Download Nucleotide sequence and corresponding amino acid sequence of

volume 9 Number 81981
Nucleic Acids Research
Nucleotide sequence and corresponding amino acid sequence of the gene for the major antigen of
foot and mouth disease virus
Christina Kurz*, Sonja Forss*, Hans Kiipper*"1", Karl Strohmaier' and Heinz Schaller*
*Mikrobiologie Universitat Heidelberg, Im Neuenheimer Feld 230, 6900 Heidelberg, and
^Bundesforschungsanstalt fur Viruskrankheiten der Tiere, Paul-Ehrlich-Strasse 28, 7400 Tubingen,
GFR
Received 17 March 1981
ABSTRACT
A segment of 1160 nucleotides of the FMDV genome has been
sequenced using three overlapping fragments of cloned cDNA from
FMDV strain O..K. This sequence contains the coding sequence for
the viral capsid protein VP1 as shown by its homology to known
and newly determined amino acid sequences from this main antigenic polypeptide of the FMDV virion. The structural gene for
VP1 comprises 639 nucleotides which specify a sequence of 213
amino acids for the VP1 protein. The coding sequence is not
flanked by start and stop codons which is consistent with the
mode of biosynthesis of VP1 by post-translational processing of
a polyprotein precursor.
INTRODUCTION
FMDV is a small animal virus which belongs to the family picornaviridae. Like other picornaviruses it has a single stranded
RNA genome of about 8000 nucleotides (for reviews see Ref. 1 , 2 ) .
In the FMDV virion this RNA molecule is encapsidated by four
structural proteins designated VP1 to VP4 . Of these VP1 is of
particular interest since it harbours the main antigenic determinants of the virion, and therefore changes in its amino acid
sequence must be responsible for the high antigenic variability
of FMDV (5).
So far only limited amino acid sequences have become available from the direct analysis of VP1 on the protein level. A more
rapid approach to elucidate its primary structure appears to be
to determine the nucleotide sequence of the corresponding structural gene in the FMDV RNA. cDNA copies of the VP1-coding segment
of the FMDV genome have recently been isolated and amplified by
molecular cloning in E. ooli (6). We here report the nucleotide
sequence of this region and the deduced amino acid sequence of
© IRL Press Umlted. 1 Falconberg Court, London W1V5FG, U.K.
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Nucleic Acids Research
the VP1 protein.
MATERIALS AND METHODS
Plasmids pFMDV-144, -715, and -1034 which contain cloned FMDV
cDNA sequences have been described (6). Plasmid DNA was amplified in and isolated from E. coli C600 by standard procedures
(7). Restriction enzymes were purchased from New England Biolabs
and Boehringer Mannheim, or purified as described (8) and used
accordingly.
DNA sequencing was carried out with 5'-terminally labelled
DNA fragments using the chemical degradative methods (9) and
thin gels for separation of the cleavage products (10). Further
improved resolution of the sequence ladders was obtained by drying of the gels prior to printing (11). Nucleotide sequences
from the individual sequence runs were stored, matched and processed using the computer programs of Osterburg and Sommer (30).
Protein analysis FMDV was grown and purified essentially as
described (12). After the precipitation of the viral RNA by 66 %
acetic acid the viral proteins were separated by ion exchange
chromatography in 6 M urea on a column of carboxy methyl cellulose (13). Cleavage of protein VP1 by cyanogen bromide was carried out for 48 h in 70 % formic acid as a solvent; the cleavage
products were separated on a column of Biogel P60 as described
(14).
Carboxy-terminal amino acid sequences were determined with
carboxy peptidase Y: About 50 nmol of protein or peptide were incubated at 37° C with 25 yg of enzyme (Pierce) in 500 yl of 0.1M
pyridine acetate, pH 6.0, 0.2 % sodium dodecyl sulfate. Samples
of 50 yl were mixed with an equal volume of formic acid and analysed for free amino acids.
Amino acid compositions of proteins and peptides were determined by standard procedure after acid hydrolysis (15).
RESULTS AND DISCUSSION
Sequence analysis of cloned FMDV cDNA
The structure of the FMDV genome and the biochemical map of
its gene products (16) are outlined in Fig. 1. An alignment of
this biochemical map with a restriction map of cDNA prepared from
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Nucleic Acids Research
4000
2000
6000
8000
—'3'RNA
An
P20
P88
P100
P55
,16 ,,10, 22, 24,26,, 34 ,20,,,
VR, VRj
VP 3
72
VP,
j precursors
20 proteins
FMDV
715
1034
cDNA
E
o
m
63
H
c
o
I
—
u
UJ
a. c
if
E
o
Fig. 1; Description of the FMDV genome and localisation of the
cloned FMDV cDNA fragments used for nucleotide sequence determination. The estimated molecular weights of the FMDV gene products are given in kilo daltons. VP1 to VP4 are the viral capsid
proteins. PolyC indicates the internal poty: tract close to the
5'-end of the RNA, and polyA is the polyadenylated 3'-end. The
restriction endonuclease map is from Kiipper et al. (6). For convenience the BamHI site is arbitrarily referred to as map position 3000, and the total length of the genome is given as 8000
bases. The biochemical map (16) has been modified with respect
to the position of the VP1 gene as determined in this work and
also at the 3'-end of the viral RNA to accomodate an untranslated region of about 0.5 kb.
FMDV strain 0^K predicted that the gene for VP1 starts close to
the BamHI site at map position 3000 and extends towards a
Hindlll site located about 900 basepairs downstream (6). Three
cloned cDNA fragments which cover that region, FMDV-144, FMDV715, and FMDV-1034, were subjected to DNA sequence analysis according to Maxam and Gilbert (9) as outlined in Fig. 2. A complete nucleotide sequence was determined from clone FMDV-1034; corresponding sequences were obtained from clone FMDV-715, and to a
lesser extent from clone FMDV-144.
As far as determined identical sequences were obtained from
all three clones except for a single variation at nucleotide 3393
which is T in clones 715 and 1034, but A in clone 144. Each of
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s>
1000
2000
3000
400,0
50
5000
6000
7000
8000
200,
4000
3500
3000
bp
•FMDV 1034x
E
o
m
MM
a
a.
I
c
z
4010
2916
FMDVU«{ J
3042
FMDV 715 <T
'3122
3000
as
3500
4000
Fig. 2: The position of restriction sites in FMDV cDNA and the
strategy used for nucleotide sequence determination. Vertical
arrows indicate the position of restriction targets used for
5'-end labelling of restriction fragments. Horizontal arrows
show the direction and the extent of sequencing runs. They are
grouped according to the source of the cloned FMDV DNA. Nucleotide positions of the end points of the cloned FMDV cDNA fragments are included.
the three cloned cDNA fragments started in oligo dG runs and
terminated in oligo dC runs of about 30 nucleotides as predicted
from the cloning technique used (6, 17). These results indicate
that the cloned cDNA fragments represent intact copies of different overlapping segments of the FMDV genome. This conclusion is
further supported by the size distribution and by the DNA sequences from Haelll cleavage products of single-stranded cDNA copies
synthesized from FMDV RNA, which were identical to those obtained
from the cloned cDNA fragments (data not shown). Altogether, a
composite nucleotide sequence of about 1200 bases was obtained
(Fig. 3 ) , which extends from the 5'-end of clone FMDV-1034 (pos.
2916) beyond its 3'-end (pos. 4010) to the Xhol cleavage site at
position 4074. This sequence represents about 15 % of the FMDV
genome.
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The VP1 coding sequence
As known for all picornaviruses the RNA genome of FMDV is
translated from a single initiation site into a polyprotein of
some 2000 amino acid residues which is subsequently processed
via a series of specific proteolytic cleavages into the final
mature viral gene products (Fig. 1 ) . This mode of protein biosynthesis predicts that, except for its untranslated terminal
segments, the nucleotide sequence of FMDV RNA contains a continuous translational reading frame of some 6000 nucleotides. In
accordance with this expectation all of the base sequence presented in Fig. 3 can be translated in one phase into a continuous amino acid sequence, whereas many stop codons occur in the
other two phases. As the limits of the individual genes are not
specified in the nucleotide sequence by start and stop codons,
these can only be located within the polyprotein with the help
of amino acid sequence data from the termini of the individual
gene products.
A sequence for the NH_-terminal fourty amino acids has been
determined previously for VP1 from the same FMDV subtype O^K as
used in this investigation (18). A nucleotide sequence that can
code for these amino acids starts at position 2977 of the cDNA
sequence given in Fig. 3. The amino acid sequence deduced from
it agrees very well with that obtained previously by protein analysis. Only two amino acids were found to differ from those determined earlier: amino acid 7 was Ser instead of Lys, and amino
acid 33 was Ser instead of Trp. These minor differences are compatible with the uncertainities in the earlier amino acid assignments in these positions (18). Thus, we conclude that the VP1
coding sequence starts with nucleotide 2977, which is eight codons ahead of the BamHI site used as reference point for the relative numbering of nucleotide positions in the FMDV RNA sequence (6).
For the carboxy-terminus of VP1 from strain C^K no amino acid
sequence data were known which would allow to locate the 3'-end
of the VP1 gene in the FMDV base sequence. Two types of analysis
were used to obtain this information: Degradation of VP1 with
carboxy-peptidase Y which degrades proteins from the carboxyend, and amino acid analysis of carboxy-terminal peptide frag-
1923
GTACTGGCTAGTGCTGGTAAAGACTTTGAGCTAAGGCTGCCGGTGGACGCCCGTGCGGAAttXACTTCTGCGGGCGAGTCAGCG
BamH!
***
R1
R2
R3
***
GATCCTGTCACrACCACCGTTGAAAACTACGGTGGCGAAACACAGATCCAGAGGCGCCAACACACGGACGTCTCGTTCATCATGGACAGATTTGTGAAGG
A-.pd oVcillhi 1 hi Tin V.ilGUAMiTyi G lyG lyG luThrG I n l leG inAigAi-gG inH I sThrAspVa ISerPhel leMet AspArgPheVa ILysVa I
R2
R3
TGACACCGCAAAACCAAATT
ThrProGlnAsnGlnlleAsnlLeLeuAspLeuMetGInlLeProSerHIsThrLeuVaIGlyA LaLeuLeuArgA laSet-ThrTyrTyrPheSerAsp
**«
*#*
»KN
CrTGGAGATAGCAGTAAAACACGAGGGAGACCTCACCTGGGTTCCAAATGGAGCGCCCGAAAAGGCGTTGGACAACACCACCAACCCAACTGCTTACCAC
LeuGlulIPAlaU*ILysHiiGluGlyAjpLeuThrTrpWaIProAsnGlyAlaProGIuLysAlaLeuAspAsnThrThrAsnProThrAlaTyrH i s
TTGCCCTGCCCTACACTGCGCCCCACCGCGTGTTGGCAACCGTGTACAACGGTGAGTGCAGGTACAACAGAAATGCTGTGC
L ysALaProLeuThi ArgUeuAlaLeuPrnTyrThrAlaProHisArqVa LLeuAlaThrVaITyrAsnGlyGluCysArgTyrAsnArqAsnAlaVaIPro
•»•
1V
.T
CCAACT1GAGAG0TGACCTTCAGGTGTTGGCTCAAAAGGTGGCACGGACGCTGCCTACCTCCTTCAAC1ACGGTGCCATCAAAGCGACCCGGGTCACCGA
AinLeuArgGlyA'.pLeuGlnUalLHuAlaGlnLysValAlaArgThrLeuProlhvSerPheAsnTyrGlyAlalleLyiAlaThrArgyalThrGlu
ItMtt
**#
GTTGCTTTACCGGATDAAGAGGGCCGAAACATACTGTCCAAGGCCCTTGCTGGCAATCCACCCAACTGAAGCCAGACACAAACAGAAAATTGTGGCACCG
Li'uLcuTyr Ai gMt't/ y.Ai gA LaG LuThi Tyi CysProArgProLeuLeuA la I LeHi sProThrG luA LaArgH i sLyrG inLysI leVa lAlaPro
0B2
•GTGAAACAGACTTTG»ATTTTGACCTTCTCAAGTTGGCGGGAGACGTCGAGTCCAACCCTGGGCCCTTCTTTTTCTCCGACGTTAGGTCGAACTTCTCCA
ValLysGlnThrLeu&jnPheAspLeuLeuLysLeuALaGLyAspValGluSerAsnProGlyProPhePhePheSerAipUslArgSerAsnPheSerLys
3701
AACTGGTGGAAACCATCAACCAGATGCAGGAGGACATGTLAACAAAACACGGGCCTGACTTTAACCGGTTAGTGTCCGCATTTGAGGAGTTGGCCATTGG
LeuUalGluThrlleAsnGLnMptGlnGluAspMetSerThrLysHlsGlyProAspPheAinArgLeuValSorAlaPheGLuGluLeiiAlalteGly
••»
»«»
3801 AGTGAAAGCCATCAGAACCGGTCTCGACGAAGCCAAACCCTGGTACA'AGCTTATCAAGCTCCTAAGCCGCCTGTCGTGCATGGCCGCTGTGGCAGCACGG
VaILysAlalleArgThrGlyLeuAspGluAlaLyrProTrpTyrLysLeuILeLysLeuLeuSerArgLeuSerCysMetAlaAlayalALaALaArg
SerLysAspProVaILeuVa LA la IleMetLeuAlaAspThrGlyLeuGIulleLeuAspSerThrPheVaIVaILysLysIleSerA
(ton
Xhol
4001
GTCTCTTTCACGTGCCGGCCCCCGTCTTCAGTTTCGGAGCACCGGTCCTGTTGGCCGGGTTGGTCAAABTTGCCTCGAG
LpuPhoHuValProAlaProValPheXerPhBGlyAlaProyalLeuLeuAlaGlyLeuValLysUalAU
Rt
R2
R3
Rl
R2
R3
VP1
Rl
R2
R3
Rt
R2
R3
Rt
R2
R3
R<
R2
R3
Rt
R2
R3
Rt
R2
R3
R1
R2
R3
Rt
R2
R3
Nucleic Acids Research
ments from VP1. Degradation of the whole VP1 protein resulted in
a preferential initial release of leucine which was followed by
threonine and glutamine as second and third amino acids (Fig. 4 ) .
All other amino acids were released with distinctly lower initial
rates and no definite order could be established for the amino
acids released thereafter. Essentially identical results were obtained with peptide CB2 which is the carboxy-terminal subfragment of VP1 obtained after cleavage with cyanogen bromide (data
not shown). These results indicate that VP1 terminates with the
amino acid sequence Gln-Thr-(Leu)^_ 2 which is similar to the sequence Gin-Ala-Leu reported for the carboxy-terminus of VP1 from
FMDV strain A12 (19). This in turn suggests that the VP1 gene
most likely terminates with the TTG codon at position 3613 - 15,
since there is no other similiar coding sequence at the appropriate distance from the beginning of the VP1 gene in our nucleotide
sequence.
Proof for this preliminary assignment was obtained by the result of an amino acid determination of peptide CB2. This peptide
is the second largest of the four peptide fragments created from
VP1 by cleavage with cyanogen bromide (Stohmaier, Franze & Adam,
in prep.). It does not contain a homoserine residue and must
therefore be derived from the carboxy end of VP1. The amino acid
composition of this peptide was determined and compared to that
predicted for the last 33 amino acids starting after the last
methionine codon in the VP1 gene (nucleotides 3517 - 3615, see
Fig. 3 ) .
As shown in Table 1, all amino acids were found to be present
in the predicted molar ratios. The somewhat low values of Val
and lie (which also result in a slight increase of the relative
amounts of the other amino acids) are explained by the often
observed high resistance of the lie - Val peptide bond to acid
hydrolysis. Several amino acids are absent in peptide CB2. Of
these the lack of phenylalanine and of aspartic acid is parti-
Fig.
3: Nucleotide sequence of cloned FMDV cDNA. The deduced
amino acid sequence of the translation product is shown for
the first phase, potential stop codons («*•) in the other two
phases. The sequence that codes for VP1 is boxed.
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nM
200
100
min
Fig.
4: Kinetics of the release of amino acids from VP1 during
degradation with carboxy-peptidase Y (for d e t a i l s see Methods).
Table 1 Amino acid compositions for VP1 and for its
carboxy terminal peptide CB2 from FMDV strain C^K.
amino
acid
Ala
Arg
CB2
VP1
(a)
21,50
14,97
Asn
Asp
19,12
Cys
(b)
(a)
21
15
4,24
3,23
11
8
<0,03
-
-
2
Gin
Glu
21,12
10
10
Gly
His
8,86
7,12
8,44
20,16
10,75
2,74
3,86
16,06
6,79
22,78
He
Leu
Lys
Met
Phe
Pro
Ser
Thr
Trp
-
Tyr
9,96
15,76
Val
Total
210*
9
7
9
21
10
3
4
15
7
24
1
10
16
213
4,20
(b)
4
3
0
0
1
2
2
0,03
2,04
1,40
2,98
4,00
<0,02
-0,05
4,26
<0,04
3,03
0
2
-
0
1
1 ,04
1,43
32»
2
3
4
0
0
4
0
3
2
33
(a) determined experimentally from protein
(b) deduced from the nucleotide sequence
*) corrected for the lack of values for Cys and Trp
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cularly important since these are the ainino acids specified by
the three codons that follow the predicted end of the VP1 gene.
Together with the results from the carboxy-peptidase degradations this defines precisely the position of the carboxy-terminal of the VP1 segment in the FMDV polyprotein. Thus, VP1 is encoded in a sequence of 639 nucleotides between pos. 2977 and 3615.
This is several hundred nucleotides closer to the 5'-end of the
FMDV genome than predicted originally from the biochemical map
and suggests that there may be an extended non-coding region at
the 3'-end of the FMDV RNA. Such a silent sequence of 562 nucleotide residues has recently been detected in the genome of poliovirus, another picornavirus which shares many structural features with FMDV (20).
Several features of the VP1 coding sequence seem worthwhile
mentioning. Firstly, as a sequence derived from an RNA virus the
FMDV sequence is relatively rich in CG dinucleotides, which rarely occur in eucaryotic DNA sequences (21). Consequently, there
are also many cleavage sites for restriction nucleases such as
Hpall (CCGG), Taql (TCGA) and Hhal (GCGC). Secondly, there is a
high preference for C or G as a third base of the codons in the
VP1 coding sequence. As shown in Table 2, this is the case in
particular for the choice between the codons for histidine (CAC/
Table 2: Codon distribution in the VP1 gene
TTT
TTC
TTA
TTG
CTT
CTC
CTA
CTG
ATT
ATC
ATA
ATG
GTT
GTC
GTA
GTG
Phe 1(6)
Phe 3(6)
Leu 0(1)
Leu 10(3)
Leu 3(3)
Leu 4(6)
Leu 1(2)
Leu 3(7)
H e 4(2)
H e 4(5)
H e 1(0)
Met 3(4)
Val 2(1)
Val 3(5)
Val 1(1)
Val 10(8)
TCT
TCC
TCA
TCG
CCT
CCC
CCA
CCG
ACT
ACC
ACA
ACG
GCT
GCC
GCA
GCG
Ser 2(0)
Ser 2(7)
Ser 2(1)
Ser 1(3)
Pro 2(2)
Pro 5(3)
Pro 6(1)
Pro 2(3)
Thr 7(0)
Thr 12(4)
Thr 3(1)
Thr 2(0)
Ala 3(3)
Ala 4(9)
Ala 7(4)
Ala 7(2)
Tyr 0(0)
Tyr 10(1)
Och 0(0)
Amb 0(0)
His 0(0)
His 7(2)
Gin 4(0)
Gin 6(2)
TAT
TAC
TAA
TAG
CAT
CAC
CAA
CAG
AAT
AAC
AAA
AAG
Asn
Asn
Lys
Lys
2(1)
9(4)
5(5)
5(6)
GAT
GAC
GAA
GAG
Asp
Asp
Glu
Glu
1(0)
702)
5(3)
5(6)
TGT
TGC
TGA
TGG
CGT
CGC
CGA
CGG
AGT
AGC
AGA
AGG
GGT
GGC
GGA
GGG
Cys
Cys
Opa
Trp
Arg
Arg
Arg
Arg
Ser
Ser
Arg
Arg
Gly
Gly
Gly
Gly
1(0)
1(1)
0(0)
1(1)
0(1)
3(1)
0(0)
4(2)
0(3)
0(2)
4(1)
4(2)
4(3)
2(0)
3(3)
0(3)
The figures in brackets are from the FMDV sequences that preceed and follow the VP1 coding sequence in Fig. 3.
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CAU), tyrosine (UAC/UAU), leucine (UUG/UUA), and aspartic acid
(GAC/GAU), but it can also be noticed for most other pairs of codons. This preferential selection for CG rich codons seems also
to hold for the coding properties of the FMDV RNA outside of the
VP1 gene (Table 2 ) , but has not yet been noticed in the genome
of other eucaryotic RNA viruses (22).
The VP1 polypeptide
From the VP1 coding sequence as defined and discussed above
an amino acid sequence of 213 residues can be deduced for the
VP1 gene product which is presented in Fig. 3. This sequence appears to be that of authentic mature VP1 since there is a high
consistency between many biochemical properties predicted and
those observed experimentally. This includes the amino acid composition of VP1 determined during this work (Table 1 ) , and the
number, the size, and the order of peptide fragments that are
produced from VP1 by various chemical and proteolytic cleavage
reactions (Strohmaier, Franze & Adam, in prep.). In addition,
close to 50 % of the amino acid sequence has been confirmed recently by direct sequencing of the various peptide subfragments
from VP1 mentioned above (Strohmaier et al., in prep.). We
therefore conclude that VP1 consists of the sequence of the 213
amino acid residues shown in Fig. 3.
In addition to the primary structure the sequence data presented here also allow to deduce amino acid sequences that flank the
VP1 polypeptide in the precursor polyprotein and which must contain (part of) the information responsible for the specific
cleavages that occur during the maturation of the VP1 polypeptide
chain. However, more sequences for other such processing signals
from FMDV or from other picorna viruses will be required to elucidate the common structural features of these sites which appear
to be recognized with high specificity by cellular and virus induced proteases.
A molecular weight of 23840 is calculated for VP1 from our sequence analysis. This is considerably lower than the values of
about 30000 that have been determined for the coat proteins of
FMDV by various physico-chemical methods (23, 24, 25). A similarly low molecular weight seems also to be correct for protein VP3
whose gene has been found to code for no more than 220 amino
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acids (R. Cattaneo, unpubl. results). These discrepancies seem
not to be due to glycosylation of the FMDV coat proteins
(Strohmaier, unpubl.) although two potential glycosylation sites
(26) are present in the Asn-Thr-Thr-Asn-Pro-Thr sequence in the
center of the VP1 chain. Therefore, it is more likely that these
inconsistencies are caused by the substantial degree of phosphorylation of the structural proteins of FMDV which have been noticed very recently by La Torre et al. (27).
Further comments
VP1 carries the main antigenic determinants of the FMDV virion
(28, 29). The detailed knowledge of its gene structure and of its
amino acid sequence represents a major step towards an understanding of its immunogenic properties. So far, the nucleotide sequence has allowed the specific construction of a fused gene that
is expressed in a bacterial cell (6). In addition, the VP1 sequence from one FMDV strain also provides a basis for a rapid
determination of the amino acid changes that lead to different
serotypes and subtypes of FMDV and thus should allow to identify
the main antigenic determinants in the amino acid sequence of
VP1. Indeed, a particularly high degree of sequence variation has
been detected in the VP1 coding region by hybridisation experiments with cloned cDNA fragments from FMDV strain 0..K and the
viral RNAs from FMDV strains A and C (Kiipper, unpubl.). Finally,
knowledge of the VP1 amino acid sequence should also help to
identify and to localize antigenic sites on the surface of the
three-dimensional structure of the protein and of the virion by
specific biochemical and immunological methods.
ACKNOWLEDGEMENTS
We thank K.H. Adam for compentent technical assistance in the
protein analysis, J. Wolters for carrying out the computer analysis of the sequence data and the Deutsches Krebsforschungszentrum
for use of computer facilities. This work was supported by
BIOGEN S.A.
+
present address: 3, rte de Troinex, 1227 Carouge/GE, Switzerland
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Nucleic Acids Research
REFERENCES
§The terminology as in Ref. 1 is used, VP1 corresponds to VP3 in
Ref. 2. VP1 has been also designated VP
or VP_.
on the basis
of its NH_-terminal amino acid threonine (3, 4 ) .
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Nucleic Acids Research
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(1978) Biochem. Biophys. Res. Comm. J35_, 1640 - 1645
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Virology 5_2, 520 - 528
20. Kitamura, N. and Wimmer, E. (1980) Proc. Natl. Acad. Sci.
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