Download Amino Acid Sequence Homology of Coat Proteins as a Basis for

J. gen. Virol. (1988), 69, 2703-2710. Printed in Great Britain
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Key words: potyviruses/taxonomy/coat protein sequence
Amino Acid Sequence Homology of Coat Proteins as a Basis for
Identification and Classification of the Potyvirus Group
By D . D . S H U K L A * AND C. W . W A R D
CSIRO, Division of Biotechnology, Parkville Laboratory, 343 Royal Parade, Parkville,
Victoria 3052, Australia
(Accepted 11 August 1988)
SUMMARY
Analysis of the 136 possible pairings of the coat protein amino acid sequences from
17 strains of eight distinct potyviruses revealed a bimodal distribution of sequence
homology. Distinct members of the group exhibited sequence homologies ranging from
38 to 71 ~ (average 54%) with major differences in the length and sequence of their N
termini and high sequence homology in the C-terminal half of the coat proteins. In
contrast strains of individual viruses exhibited sequence homologies of 90 to 99%
(average 95 9/o)and had very similar N-terminal sequences. These findings cast doubt
on the currently held 'continuum' hypothesis proposed to explain the unsatisfactory
taxonomy of the potyvirus group. The coat protein sequence data, in combination with
information on the nature of the potyvirus particle assembly, can be used to develop
rationally designed, simple serological techniques that appear to be more useful and
more easily applied than those properties previously used for potyvirus identification
and classification.
INTRODUCTION
The potyvirus group is the largest and economically the most important of the 28 plant virus
groups and families (Matthews, 1982; Brown, 1986). Some members of the group are very
successful pathogens and in 1974 were reported to infect 1112 species of 369 genera in 53 plant
families (Edwardson, 1974). The group contains at least 152 definitive and possible members
(Matthews, 1982; Francki et al., 1985) accounting for about one-quarter of all viruses known to
infect plant species around the world. Potyviruses exist in numerous strains or pathotypes which
differ mainly in biological properties, such as host range or pathogenicity (Hollings & Brunt,
1981 b). Definitive potyviruses are transmitted in the non-persistent manner by many aphid
species while some possible members have fungus, mite or whitefly vectors. Definitive and
possible members of the group investigated so far have all been found to induce characteristic
'pinwheel' cytoplasmic inclusion bodies in infected plant cells. Potyvirus particles are flexuous
rods, 680 to 900 nm long and 11 nm wide, consisting of a single protein species of Mr ranging
from 30000 to 37000 and a single molecule of ssRNA of Mr 3"0 x 106 to 3"5 × 106 (Hollings &
Brunt, 1981b; Francki et al., 1985).
It has been consistently pointed out by taxonomists and reviewers that the taxonomy of the
potyvirus group is in a very unsatisfactory state and that successful resolution of potyvirus
taxonomy presents a major challenge for plant virologists (Hollings & Brunt, 1981 a, b; Francki,
1983; Francki et al., 1985; Harrison, 1985). As Francki (1983) pointed out, the current
unsatisfactory state of potyvirus taxonomy is due in part to the large size of this group, but
mainly to the vast variation among the viruses in the group and the inability of the workers to
understand the taxonomic significance of this variation. It is currently believed, on the basis of
comparative biological properties and inconsistent serology, that strains of potyviruses form a
'continuous' array between two or more distinct viruses in such a way that the borderlines
separating individual potyviruses cannot be sharply defined (Bos, 1970; Hollings & Brunt,
0000-8438 © 1988 SGM
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D. D. SHUKLA AND C. W. WARD
1981 a, b; Harrison, 1985). In this way a distant strain of one virus could appear more closely
related to the distant strain of a second virus than either are to their homologous viruses
(Hollings & Brunt, 1981a; Harrison, 1985).
During our investigations on the structural characterization of potyvirus coat proteins (Gough
& Shukla, 1981 ; Skukla et al., 1986, 1987, 1988 b, c, e) we observed that the amino acid sequences
of coat proteins could clearly differentiate individual potyviruses and their strains. In this paper
we have compared the coat protein sequences from 11 strains of four distinct potyviruses
determined in our laboratory with additional data from nine strains of five distinct potyviruses
determined by other workers. The results show that amino acid sequence homology of coat
proteins can serve as a basis for identification and classification of the potyvirus group.
METHODS
The potyvirusesused for comparison of coat protein structural properties were four strains of potato virus Y
(PVY), the type member of the potyvirus group (Shukla et al., 1986, 1988e), pepper mottle virus (PeMV;
Dougherty et al., 1985), Johnson grass mosaic virus (JGMV; Shukla et al., 1987), three strains of passion-fruit
woodiness virus (PWV; Shukla e t al., 1988c), two strains of tobacco etch virus (TEV; Allison et al., 1985a, b),
tobaccovein mottling virus (TVMV; Domieret al., 1986),three strains of plum pox virus (PPV; Ravelonandroet
al., 1988; Maiss et al., 1988; E. Maiss, personal communication), two strains of soybean mosaic virus (SMV;
Gunyuzluet al., 1987;Eggenbergeret al., 1988)and three strains of sugar-canemosaicvirus (SCMV; Shuklaet al.,
1987). Percentage homologiesbetween the coat protein amino acid sequences of the distinct viruses and strains
were obtained by computer analysis of all 136 possible pairings using the ALIGN program of the National
Biomedical Research Foundation, U.S.A., with modificationmade by Dr A. Kyne for the VAX/VMScomputer
system.
RESULTS AND DISCUSSION
When the 136 possible comparisons of complete coat protein sequences of the 17 strains of
eight distinct viruses (Table 1) are graphed as a frequency distribution (Fig. 1) the results clearly
reveal a bimodal distribution of sequence homologies. In almost all cases the sequence homology
between distinct members ranged from 38 to 71 ~ (average 54~o) while that between strains of
the one virus from 90 to 99 % (average 95 ~). The only exceptions to this pattern were the two
SMV isolates which were as different from each other as they were from other distinct members,
and PeMV which was as closely related to the four strains of PVY as other known strain
groupings were to each other (Table 1, Fig. 2 and 3). The very close structural relationship
between the coat proteins of PeMV and the four strains of PVY (Table l, Fig. 2) compared to the
much lower sequence homology between distinct members of the potyvirus group suggests that
PeMV should be considered a strain of PVY (Shukla et al., 1988e). This analysis also indicates
that SMV-N and SMV-V, with a sequence homology of 58%, should be considered as two
distinct potyviruses rather than strains of SMV. Although both are known to cause similar
mosaic diseases of soybean in the U.S.A., SMV-N is characterized as a severe isolate of SMV
originating in Illinois and is not transmissible by aphids (Vance & Beachy, 1984) while SMV-V
is a common isolate of SMV in Virginia and is aphid-transmissible (Hunst & Tolin, 1982).
Comparison of the two isolates in soybean cultivars showed symptom differences typical of
those generally found between strains of one virus (D. D. Shukla, unpublished results).
However, on the basis of sequence homology SMV-N is more closely related to PWV (70-71 ~)
than to SMV-V. In fact SMV-N has 127 of the 150 C-terminal amino acid residues identical to
PWV in the same C-terminal region and both viruses have one deletion each in their sequences
at exactly the same position (position 44 in PVY) relative to other coat proteins. Similarly,
SMV-V is more closely related to TEV (68 to 69%), PVY (65 to 68%) and TVMV (65%) than to
SMV-N.
The findings reported here (Fig. 1) are not consistent with the 'continuum' hypothesis and
show a clear demarcation of sequence homologies between distinct individual viruses and
between strains. The sequence homology between distinct members was little affected by the
choice of strain used to make the comparison (Table 1), indicating that, at least for the viruses
studied here, the boundaries between peripheral virus strains are not blurred.
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-
95"5t
-
90-6
94.2
-
PPVNAT
44.8
47-6
49.5
-
43.2
45.6
48.1
51.5
-
J G M G - PWVJG
TB
43.2
45.6
48-1
51.5
98.9
-
PWVS
42-6
44.7
44.7
50.2
95.9
95.5
-
PWVM
45.2
48-8
51.1
47-7
60.0
59.6
58.9
-
PVYD
45.2
48.5
50.8
47.7
60.0
59.6
59.3
99.3
-
PVY10
45-5
49.1
51-4
49.7
60.4
60.0
59-3
96.3
96.3
-
PVY18
46.1
49.1
51.4
49.0
59.3
59.3
58.5
97.0
97.8
96.6
-
PVY43
-
47.0
49.7
51.7
49.3
61.5
61.9
60.0
91.4
92.1
92.1
92.9
PeMV
43-2
45.3
47-8
53.8
70.7
70.7
70-0
59.3
59-3
59.0
59.3
60.4
-
SMVN
43.0
46.1
47-0
46-7
55.2
54.8
53-7
65-2
65.9
66-7
66.7
67.8
58.3
-
SMVV
37.9
40.9
40.6
44.4
52.6
52.2
51.5
51.3
51.3
52.8
51-7
53-6
53:4
64.5
-
TVMV
46-7
49.1
51-1
48.5
58-9
58-5
56.7
61.4
61.0
62.5
61.8
61.4
56-8
68.3
57.4
-
TEVNAT
46-4
48.5
50.5
48.8
58.5
58.5
56.7
61.4
61.0
62-5
61.4
61.4
57.9
69-4
58.5
97-7
TEVHAT
* The sources of sequence data were: PPV-D (Ravelonandro et at., 1988); PPV-AT and P P V - N A T (Maiss et al., 1988; E. Maiss, personal communication);
J G M V - J G (Shukla et al., 1987); PWV-TB, PWV-S and PWV-M (Shukla et aL, 1988c); P V Y - D (Shukla et al., 1986); PVY-10, PVY-18 and PVY-43 (Shukla et al.,
1988e); PeMV (Dougherty et al., 1985a); SMV-N (Eggenberger et al., 1988); SMV-V (Gunyuzlu et al., 1987); T V M V (Domier et al., 1986); T E V - N A T and TEVH A T (Allison et al., 1985a, b). The three strains of SCMV (Shukla et al., 1987) were not included in the full matrix analysis as their sequence data are incomplete.
i" Related strains with a sequence homology of 90 to 100% are indicated by bold type-face.
PPV-D
PPV-AT
PPV-NAT
JGMV-JG
PWV-TB
PWV-S
PWV-M
PVY-D
PVY-10
PVY-18
PVY-43
PeMV
SMV-N
SMV-V
TVMV
TEV-NAT
TEV-HAT
PPVAT
Table 1. Percentage amino acid sequence homology between coat proteins of distinct potyviruses and their strains*
PPVD
t-o
-..o
2706
D.D.
I
I
16
S H U K L A A N D C. W . W A R D
I
I
I
/
15
!
r,'--'
I
10
20
30
80
90
\
!
s/I
70
\
/
8
'4
x
/
*.,, 10
i
\
/
12
i
b
\
/
14
I
I
/
\
ill
40
'
50
60
Homology (%)
100
Fig. 1. Frequency distribution of the a m i n o acid sequence homologies for the coat proteins f r o m 17
strains of eight distinct potyviruses. The sequence homologies are s h o w n in Table 1. T h e homologies
between distinct viruses had a m e a n value of 54.1% and a standard deviation of 7.29% while the
homologies between strains of individual viruses showed a m e a n of 95-4% and standard deviation of
2.56%. The dotted curves show that all values for distinct viruses and strains fall within + 3 standard
deviations from their respective m e a n values.
IllgllllllIHIIllllgllHIHllll[lllllllllllllllllllllllllnlllllllnllllllllllllI Ill I lllllll IIIIUIIIllE Ill Ill]IlllIllll
IlglllllllmmlllglIHl[ll[llllIWIrllllllllgllHI IIII[llllllll IMInlllIIIIU IIII HIllflllllll[I
JGMV-JG
IIIIllIllIIIlllIIIIIIIIIl IU
IllIl Ill I ltll II Ill llIII II I I~llll~lllllltl
PWV-TB
[
PVY-D
I fit tI 1111
fill
I
I
PeMV
IIIIIrl[111 III [Him[
Ill [ill Ill Ill II II I I I [ I n II ii I 1 II H II [
SMV-V
111111111Imllnl
[ 111 I1111111111111111 II 11111 I 11111111111111111111
TVMV
IIIIIIIII1111111i111111 I
11 Ili1III 111111 II I I II IIII II 11111111111
TEV-NAT
PPV-D
I
I
I
I
I
I
50
I
I
I
I
I
I
I
I
I
I
I
I
I
II TI Illlll Illl IlIIll[
IIlll Ill
rl I V
III
TI
III
I7
I
II1[
IIIIII
ffllll
nil
gill[
JI l]1114LJ
LIJ
II
IIII II Iil111 II1111 tt it1 ILI~
II
I I
I
[
I I
I [
II I
IlIIfll
II
I
I I
I III
I
I
I11
Ill III
I II I t1111 IIIIIII
I 1 ~ 1 1
I
I
100
150
200
A m i n o acid residue n u m b e r (PPV-D)
I
I
Illl
I]
I
250
I
I
Itll
J
II
I II
Illllfllfl[ Ill
li IJll
111111
I
1111 I II I~l
I
I
U
1I
I J
300
Fig. 2. Schematic d i a g r a m showing the location of sequence differences between distinct m e m b e r s of
the potyvirus group. The sequences were c o m p a r e d with strain D of PVY, the type m e m b e r . T h e
sources of their sequences are listed in Table 1.
Although it may be argued that we have based our conclusion on less than 10~ of the total
number of viruses in the group, the results reported here indicate a definite trend which is
further reinforced by partial sequence data for the three strains of SCMV (Shukla et al., 1987). It
is expected that other members of the group would exhibit similar coat protein properties
because the nine viruses compared in this paper already represent diverse subgroups in terms of
their biological properties and previous serological relationships. For example PVY and TEV
are viruses that have overlapping host ranges and whose coat proteins and cylindrical inclusion
proteins have been reported to be serologically related (Purcifull et al., 1973, 1975; Nelson &
Wheeler, 1978), and therefore their strains could have been expected to conform to the
'continuum' hypothesis. As shown in this paper they do not.
The bimodal distribution of sequence homologies is similar to that found for the coat proteins
of subtypes (39 to 70~) and strains (83 to 99~) of influenza virus (Ward, 1981 ; Colman & Ward,
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Coat protein as basis for potyvirus taxonomy
PPV-D
PPV-AT
PPV-NAT
I
II
[
I
I
I
IIIIIIIllllllll
if
IIII II
III
II 1
i
)(
SCMV-SC
SCMV-BC
SCMV-Sabi
PWV-TB
PWV-S
PWV-M
IlL
11 li
PVY-D
PVY-10
i
]
i
[
PVY-18
PVY-43
Iiii
i
II
[
lii
il
SMV-V
SMV-N
JJJJJJJIJlJlJllllJlJJJJJIJJJJJIJ5JJJJJJJllJJJJJJJJJ IJ I II I II IIIIIIIII]1 I II I
TEV-NAT
[
TEV-HAT
I II
I
I
I
I
I
I
50
I
I
I [ I I
I
I
I
I
I
I
II
J Ill Ill I I II Illllll It II II If IIIII
I
I
I
I
I
I
I
I
I
I
I
I
100
150
200
Amino acid residue number (PPV-D)
I
I
250
I
I
I
I
I
I
300
Fig. 3. S c h e m a t i c d i a g r a m s h o w i n g t h e l o c a t i o n o f s e q u e n c e d i f f e r e n c e s b e t w e e n s t r a i n s o f P P V ,
S C M V , P W V , P V Y , S M V a n d T E V . T h e s o u r c e s o f t h e i r s e q u e n c e d a t a a r e l i s t e d in T a b l e 1. T h e w a v y
lines indicate that the sequences are incomplete.
1985; Raymond et aL, 1986; Dale et al., 1986; Nestorowicz et al., 1987; V. Harley, personal
communication).
Although amino acid sequence homologies between coat proteins of potyviruses clearly
differentiate between distinct members and strains, their value as a taxonomic criterion would
be greatly enhanced if this information can be exploited by simple techniques such as serology.
Recent results on potyvirus serology from our laboratory suggest that such an exercise is possible
(Shukla et al., 1988a, d, 1989). First, the sequence data show that the N terminus is the only large
region in the entire coat protein of a potyvirus that is virus-specific. Therefore, epitopes
contained in this region should generate antibodies specific for the virus and its strains. On the
other hand, epitopes contained in the internal conserved region should generate potyvirus
group-specific antibodies. Secondly, using six biologically and serologically distinct potyviruses
we found that 30 to 67 amino acid residues (depending on the virus) from the variable N
terminus and 18 to 20 residues from the C terminus of the coat proteins are exposed on the
particle surface and that the N-terminal region constitutes the most immunodominant region in
the potyvirus coat proteins (Shukla et al., 1988 d). Using this information we were able to show
that highly specific antibodies could be readily prepared by adsorption of crude antisera on an
affinity column of virus particles from which the surface-exposed N-terminal epitopes had been
removed. Antibodies that did not bind to the column were found to recognize the homologous
viruses and their strains. In contrast, the original unfractionated antisera contained antibodies
to conserved core regions of the coat proteins and reacted with several distinct potyviruses
(Shukla et al., 1988a, 1989). The results clearly demonstrated that virus-specific polyclonal
antisera can be readily obtained by this method.
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I
2708
D. D. SHUKLA
AND
C. W.
WARD
We believe that criteria based on structural properties of coat proteins may be more useful
than other properties for identification and classification of potyviruses for three main reasons.
Coat protein is a unique gene product
Analysis of all available data on amino acid composition of coat proteins of members in
different plant virus groups including potyviruses has convincingly demonstrated that the
amino acid composition of coat proteins is characteristic of the virus group (Fauquet et al., 1986a,
b). Also, known coat protein amino acid sequences of plant viruses have so far shown very little
or no sequence homology between different groups of plant viruses. Comparison of amino acid
sequences of the gene products of potyviral genomes revealed that except for the coat protein all
other gene products have homology with gene products of viruses in other plant or animal virus
groups (Domier et al., 1987). These findings show that coat protein is the only unique gene
product characteristic of the potyvirus group. Therefore, any classification scheme based on
coat protein properties may be more appropriate for differentiating members within the
potyvirus group than those based on properties of other gene products.
Coat protein is the only major gene product in the virion
The potyvirus genome is translated into eight different gene products (Domier et al., 1987) of
which the coat protein and genome-linked protein are the only gene products in the virion. The
other gene products are present in the infected cells. Some of these gene products have not yet
been isolated (Domier et al., 1987) and methods to obtain others in pure form are either difficult
or time-consuming (Hiebert et al., 1984; Thornbury et al., 1985). This makes the coat protein an
attractive candidate for use in taxonomy of potyviruses as particles of most potyviruses can now
be obtained in a sufficiently pure form. The coat protein accounts for approx. 95 ~ of the weight
of potyvirus particles. There are about 2000 coat protein subunits per RNA molecule (Hollings
& Brunt, 1981 b). It has been argued in the past that coat proteins represent only a fraction of the
total coding capacity (about 12~ in the case of potyviruses; Domier et al., 1987) of the viral
genomes and therefore that taxonomic criteria based on coat proteins will compare only a
fraction of the total genomic information. However, as has been pointed out by Van
Regenmortel (1986), successful classifications based on very few parameters have been
developed, such as the reconstruction of phylogenetic trees of higher organisms on the basis of
the degree of sequence homology found in a small number of proteins (Wilson et al., 1977).
Serology reflects protein structure
To date serological techniques are most popular among the various methods used for
identification and classification of viruses in plant virus laboratories around the world because
they are simple, reproducible, efficient and relatively inexpensive in terms of reagents and
space. Members of several plant virus groups, tobamoviruses (Gibbs, 1977) and tymoviruses
(Koenig & Lesemann, 1979) in particular, can be accurately classified on the basis of their
serological properties. Although serology has so far failed to provide a useful taxonomy for the
potyvirus group (Hollings & Brunt, 1981a; Francki et al., 1985), we have recently shown that
coat protein sequence data, in combination with information on the nature of potyvirus particle
assembly (Shukla et al., 1988d), can be used to develop rationally designed, simple serological
techniques (Shukla et al., 1988a, 1989) that appear to be more useful and more easily applied
than other properties for potyvirus identification and classification. Such technology was
employed recently to show that 17 potyvirus strains infecting Poaceae were not all closely related
strains of SCMV as previously believed but were forms of four distinct potyviruses namely
SCMV, JGMV, maize dwarf mosaic virus and sorghum mosaic virus (Shukla et al., 1989). This
finding along with that reported here for SMV-N and SMV-V suggest that the number of
distinct potyviruses recognized is going to increase. In addition the belief that many viruses
included in the group may be synonymous (Francki, 1983) is now testable and may prove false.
We are most grateful to Dr R. N. Beachy, Washington University, St Louis, Mo., U.S.A., Dr S. A. Tolin,
Virginia PolytechnicInstitute and State University, Blacksburg, Va., U.S.A., Dr M. Ravelonandro, Station de
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Coat protein as basis for potyvirus taxonomy
2709
Pathologie V6g~tale, I N R A , Pont-de-la-Maye, France and Dr E. Maiss, Institute ffir Viruskrankheiten der
Pflanzen, Braunschweig, F.R.G. for their generous provision of sequence data on SMV-N, SMV-V and PPV
strains, respectively, before publication. We thank M s L. M o n a r c h for the photographs.
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