Download Surface Structure and RNA-Protein Interactions of Foot-and

J. gen, Virol, (1987), 68, 1649-1658. Printed in Great Britain
1649
Key words: FMDV/RNA-protein interactions/surface structure
Surface Structure and RNA-Protein Interactions of Foot-and-Mouth
Disease Virus
By D. J. M O R R E L L , t E. J. C. MELLOR,~: D. J. R O W L A N D S § AND
F. B R O W N * §
The Animal Virus Research Institute, Pirbright, Woking, Surrey GU24 ONF, U.K.
(Accepted 27 February 1987)
SUMMARY
The surface structure of foot-and-mouth disease virus (FMDV) and the interaction
of the individual capsid proteins with the virus RNA have been examined using
modification reagents. By measuring the extent of modification of the lysine residues of
intact and disrupted virus particles and the 12S protein subunit with Bolton & Hunter
reagent it was found that 54~o of the residues of VP 1, 15 ~o of the residues of VP2 and
37~ of the residues of VP3, equivalent to five, two and four lysine residues
respectively, are on the surface of the intact virus particle. Polypeptide VP4 was not
modified in intact virus particles, indicating that it has no lysine residues on the surface
of the virus. Modification with sodium metabisulphite, which causes a specific
transamination reaction between cytidylic acid residues in ssRNA and closely
associated basic amino acids, cross-linked all four structural proteins to the virus RNA.
Both fragments of VPI, produced by treatment of the virus particle with trypsin, are
also cross-linked to the RNA. These observations have been combined with the
evidence that the immunogenic activity of VP 1 may be contained in two discontinuous
sites, at amino acids 141 to 160 and 200 to 213, in proposing a model for the
arrangement of this polypeptide in the virus particle.
INTRODUCTION
Foot-and-mouth disease virus (FMDV) forms the genus aphthovirus of the family
Picornaviridae (Cooper et al., 1978). The virus particles are composed of 60 copies of four
structural proteins, VPI to 4 (tool. wt. values: VP1 to 3 approx. 24 × 103, VP4 approx.
8.5 x 103; Boothroyd et al., 1982), which form a protein capsid surrounding a single positivestrand RNA molecule, mol. wt. 2.8 × 106. The aphthoviruses break down under mild acid
conditions (pH 6.5) to give VP4, the virion RNA and a 12S subunit containing five copies of the
structural proteins VP1 to 3 (Vasquez et al., 1979; Morrell, 1983).
The use of protein modification reagents to examine the surface of picornavirus particles can
give important information concerning the virus structure. Such studies have been performed on
FMDV using lactoperoxidase-catatysed iodination (Laporte & Lenoir, 1973; Talbot et al., 1973).
However, comparison of the results of chemical modification of proteins with their threedimensional structures obtained by X-ray diffraction studies has shown that lysine modification
gives a better indication of surface exposure than tyrosine modification (Glazer, 1982). To
obtain a greater understanding of the structure of the FMDV particle the surface exposure of the
structural polypeptides of the virus and 12S particles has been examined with Bolton & Hunter
reagent [N-suecinimidyl-3-(4-hydroxy-5-iodophenyl)propionate; Bolton & Hunter, 1973], a
small lysine-modifying reagent.
t Present address: Department of Growth and Development, Institute of Child Health, 30, Guilford Street,
London WC1N 1EH, U.K.
Present address: Department of Biochemistry, South Parks Road, Oxford OX1 3QU, U.K.
§ Present address: Wellcome BiotechnologyLimited, Ash Road, Pirbright, Woking, Surrey GU24 0NQ, U.K.
0000-7391 © 1987 SGM
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D.J.
MORRELL AND OTHERS
The presence of intact RNA within the virus particle stabilizes the capsid against degradation
(Bachrach, 1968; Denoya et al., 1978a) suggesting that the RNA is involved in interactions with
the protein components of the virus capsid. These interactions have been studied by covalently
cross-linking basic amino acids of the capsid proteins to cytidylic acid residues of the virus RNA
in situ using sodium metabisulphite (Shapiro & Gazit, 1977). In addition, production of RNAfree 'artificial empty' or 12S particles from mature FMDV particles results in a change of
antigenicity (Rowlands et al., 1975; Cartwright et al., 1982). Thus, maintenance of the correct
conformation of the antigenic determinants appears to be RNA-dependent. The antigenic
determinants of FMDV have been shown to be located within VP1 (Laporte et al., 1973;
Strohmaier et al., 1982; Bittle et al., 1982). To locate more precisely the relationships between
the antigenic and RNA-binding sites of VP1, advantage was taken of the fact that trypsin
cleaves the protein in situ at defined positions to give two major fragments L and S (Burroughs et
al., 1971), whose participation in RNA-protein cross-links following bisulphite treatment was
examined.
The interactions of the structural proteins of FMDV with the internal and external
environments have been studied. The implications of this work for virus structure, stability and
antigenicity are discussed.
METHODS
Virus growth and purification. Monolayers of BHK-21 cells in Roux flasks (about 108 cells/flask) were infected
with FMDV of serotype A, subtype 10, strain Argentina 1961 (A61) and the virus particles were radioactively
labelled by the addition to the culture medium of [35S]methionine (150 ~tCi/flask), [3H]leucine (300 ~tCi/flask) or
[3H]uridine (500 ~tCi/flask). For labelling with amino acids, medium lacking the appropriate amino acid was used.
All radiochemicals were obtained from Amersham. Virus was purified by differential centrifugation, treatment
with l~ o (w/v) SDS and sucrose density centrifugation (Brown & Cartwright, 1963; Harris & Brown, 1977).
Preparation of l2S particles. An equal volume of 0.2 M-citric acid, 0.1 M-NaC1 was added to 0.5 ml of a sucrose
density gradient fraction containing purified [3H]leucine-labelled virus particles (approx. 150 ktg/ml). Pancreatic
RNase A (Sigma), 2 mg/ml in 0.3 M-sodium acetate buffer pH 5.0, was added at a final concentration of 100 ~tg/ml
and the mixture was incubated at 37 °C for 30 min. After filtration through a l0 ml Sephadex G-50 column
equilibrated with TN buffer (0.1 M-NaCI, 0.I M-Tris-HC1 pH 7.4), the crude 12S particle preparation was
centrifuged through a 20 ml 15 to 25~o (w/v) sucrose gradient in TN buffer for 16 h at 30000 r.p.m. (MSE 3 × 23
rotor). Fractions (1 ml) were collected and the 12S particles were detected by liquid scintillation counting of
samples of the fractions.
Modification with 1,.SI_labelled Bolton & Hunter reagent. Purified virus or 12S preparations were filtered through
Sephadex G-25 columns equilibrated with 0.1 M-NaCI, 0.2 M-sodium borate buffer pH 8.2, concentrated by
pressure dialysis against the same buffer and adjusted to 1 nag protein/ml. Disrupted virus was prepared by the
addition of SDS and 2-mercaptoethanol (final concentrations 1 ~ w/v and 2 ~ v/v respectively) to a concentrated,
purified virus preparation and boiling for 5 rain.
125I-labelled Bolton & Hunter reagent (1000 Ci/mmol; Amersham) was transferred to siliconized Eppendorf
tubes (300 ~tCi/tube) and the solvent was removed by evaporation in a stream of dry nitrogen. Samples (25 ~tg)to be
iodinated were added, with mixing, and the tubes were incubated at 20 °C for 30 min. The reaction was stopped by
the addition of 100 p.l 0.1 M-glycine in TN buffer. Following a further 30 rain incubation the samples were passsed
through 10 ml Sephadex G-25 columns equilibrated with TN buffer to remove unreacted 125I-labelled Bolton &
Hunter reagent. Modified disrupted virus particles were precipitated with acetone. Modified virus or 12S particles
were repurified by sucrose density centrifugation and precipitated with acetone.
Treatment with sodium metabisulphite. One M-sodium metabisulphite (1.4 ml), buffered with sodium sulphite and
sodium hydroxide to pH 7.2 and containing 0.005 M-quinol as a free radical scavenger, was added to a siliconized
Eppendorf tube containing 30 ~tl of a purified virus particles preparation labelled with both [35S]methionine and
[3H]uridine. After incubation at 37 °C for 4 h the reaction mixture was passed through a l0 ml Sephadex G-25
column equilibrated with TN buffer. Intact virus particles were repurified by sucrose density gradient
centrifugation.
Trypsin digestion ofbisulphite-treated virus particles. Repurified, bisulphite-treated particles were passed through
a Sephadex G-25 column equilibrated with 0.04 M-sodium phosphate buffer pH 7.6, and incubated at 37 °C for 30
min in the presence of 100 ~tg/ml TPCK-trypsin (Worthington). The digestion was stopped by the RNA
extraction procedure.
Extraction and purification ofprotein-RNA complexes. Bisulphite-modified virus particles were incubated at
56 °C for 5 min in 0.1 M-sodium acetate buffer pH 5-0, containing 0.1 ~o (v/v) 2-mercaptoethanol and 0.05~o (w/v)
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RNA-protein interactions in F M D V
80
(a) vPI
3
2D ~,14/~F..]
(b)
WP
3
'2
14/SF!
1651
I
I
I
(c)
"~ 60
e,o
VP
40
3
2
4/
1 SF
×
20
I
20
40
60
20
40
Slice number
60 1
20
40
60
Fig. 1. SDS-PAGE of proteins from (a) disrupted virus particles, (b) 12S particles and (c) intact virus
particles treated with t25I-labelled BoRon & Hunter reagent. Electrophoresis was from left to right.
SDS and centrifuged through 36 ml 5 to 25 ~ sucrose density gradients in 0.14 M-NaC1, 0.005 M-EDTA, 0.5 ~ (w/v)
SDS, 0.05 M-Tris-HCI pH 7.6, for 16 h at 18000 r.p.m. (Beckman SW28 rotor) at 22 °C. Yeast tRNA was added to
RNA sedimenting at about 37S and the mixture was precipitated with 2.5 vol. of ethanol at - 20 °C overnight.
Preparation of samples for gel electrophoresis. Acetone-precipitated samples were resuspended in gel sample
buffer (Laemmli, 1970) and boiled for 5 rain. Ethanol-precipitated samples containing RNA were resuspended in
0-005 M-EDTA, 0.05 M-Tris-HCI pH 7-6. Half of each sample was treated with 500 units/ml RNase T1 (Sankyo)
and 700 units/ml RNase A (Worthington) at 37 °C for 30 min. An equal volume of 2 × concentration gel sample
buffer was added to the samples which were then incubated at 56 °C for 15 rain.
SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Samples were loaded onto 10~ discontinuous SDSpolyacrylamide gels (Laemmli, 1970) containing 8 M-urea (Vande Woude et al., 1972) in 0.5 x 12 cm tubes.
Electrophoresis was at 20 mA until the bromophenol blue dye had migrated approx. 10 cm. The gels were fixed in
10% acetic acid, frozen and cut into 1 mm sections using a gel slicer (Mickle Instruments Ltd). Slices from gels
containing '~sI-labelled proteins were counted in a gamma counter. Slices from gels containing 3sSqabelled
proteins were digested in 0.2 ml NCS Tissue Solubilizer (New England Nuclear) at 37 °C for 4 h. Scintillation fluid
was added and the slices were counted in a liquid scintillation counter.
RESULTS
Modification with l zSI-labelled Bolton & Hunter reagent
Modified virus and 12S particles were repurified by sucrose density centrifugation. No
evidence of particle breakdown was observed (results not shown). Fig. 1(a) shows the
incorporation of '25I-labelled Bolton & Hunter reagent into the proteins of totally disrupted
virus particles. The major structural proteins, VP1 to 3, were labelled in proportion to their
lysine content (Table 1). It was not possible to quantify the incorporation of label into VP4 as
this protein ran close to the solvent front. The label detected at higher mol. wt. is due to
incorporation into minor structural components of the virus particle (Sangar et al., 1976).
Fig. 1 (b) shows the incorporation of 125i.labelled Bolton & Hunter reagent into the proteins of
12S particles, which do not contain VP4. The extent of modification is essentially the same as in
totally disrupted virus particles, demonstrating that all the lysine residues of 12S particles are
externally located.
Fig. 1(¢) shows the incorporation of 125I-labelled Bolton & Hunter reagent into the proteins of
intact virus particles. The extent of modification is quantitatively and qualitatively different
from that of 12S and totally disrupted virus particles. There is generally less incorporation and,
in particular, the incorporation into VP2 is much decreased. After taking into consideration the
level of radioactivity associated with the solvent front, derived from the labelling profile of 12S
particles (Fig. 1 b) which do not contain VP4, there is no detectable incorporation of Bolton &
Hunter reagent into VP4 in intact virus particles.
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D. J. MORRELL AND OTHERS
Table 1. Incorporation o f t25I-labelled Bolton & Hunter reagent into the structural proteins o f
FMD V
Preparation
Disrupted virus particles
12S particles:~
Intact virus particles
Protein
VP1
VP2
VP3
VP4
Label incorporated
into disrupted
virus particles (~o)*
100
100
100
ND§
No. of lysine
residues in
protein~
9
13
10
1
Lysine
residues
labelled
9.0
13'0
10'0
ND
VP1
VP2
VP3
VP1
VP2
VP3
VP4
95
94
97
54
15
37
011
9
13
10
9
13
10
1
8.6
12.2
9.7
4.9
2.0
3.7
011
* Derived from Fig. 1.
~"From Boothroyd et al. (t982).
:~ 12S particles do not contain VP4.
§ ND, Not determined.
II None detected.
Table 2. Cross-linking o f the structural proteins o f F M D V to the virus R N A
Lysine
Methionine
Protein
residues
Radioresidues
molecules
available
activity in
cross-linked
Number of
cross-linked
for crossEfficiency
virus crossto RNA
methionine
to RNA
linking
of crosslinked to
per virus
residues in
per virus
per virus
linking
Protein RNA (~ total)*
particle~
protein:~
particle
particle§
x 10311
VP1
0'45¶
3'2
3
1'07
240
4"5
VPI(L)
0"32**
2"3
2
1-15
VPI(S)
0"18'*
1"3
1
1"30
VP2
1"67¶
12"0
4
3"00
660
4"5
t"68"*
t2-1
3-03
4"6
VP3
0.38¶
2-7
3
0-90
360
2"5
0.40**
2.9
0.97
2'7
VP4
0"63¶
4-5
2
2-25
~<60
> 37.5
0"58**
4.2
2.1
> 35-8
* Derived from Fig. 2; the radioactivity cross-linked was 3.1 ~ and 3.2 ~/oof the total radioactivity in the virus
preparation.
~"Each methionine residue is equivalent to 0.14~o of the total radioactivity.
From Boothroyd et al. (1982).
§ [Lysine residues in protein (Boothroyd et al., 1982) minus lysine residues of protein on the surface of the virus
(from Table 1)] x 60.
tl Protein molecules cross-linked to the RNA per lysine residue.
¶ Experiment 1.
** Experiment 2.
A s s u m i n g the same i n c o r p o r a t i o n per lysine residue per p r o t e i n molecule in virus particles,
12S particles a n d totally disrupted virus particles, the results indicate that 5 4 ~ of the lysine
residues of VP1, 15~o of the lysine residues of VP2 a n d 37°/o of the lysine residues of VP3 are
available for modification on the surface of the virus particle. T h i s is e q u i v a l e n t to five lysine
residues of VP1, two of VP2 a n d four of VP3 (Table 1). T h e results suggest that VP4 is not
available for reaction on the surface of the virus particle.
The structural proteins VP1 to 3 have lysine residues exposed on the surface of 12S particles
that are not exposed o n the surface of the intact virus particle. T h e increased modification of 12S
particles gives a measure of the n u m b e r of lysine residues available for cross-link f o r m a t i o n with
the virion R N A u p o n bisulphite t r e a t m e n t (Table 2).
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RNA-protein interactions in FMD V
I
I
(a) VP
l
4
I
(b)
I
1653
I
I
12
VP
x
3 2 ! 1L
4
IS
1
eq
20
40
60
80
20
40
60
80
Slice number
Fig. 2. SDS-PAGE of [3sS]methionine-labelledproteins associated with RNA from (a) virus particles
treated with bisulphite and (b) virus particles treated with bisulphite and subsequentlywith trypsin. The
proteins were analysedwith (
) or without (...) ribonuclease digestion of the RNA. Electrophoresis
was from left to right. The d.p.m, in the protein covalentlylinked to the RNA remain in the stacking gel
which does not appear in the figure. Bands 1L and IS are the trypsin cleavage products of VPI.
Protein-RNA cross-linking with sodium metabisul#hite
Incubation at 37 °C for 4 h caused some degradation of virus particles in both untreated and
bisulphite-treated preparations. A substantial proportion of the RNA from intact, repurified
virus particles was also degraded (results not shown). Analysis by SDS-PAGE of the protein
associated with the virus RNA was confined to full length RNA from intact virus particles. This
constituted approx. 15Yo of the initial RNA.
To differentiate between proteins covalently cross-linked to the RNA and those noncovalently associated with the RNA, samples of RNA-protein complexes were analysed by
SDS-PAGE either (i) directly, to show non-covalent association, since proteins linked to the
RNA do not enter the gel, or (ii) after ribonuclease treatment, to show the total of covalently
linked and non-covalently associated protein. The difference between the two profiles shows the
extent of cross-linkage.
PAGE analysis of the RNA from untreated virus showed some non-covalently associated
protein, but the level was very low (less than 300 d.p.m, per slice). This did not increase when the
RNA was treated with ribonuclease before analysis (results not shown), indicating that direct
analysis is a good measure of the levels of protein non-covalently associated with the RNA.
Figure 2(a) shows the profiles obtained with RNA of bisulphite-treated virus particles. There
was a 20-fold increase in the levels of protein associated with ribonuclease-treated R N A
compared to RNA analysed directly. This is due to protein covalently cross-linked to the R N A
during bisulphite treatment and is equivalent to approx. 3 ~ of the total capsid protein. All four
structural proteins, VP1 to 4, were covalently cross-linked to the virus RNA. Estimates of the
number of copies of each of the structural proteins cross-linked to the R N A per virus particle,
derived from Fig. 2(a), are shown in Table 2.
The bisulphite-mediated cross-linking of proteins to R N A occurs via basic amino acids.
Table 2 shows the number of protein molecules cross-linked to the RNA per virus particle per
lysine group predicted to be on the internal surface of the 12S particle. This gives an
approximate measure of the efficiency of the cross-linking reaction but neglects the contribution
of arginine residues to cross-link formation. The extent of cross-linking of VP1 to 3 broadly
reflects the predicted number of available lysine residues and the results imply that cross-linking
is relatively inefficient. The efficiency of cross-linking of VP4 was at least tenfold higher but
cannot be quantified accurately due to the imprecision regarding the number of lysine residues
available for cross-linking. However, the results indicate that VP4 may be closely associated
with the virus RNA.
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D. J. M O R R E L L A N D O T H E R S
Fig. 2(b) shows the profiles obtained with RNA from bisulphite-treated virus particles that
were subsequently treated with trypsin. The structural proteins VP2 to 4 are present in amounts
similar to those observed in virus particles treated with bisulphite only (Fig. 2a), the difference
in d.p.m, observed being due to the difference in specific activity of the [3SS]methionine used.
Table 2 shows the close agreement between experiments when this difference is taken into
account. Both of the fragments generated by trypsin digestion of intact virus particles, VPI(L)
and VP 1(S) (Burroughs et al., 1971), are cross-linked to the virus RNA in similar amounts (Table
2). It was not possible to judge the efficiency of the cross-linking as the number of lysine groups
available for cross-linking could not be determined precisely.
DISCUSSION
Laporte & Lenoir (1973) and Talbot et al. (1973) concluded, using lactoperoxidase- or
chloramine T-catalysed iodination of tyrosine residues of FMDV, that VP1 was the
predominant surface protein, with a much smaller contribution (5 ~) from VP3. These results
are consistent with data from other picornaviruses where tyrosine modification results in the
predominant labelling of VP1 in mengovirus (Lund et al., 1977) and poliovirus (Beneke et al.,
1977). However, when small, lysine-modifying reagents are used, VP1 to 3 of poliovirus (Wetz &
Habermehl, 1979) and rbinovirus (Lonberg-Holm & Butterworth, 1976) are labelled. There
appears to be a consistent difference in the results obtained from the two types of modification
reaction. In general, it is thought that the results obtained by lysine modification give a better
reflection of surface exposure than those obtained from tyrosine modification (see
Introduction). It would therefore appear that regions of VP1, VP2 and VP3 are present on the
surface of the FMDV particle and, more generally, on the surface of all picornaviruses as the
recent X-ray crystallographic studies by Hogle et al. (1985) and Rossmann et al. (1985) with
poliovirus and rhinovirus respectively show unequivocally that VP1, VP2 and VP3 of these
viruses are partly exposed on the surface of the virus particles.
Assuming that the number of lysine residues modified per unit area is roughly constant, the
results indicate that more regions of VP1 than of VP2 or VP3 are exposed on the virus surface.
This is compatible with the role of VP1 in cell attachment (Cavanagh et al., 1977) and in the
stimulation of the immune response (Laporte et al., 1973).
VP4 does not appear to be modified by 12SI-labelled Bolton & Hunter reagent in the intact
virus particle, suggesting an internal location. This view is supported by the findings of Talbot et
al. (1973) who observed iodination of VP4 after cleavage of VP1 with trypsin, suggesting that
VP4 underlies VP1. The X-ray studies on poliovirus and rhinovirus referred to above (Hogle et
al., 1985; Rossmann et al., 1985) provide conclusive evidence that VP4 is entirely internal in
these viruses also.
The increased level of modification of 12S particles compared to intact virus particles
indicates that there are lysine residues exposed on the internal surface of the 12S particles. These
residues are available for interactions with the virus RNA and may, in addition, account for the
increased degradation of VP1 and the degradation of VP2 observed when 12S particles, as
opposed to virus particles, are digested with trypsin (King & Newman, 1980).
There was substantial degradation of the virus RN A and breakdown of the virus particles at
37 °C, both in the presence or absence of bisulphite. This may be attributed to the activation of
the endogenous endoribonuclease of the virus particles (Denoya et al., 1978b) resulting in
degradation of the virus RNA and consequent virus breakdown. Since RNA degradation within
the virus particle has been correlated with their increased disruption at a lower temperature
(Bachrach, 1968; Denoya et al., 1978a), intact RNA may contribute to capsid stability.
The results show that the structural proteins VP1 to 3 can be cross-linked to the virus RNA
and thus they have regions both on the surface of the virus and regions in contact with the RNA.
VP4 appears to be primarily involved in RNA interactions: it does not react with surface
labelling reagents, indicating an internal location and the efficiency of cross-linkage to the RNA
is significantly higher than that of the other structural proteins; it may therefore be considered
as the 'core' protein of FMDV.
Nucleic acid-binding regions of proteins appear to have common features, namely a high
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RNA-protein interactions in F M D V
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content of basic amino acids, a significant number of acidic amino acids and pairs of adjacent
basic and acidic residues (Ehresmann et al., 1980). In addition, clusters of basic and acidic
amino acids with proline residues appear to be involved in nucleic acid-binding (Garoff et al.,
1980). Analysis of the amino acid sequences of VP 1, VP2 and VP3 (Boothroyd et al., 1982) shows
that they contain a number of such regions which could be implicated in the interactions of these
proteins with the RNA. In contrast, VP4 contains only one lysine residue, has a blocked amino
terminus and does not contain any regions of the type described. However, amino acids 1 to 30 of
VP4 contain a high proportion (37 ~o) of glutamine and asparagine residues which may form
hydrogen bonds with the phosphate groups of the RNA and amino acids 1 to 17 of VP4 contain a
very high proportion (65~o) of helix-breaking amino acids (Chou & Fasman, 1974) which may
be capable of intimate interaction with the RNA due to the high flexibility of the polypeptide
chain.
Wetz & Habermehl (1982) have shown that VP4 of acid-stable poliovirus can be covalently
linked to the RNA during u.v. irradiation, suggesting that VP4 may have a 'core' role in
poliovirus. However, the amino acid sequence of poliovirus VP4 (Nomoto et al., 1982) is
strikingly different from that of FMDV VP4: poliovirus VP4 is basic and it does not have
regions with high proportions of helix-breaking amino acids or amide-containing amino acid
residues. However, it does contain the clusters of charged amino acids predicted to be nucleic
acid-binding sites. This difference may be a factor in the different forces stabilizing the two
viruses below pH 7 and it is possibly relevant that VP4 (6) of the acid-labile encephalomyocarditis virus, like that of FMDV, is acidic and has a high proportion of helix-breaking and amidecontaining amino acids (Palmenberg et al., 1984).
The picornaviruses appear to be stabilized by a combination of protein-protein and R N A protein interactions, but the relative contributions of the interactions appear to vary within the
group. In poliovirus the stabilizing forces seem to involve largely, but not exclusively, proteinprotein interactions between the major subunits of the capsid. This also appears to be the case in
FMDV 'natural empty' particles, RNA-free capsids which contain 60 copies of VP1, VP3 and
VP0 (the precursor of VP2 and VP4) (Rowlands et al., 1975). However, after encapsidation of
the RNA and concurrent cleavage of VP0 to VP2 and VP4, RNA-protein interactions appear to
contribute to the stability of the FMDV capsid and protein-protein interactions are weakened.
Breakdown to 12S particles, rather than 'artificial empty' particles, now occurs on
destabilization. The changes in stabilizing interactions may be due to the transfer of some of the
interactions of VP2 and VP4 from the other structural proteins to the virus RNA.
Two regions of FMDV VP1, amino acids 140 to 160 and 200 to 213, have been shown to
stimulate the production of ~aeutralizing antibodies (Strohmaier et al., 1982; Bittle et al., 1982)
and these regions may be a~sumed to be on the surface of the virus particle. Trypsin cleaves
FMDV VP1 at, or near, the first of these regions to produce the VP1 (L) and VPI (S) fragments.
Therefore, the interactions observed between the VPI(S) fragment and the virus RNA must
occur in a region between the two sites capable of stimulating the production of neutralizing
antibodies. Two explanations can be put forward : either the RNA passes through the capsid to
react with the interacting regions near the surface of the virus particle or the interacting region
of VPI passes through the capsid to interact with the RNA within the virus particle. Neutron
scattering studies (Jacrot et al., 1977) and X-ray diffraction analysis (Harrison et al., 1980; Hogle
et al., 1985; Rossmann et al., 1985) have shown that the protein and nucleic acid regions of virus
capsids tend to be discrete. Furthermore, hydrophilicity analysis by the method of Hopp &
Woods (1981) of the amino acid sequence of FMDV VPI has shown that there are very
hydrophobic regions of the protein between the two antigenic regions, compatible with the
passage of VP1 across the protein core of the capsid. The length of these regions, about seven
amino acids, is sufficient to cross the capsid which has been shown to be approximately 4 nm in
width (Vasquez et aL, 1979). In between the hydrophobic regions is a cluster of charged amino
acids (at amino acids 176 to 179) which may be proposed as an RNA-binding site. These data
suggest that the second explanation proposed above is more likely.
The iodination of tyrosine residues of VP4 after in situ cleavage of VP1 by trypsin (Talbot et
al., 1973) suggests that VP4 underlies VP1 near the site of the interaction between VPI and the
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1656
D. J. M O R R E L L A N D O T H E R S
!iii iiilVPllL~iii!iiii!iii iiiii iiV ::aSii iii!iiiil
.... , ....... i ..........
iii#i , ;tl
iiil iiii;iiiiiiiiiiiiiiili4iiil iiiii iljiiliiliilliiii iiiiiii;i
Fig. 3. Model for the interactions of VPI with the virus RNA, The antigenic regions (hatched), the
hydrophobic core of the capsid (stippled) and a possible position for VP4 are indicated. The antigenic
regions may lie alongside each other.
virus RNA. From this observation and the discussion above, a model can be proposed for the
virus capsid in the proximity of the major antigenic determinants of VP 1 (Fig. 3). The model has
a number of features: during production of 12S particles or 'artificial empty' capsids the
disruption of the proposed interactions of the RNA and/or VP4 with a region of VP1 in close
proximity to the antigenic regions of the protein may cause changes in its antigenic
determinants, resulting in the different antigenicity of these particles (Rowlands et al., 1975;
Cartwright et aL, 1982).
The model also proposes that the two antigenic regions of VP1 are close together, possibly
combining to form a single antigenic region. This proposal is supported by recent evidence that a
peptide corresponding to amino acids 141 to 160 and 200 to 213 joined together is more active
than either of the sequences alone in blocking the reaction of virus particles with a neutralizing
monoclonal antibody and in eliciting neutralizing antibody (Parry et al., 1985; DiMarchi et al.,
1986).
D.J.M. was supported by a Wellcome Foundation Studentship and E.J.C.M. by an Agricultural Research
Council Studentship.
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(Received 12 August 1986)
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