS I4~NKFRNP TVAP THDVTTD R$ QRLTLRF IPVDREDT I
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40 ATPSMMPQWSYMHI~QDASEYLSPGLVQFARATDTYFSI~NKFRNPTVAPTHDVTTDRSQRLTLRFVPVDREET 1 75

2 AYS YKARFTLAVGDNRVLDMAS TYFD IRGVLDRGP TFKP Y S G T A Y ~ L A P ~ C~--'~QTEDS GRAVAEDEEE 1~0

5 AYSYKARFTLAVGDNRVLDMAS TYFD IRGVLD RGPTFKP Y S G T A Y ~ L A P I ~ P ~ C ~ D E A A T A L E INLE EEDD
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40 AYS YKVRFTLAVGDNRVLDMAS TYFD IRGVLDRGP S F K P Y S G T A Y N ~ L A P ~ S~.~NQN ............ 138

2 EDEDEEEEEEEQNARDQAT~THV~--'~L@@K- ~ A E TQAKPi~YAD~ YQP EP Q IGESQ~qE- 223

5 DNEDEVDEQAEQQ ...... -~THV~GQAI~YS~I I~IFK- E~31Q IG ~ G Q T .... PI~YAD~FQP EPQ IGE S Q~YE-
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2 IVDLQDRhlTELSYQLLL~GDRTRYFSMWq,,IQAVDSYDPDVRIIENHG~EDELPNYCFP~T~/~'~G 444
5 [VDLQDRNTE LS YQLLLE~GDRTRYF SMWNQAVD S YDPDVR I IENHG~IEDE LPNYCFP I ~ I ~ E T L [ f K ~ K ~ K T
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2 NG SGDNGD T # ~ T - F AT RN - ~ - ~ i ~ - ~ AME IN L~AN L W ~ F LY'SN IALY L P I ~ K ~ h ~ ~ 517

5 GQ. . . . . EN6~E~%T EFSDKN~EI~Gh~FAMEI NI~%NLW~F Ly SNI ALYLPD~L~ S~S~V~IW h~NT~D

40 G ....... Tb~m]SQ~qTDVSSNN-~_~SI~__b~AMEINI4A~qLW~FLYSNVALYLPD~Sy~_]I ~ I 472

I
41 I ~ V A V l I ~ L I ~ ] Y V N IGARWS~D~4DNVNPFNHHRNAGLRYRSMLLGNGRYVP FH IQVp QKFFAI KNLLLLP G ~
40 Y~M.M.M.M.M.M.M.M~VA~L~]YVNIGARW~DNVN~FNHHRNAGLR~RSMLLGNGRYVPFHIQVPQ~FFAIKNLLLLPG~ 547

2 SYTYEWNFRKDVNMVLQS S LGNDLRVDGAS IKFD S ~ F P M A H N T A S T LEAMLRND TNDQS FNDYI~h~NM 1 667

5 SYTYEWNFRKDVNMVLQS S LGND LRVDGAS IKFD S I~y;~FFPMAHNTAS T LEAMLRNDTNDQSFNDYL~AANM I
41 SYTYEWNFI~VNMILQSSLGNDLRVDGASVRFDSI~LYA~FFPMAHNTASTLEAMLKNDTNDQSFNDYI~AANMI
40 SYTYEWNFRKDVNMILQSSLGNDLRVDGASVRFDS~FPMAHNTASTLEAMLRNDTNDQSFNDYI~ANM] 622

5 ~LYP I P ~ N A ~ / P ISIP S R N W A A F R G ~ T R L K T K E T P S LGSGYDP yyTYSGS IP YLDGTFYLNHTFKK~I TFD S ~

41 ~LYP I P ~ q A ~ V P I S IP SRNWAAFRGW~TRLKTKETP S LGSGFDPYFTYSGSVPYLDG~FYLNHTFKK~,~IMFDS ]
40 lLYP~:~AT~[~/PISIPS~WAAFRG~S~TRLKTKETPSLGSGFDPYFTYSGSV~YLDGTFYLNHT~V~MFDS~ 697

2 ISVSWPGNDRLLT~FEIKRSVDGEGYNVAQCNMTKDWFLVQMI~N~NIGYQGFYIPESYKDI:tMYSFFRNFQPMSI 817
5 ~SVSWPGNDRLLTP~FE IKRSVDGEGYNVAQCNMTKDWF LVQMI~hIyN IGYQGF y Ip E S YKDRMYS F FRNFQPMS I
41 ~SVSWPGNDRLLTP~FE IKRTVDGEGYNVAQCNMTKDWF L IQMI~SH~N IGYQGFYVP ES YKDRMYSFFRNFQPMS I
40 ~SVSWPGNDRLLTP~FEIKRTVDGEGYNVAQCNMTKDWFLIQM~S~yNIGy~GFHVPESYKDKMYSFFRNF~PMS 1 772

5 IRQ~YQQ%~IQHNNS GFV(BYLA~4REC-QAyp AI~Ipyp L IG ~ A % ~ S ITQKKF LCDRT LWR Ip F $ S I

41 [RQVV~/~YQN%~QHNNSGFVGYMG~MREGQAYPA b ~ yp LI G~TA%~SLTQKKFLCDRTMWRI PFSS[
40 ]RQVVI~I~YQN%~QHNNSGFVGYMGP~IP~GQAYPA~PYpLI~TA~SLT~KKFLCDRTMWRIPFSS] 847

2 INFMSMGALTDLGQNLLYANSAHALDMTFEVDPMDEPTLLYVLFEVFDVVRVH~HRGVI~YLRTPFSAGNATT[ 967
5 [NFMEM~ALTDL~LLMANSAHALDMTFEVDP~EPTLLYVLFEVFDVVRVH~ HRGVI E~YLRT p F SAGNATT [
41 ~NFM~C, ALTDLC~NMLYANSAHALDMTFEVDPMDEPTLLYVLFEVFDVVRI~ HRGVI E~YLRTP F S AGNATT I
4(} ~N~TDL(~NMLYANSAMALDMTFEVDPMDEPTLLYVLFEVFDVVRIH~HRGVIE~YLRTPFSAGNATT~ 922
Fig. 3. Sequence alignment of the four hexons from adenovirus types Ad2, AdS, Ad40 and Ad41.
Residues conserved in all four serotypes are boxed. Residues are considered conserved on the basis of
the following assumptions: exchanges between hydrophobic residues (Ala, Gly, Leu, Ile, Val, Cys and
Met) are considered as conserved, as are exchanges between aromatic residues (Phe, Tyr, Trp and His);
exchanges between hydrophilic residues of similar bulk (Glu, Asp, Gin and Ash; Arg for Lys; and Thr
for Set) are considered as conserved. All exchanges involving Pro are treated as significant. Amino acid
sequence numbering is shown for both Ad2 and Ad40.

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3208 C. I. A. TOOGOOD AND OTHERS
11 12 14 I1 12 14

100

80

60

40

20

0
250 500 750 1000 250 500 750 1000
o=

100

80

60

40

20.

o.
250 500 750 1000 250 500 750 1000
Amino acid residue number
Fig. 4. Histogram representing paired homology comparisons between known human adenovirus
hexon sequences. Sequences were aligned and the percentage identity calculated for each consecutive
group of 25 amino acids. Deletions and insertions were interpreted as mismatches. 11,12and 14indicate
the position of the surface loops in the Ad2 hexon structure.

Alignment of the hexon sequences of Ad2, Ad5, Ad40 and Ad41 (Fig. 3) indicates that a
greater degree of sequence conservation exists within the members of subgroups. Thus Ad40
and Ad41, which are members of subgroup F, are highly homologous, as are the subgroup C
viruses Ad2 and Ad5. The most striking difference between the subgroup C and F viruses is the
absence, in Ad40 and Ad41, of a highly acidic region that is present in the subgroup C viruses
Ad2 and Ad5 (amino acids 139 to 170 in the Ad2 hexon). Apart from this region, it is clear that
the differences between the four serotypes are restricted to defined areas, with the remainder of
the sequence being highly conserved between the four serotypes.
To illustrate the patterns of homology between hexons, pairwise comparisons have been made
between Ad40 and Ad2, Ad41 and Ad2, Ad40 and Ad41, and Ad2 and Ad5. In this analysis, the
paired hexon sequences have been maximally aligned, divided into runs of 25 amino acids and
the percentage of identical amino acids plotted (Fig. 4). On each histogram the sequences which
compose the three surface loops 11, 12 and 14 are indicated. These were identified from the X-ray
crystallographic structure of the Ad2 hexon (Roberts et aL, 1986). The homology profiles,
obtained by comparing each of the enteric adenoviruses with Ad2, are almost superimposable.
Both loops 11 and 12 are composed of two relatively non-homologous domains separated by a
region containing conserved amino acids. An additional region where the non-identical amino
acids are concentrated is identified as loop 4 (Fig. 4). The overall pattern is similar when the
hexon of Ad40 is compared to that of Ad41. In this case however, the divergence in sequence is
not so dramatic and loop 2 contains only a single region of non-conserved amino acids. This

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 g~

g~

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Fig. 5. (a) A C~t trace of the Ad2 hexon subunit in its standard orientation looking from the outside of the trimer. The subunit contains two basal domains, P! (yellow)
and P2 (cyan), each of which is formed by an eight-stranded fl-barrel.The top of the subunit is formed from loops 1~ (lilac) and 12 (green) emerging from the P1
domain and loop 14 (orange) from the P2 domain. The blue lines indicate three untraced portions of the molecule that are not visible in the electron density map. (b)
Differences in the sequences of the Ad40 and Ad2 hexon gene products are shown on the structure of the Ad2 hexon. Deletions (that is, residues present in Ad2 that
are absent from the Ad40 sequence) are shown in red and insertions as pink spheres. The bonds following residues in the Ad40 sequence that differ (that is, non-
conserved residues, based on criteria outlined in Fig. 3) from the corresponding residues in Ad2 are shown in green. All three insertions are located in the loops. Two
are in the 11loop with one near the top and the other near the junction with the P 1 domain. The third insertion is in the 12loop near the top of the molecule. The yellow
lines indicate the untraced portions of the structure. (c) A space-filling CPK model viewed from the inside of the trimer to show changes in the surface residues.
Atoms in green, red and pink indicate amino acid changes, deletions and insertions. Brown atoms indicate the modelled part of the D-strand. One of the insertions I,o
on loop 1~ is invisible in this representation as it is buried inside the molecule.
3210 C. I. A. T O O G O O D A N D O T H E R S

(~,'} It,)

, ~ '~ ,~
~-,,

-.~-~ ....

~m

Id)

~tL "

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 Characterization o f adenovirus type 40 hexon 3211
homology profile is strikingly similar to that previously described when hexon sequences of two
viruses within the same subgroup (Ad2 and Ad5) were compared (Kinloch et al., 1984).
Closer examination of the sequences of the four serotypes in the region between 453 and 498
(numbering with respect to Ad40) reveals that this region is subgroup-specific. Within this 46
amino acid sequence, 23 amino acids are common to all four virus types. In the 23 variable
positions there is only one difference between Ad40 and Ad41 and three differences between
types Ad2 and Ad5, but in each case the sequence of the subgroup F virus is different from that
of the subgroup C virus. It is also evident that even within the areas of greatest variation in
amino acid sequence between the four serotypes, certain positions in the loops are occupied by
highly conserved amino acids. These may be important in maintaining a common framework
within the hexon polypeptide chain, and thereby conserving the overall structure of the hexon.

Modelling of adenovirus type 40 hexon structure

The strong sequence homology among the hexons of Ad2, Ad5, Ad40 and Ad41 suggests that
the hexons are also structurally homologous. The Ad2 hexon is a trimer of three identical
polypeptide chains (Griitter & Franklin, 1974). The three-dimensional structure of the Ad2
hexon trimer has been determined by X-ray crystallography and shown to consist of two distinct
parts: a triangular top 6.4 nm in height containing three 'towers', and a pseudo-hexagonal base
5.2 nm in height containing a central cavity (Burnett et al., 1984). Later work at 0.29 nm
resolution (Roberts et al., 1986), showed that the trimer is formed from three copies of each of
the two pedestal domains P1 and P2 in the base, and of the tower domain T at the surface. Each
tower domain is formed by three loops, I t and 14 rising from P1 and P2 in adjacent subunits at
their interface, and loop 12 rising from P1 in the opposed third subunit (the locations of domains
P 1, P2,1~, 12 and 14 are shown in Fig. 5 a). Domains P 1 and P2 are very similar, each consisting of
an eight-stranded flattened fl-barrel arranged in the same 'jelly roll' topology (Richardson, 1981).
This topology is also found in the coat proteins of small spherical R N A plant viruses (Olson et
al., 1983; Rossmann et al., 1983) and human R N A viruses (Rossmann et al., 1985; Hogle et al.,
1985). Although this topology is not unique to viral proteins, it is a consistently observed feature
of viral architecture (Liljas, 1986).
An attempt was therefore made to predict the structure of the Ad40 hexon by aligning its
sequence with that of the three-dimensional structure determined for the Ad2 hexon (Roberts et
al., 1986). A Cot trace of the Ad2 hexon displays the various domains contained within the hexon
monomer (Fig. 5 a). Non-conserved residues in Ad40 were superimposed on this structure and
are represented in Fig. 5 (b) and (c). In the Ad40 hexon the altered residues, deletions and
insertions with respect to Ad2, are generally located in the surface loops 1~ and 12 and to a lesser.
extent 14 (Fig. 5). Since these are considered to be the portions of the molecule subject to change,
any differences between the serotypes would be predicted to occur in these regions. Few
differences exist between the various serotypes in the 13 region as this loop is sandwiched
between P1 and P2 and is required to maintain the integrity of the structure.
Since the adenovirus hexon is a trimer with extensive interactions between loops arising from
different subunits, the alterations described above have been displayed on space-fillingmodels
of the complete trimer (Fig. 6). Amino acid changes are mainly confined to the top of the
molecule with most deletions and insertions buried inside the molecule, except for the deletion in
the D strand which is partly exposed (Fig. 6a and b). A section through the hexon trimer at the
junction of the top and the base confirms this observation and further demonstrates that amino
acid changes are on the periphery with the bulk of the core residues being conserved (Fig. 6 c). A
further section through the fl-barrels reveals the very strong conservation in the base formed by
the domains P1 and P2.
The absence in the Ad40 hexon of the 32 mainly acidic amino acids present in the Ad211 loop
indicates that this loop must be short in the Ad40 hexon and may fold differently from that

Fig. 6. Space-fillingmodelsof the hexon trimer with colour coding as in Fig. 5 (c). (a) Side view, (b) top
view. (e) Sectionof the moleculeto show internal changes. The top of the moleculeformed by the three
loops has been removed to show the junction between top and base. (d) A section has been taken
through the ~-barrels in the base of the molecule and the upper part again removed.

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3212 C. I. A. TOOGOOD AND OTHERS

(!,I

.... (
Y,L - ~--L,, / ....

L - . ; .; '

1.> L..... ~>< .... L

, i ,, ,

. / ,..~.< ] i [
" \ ~1. J -. \ ";. ~]}<:~E
)!.....
J J
J . ,..... --/= ....

• .J
[] . _ ( ,/,-

Fig. 7. A stereoview of the D-strand of the P1 fl-barrelwith the modelledresiduesfromAd41 in yellow.

Residues in the D-strand of the Ad2 hexon were changed for the correspondingresidues in Ad41 and
those side-chains were fitted as described in the text. Note that the strand cannot continue above
residue 138 due to the bulky histidine side-chain, seen just above residue 139.

of Ad2. It is also clear that this 'deletion' would extend into the D-strand of the P1 domain,
which is part of the eight-stranded fl-barrel. However since all virus coat proteins for which
structures are available contain domains with the 'jelly roll' topology, it is reasonable to assume
that the fl-barrel should be conserved in Ad40 and Ad41. Primary sequence comparison shows
that the D-strand in the P1 domain of Ad40 is altered at 135 to 138, followed by the 'deletion'
from 139 to 172 (Ad2 amino acid sequence numbering). The question then arises as to whether
the residues following 134 could be accommodated in the fl-barrel without disrupting the
structure.
Recently the structure of the Ad2 hexon has been refined (R factor of 21 ~o; F. K. AtJaappilly,
R. Murali, Z. Cai & R. M. Burnett, unpublished results) which allows the structural question to
be investigated on a stereochemical basis. Molecular modelling was used to build a D-strand for
the P1 domain of type Ad40 and Ad41 based on the structure of the Ad2 hexon. The primary
sequences in the D-strand for Ad40 and Ad41 are homologous except at position 135 where
Lys in Ad41 is substituted for Thr in Ad40. Hence the alterations at positions 135 to 138
corresponding to Ad41 were built for the D-strand with the assumption that if the bulkier Lys
could be accommodated in Ad41, then the smaller Thr could be accommodated in Ad40. The
backbone of Ad2 was held constant and the altered D-strand side-chains displayed in a standard
conformation and inspected, with adjustments being made in the side-chains to accommodate
the Ad41 sequence. When a side-chain suffered a~steric contact with its neighbouring atoms, it
was rotated by varying the torsion angles so that it could be fitted without violating van der
Waals' contacts. Torsion angles were always held well within statistically allowed values.
Residues 135 to 138 fit well, but the D-strand cannot continue beyond residue 138 due to the
large lysine side chain of both Ad40 and Ad41 and the constricted space due to the presence of
the bulky histidine side-chain, which is seen just above residue 139 (Fig. 7). There is enough
space for a small residue, but not enough to fit the lysine residue in the Ad41 sequence. The
requirement for a small residue at this site is emphasized by the reduction in available space
when~he top of the D-strand is buried upon formation of the trimer. As a consequence, loop 11 in

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 Characterization of adenovirus type 40 hexon 3213

Ad40 and Ad41 must thus commence earlier than in Ad2 and Ad5, with a corresponding
shortening of the D-strand to nine residues. In Ad2 and AdS, the corresponding residues are
small enough to allow the D-strand to continue past the histidine constriction. The fact that
these changes could be accommodated in the eight-stranded fl-barrel, thus conserving this
common structural feature in viral architecture, implies that the structures of both Ad40 and
Ad41 hexons should closely resemble that of Ad2 apart from the decoration of their towers.
It has been suggested that type-specific antigenic determinants are located predominantly on
the surface of the adenovirus hexon (Norrby, 1969; Willcox & Mautner, 1976). By comparing
the sequences of the Ad40 and Ad41 hexons and locating the variable amino acids on the
predicted structure of the molecule it is clear that almost all of the differences between the two
virus types are confined to the la and 12 surface loops of the molecule. Since precisely the same
regions vary between Ad2 and Ad5, the strong prediction is that these regions of the molecule
are the determinants for the antibodies that can distinguish between the large number of
adenovirus serotypes. Mapping the binding sites for type-specific monoclonal antibodies will
test the accuracy of this prediction.

This work was supported by the Wellcome Trust.

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(Received 30 March 1989)

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