Download Location of neutralizing epitopes on the G protein of bovine

Journal
of General Virology (1998), 79, 2573–2581. Printed in Great Britain
...................................................................................................................................................................................................................................................................................
Location of neutralizing epitopes on the G protein of bovine
ephemeral fever rhabdovirus
Kritaya Kongsuwan,1 Daisy H. Cybinski,1 Juan Cooper2 and Peter J. Walker1
1
2
CSIRO Tropical Agriculture, Long Pocket Laboratories, PMB 3, Indooroopilly, Qld 4068, Australia
Queensland Institute of Medical Research, The Bancroft Centre, PO Royal Brisbane Hospital, Qld 4029, Australia
The surface glycoprotein G is the major neutralizing
and protective antigen of bovine ephemeral fever
rhabdovirus (BEFV). Twelve neutralizing MAbs
against BEFV strain BB7721 were used to select 33
neutralization escape mutants. The mutants had
been classified previously into three major antigenic
sites (G1–G3) based on their cross-neutralization
patterns. The nucleotide sequence of the entire
extracellular domain of the G protein gene was
determined for all mutants. Each contained a single
nucleotide change leading to a single amino acid
substitution. The 16 mutants assigned to the linear
antigenic site G1 mapped to aa 487–503 of the 623
aa G protein. Results of antibody binding to several
overlapping octapeptides covering this region mapped the sequence of two common minimal B cell
epitopes recognized by the five G1 MAbs to
Introduction
Bovine ephemeral fever virus (BEFV) is an arthropod-borne
rhabdovirus which causes a disabling febrile infection of cattle
and water buffalo. The disease is common in tropical and
subtropical regions of Africa, Asia, Australia and the MiddleEast and is of major economic importance to dairy and grazing
industries. BEFV is the type species of the genus Ephemerovirus
which also presently includes Adelaide River virus and
Berrimah virus, and tentatively Kimberley, Malakal and
Puchong viruses (Wunner et al., 1995). All of these viruses have
been isolated from insects or associated with insect vectors.
However, only BEFV is known to cause disease in vertebrates.
Author for correspondence : Peter Walker.
Fax ­61 7 3214 2882. e-mail Peter.Walker!tag.csiro.au
The nucleotide sequence data reported in this paper have been
deposited with the EMBL/GenBank data libraries under accession
numbers AF058321–AF058325.
0001-5640 # 1998 SGM
(488)EEDE(491) and (499)NPHE(502). Site G2
mutations mapped either at aa 169 or 187. The 12
mutants representing antigenic site G3 (G3a and
G3b) mapped to aa 49, 57, 218, 229 and 265,
indicating that this site is likely to combine complex
discontinuous epitopes. Comparison of the deduced
amino acid sequence from five BEFV field isolates
and BB7721 identified aa 218 to be critical for the
site G3a neutralization. Alignment of the glycoproteins of rabies virus, vesicular stomatitis Indiana
virus, vesicular stomatitis New Jersey virus, infectious haematopoietic necrosis virus and BEFV revealed similarities in the location of the neutralizing
epitopes and extensive conservation of cysteine
residues, suggesting that basic elements of the
folded structure of these glycoproteins are preserved.
Like other rhabdoviruses, BEFV has a (®)ssRNA genome
and five structural proteins including a nucleoprotein (N), a
polymerase-associated protein (P), a matrix protein (M), a large
RNA-dependent RNA polymerase (L) and a surface glycoprotein (G) (Walker et al., 1991). The 81 kDa BEFV G protein
is a class I transmembrane glycoprotein which contains typespecific and neutralizing antigenic sites (Cybinski et al., 1990)
and induces protective immunity in cattle (Uren et al., 1994 ;
Hertig et al., 1996). BEFV and other ephemeroviruses also
encode a second non-structural glycoprotein (GNS) and several
other small polypeptides (Walker et al., 1992 ; Wang & Walker,
1993 ; Wang et al., 1994 ; McWilliam et al., 1997). The GNS
glycoprotein is also a class I transmembrane glycoprotein
which is structurally related to the G protein but has not been
detected in virions and does not induce a neutralizing or
protective immune response (Walker et al., 1991, 1992 ; Hertig
et al., 1996).
Four distinct neutralizing antigenic sites (G1, G2, G3 and
G4) have been identified on BEFV G protein by competition
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K. Kongsuwan and others
binding studies using neutralizing monoclonal antibodies
(MAbs) and cross-reactivity analysis of neutralization-resistant
escape mutants (Cybinski et al., 1990, 1992). Although BEFV
appears to exist as a single serotype worldwide, an analysis of
a number of Australian and Chinese isolates has revealed some
antigenic variation at these sites (Cybinski et al., 1992 ; K.
Kongsuwan and associates, unpublished). Determining the
nature of this variation will be important in understanding the
epidemiology of BEFV infection and in the design of broadly
protective vaccines. In this paper, we further characterize BEFV
neutralization epitopes and locate amino acids which are
critical for virus neutralization by an analysis of the structure of
neutralization escape mutants and field strains isolated from
various hosts and geographical locations in Australia. Overlapping octamer peptides encompassing the region identified
as antigenic site G1 have also been examined to define two
distinct B cell epitopes in this major linear site.
Methods
+ Growth of BEFV viruses and selection of escape mutants.
The production of BEFV G protein MAbs and the selection of
neutralization escape mutants have been described elsewhere (Cybinski et
al., 1990, 1992). Briefly, the MAbs were prepared following injection of
BALB}c mice with clarified BEFV (BB7721)-infected mouse brain.
Neutralization-resistant escape mutants were selected from the same
BEFV isolate following adaptation to cell culture. The mutants were
selected from the natural virus population (i.e. without mutagenesis)
following pre-incubation with and plaque assay in medium containing the
selecting MAb. The five field strains of BEFV studied here were obtained
from the virus collection of CSIRO Tropical Agriculture, Indooroopilly,
Australia. The details of the passage level, host origin, place and year of
collection have been described (Cybinski et al., 1992). Viruses were
propagated by infection of confluent monolayers of BHK-21 cells at an
m.o.i. of 1–10 p.f.u. per cell. After 1 h absorption, BME medium
containing 5 % FCS was added and cultures were incubated at 37 °C for
24–48 h before RNA extraction.
+ Viral RNA isolation and PCR amplification. Sequences of the
mutants were determined from PCR-amplified cDNA produced from
cytoplasmic RNA from BEFV-infected cells. Total cytoplasmic RNAs
were prepared from infected cells using RNAzol (Bresatec). cDNA from
the G coding region was synthesized using approximately 1 µg of total
RNA with primer EG2 (5’ TACAACAGCAGATAAAAC 3’) and 5 units
of MMLV H− reverse transcriptase (Promega). The EG2 primer binds to
the sequence at the 3’ end of the G ectodomain (Walker et al., 1992). The
cDNA was amplified by Taq polymerase (Promega) and a hot start
programme with EG1 and EG2 (5’ AAGATTCATTTGGAGAAA 3’)
primers. The EG1 primer sequence was derived from the sequence at the
5’ end of the G gene. After the initial denaturation at 94 °C for 4 min, the
amplification proceeded through a total of 30 cycles consisting of
denaturation at 94 °C (1 min), annealing at 55 °C (1 min), primer
extension at 72 °C (1±5 min) and the final extension of 10 min at 72 °C.
PCR products were purified with the Wizard PCR Clean-Up kit (Promega)
and either subcloned into Srf I-digested pPCRscript SK vector (Stratagene)
or pGEM-T vector (Promega) using standard techniques, or sequenced
directly.
+ DNA sequencing. PCR products or plasmids containing inserts
were sequenced using PRISM Cycle Sequencing (Applied Biosystems)
CFHE
with EG1, EG2 and internal primers covering the G ectodomain.
Reactions were processed by using an Applied Biosystems automated
DNA sequencer and analysed using the Applied Biosystems Seqed
program. For each escape mutant two or more RT–PCR products or
subclones were sequenced to eliminate errors introduced during
amplification. Multiple sequence alignments were done with the
CLUSTAL W software package (Thompson et al., 1994) and adjusted to
account for obvious sequence similarities.
+ Mapping of epitopes with synthetic peptides. Fourteen
octamer-peptides representative of the predicted G1 antigenic site
covering aa 485–505 in the native BEFV G sequence were synthesized
using the multipin peptide synthesis method. The peptides overlapped
the preceding and subsequent peptides by one residue. The MAbs were
assayed as culture supernatants or ascitic fluids diluted at 1 : 20 (MAbs
9C5 and 13C6) or 1 : 50 (MAbs DB5, 13A3 and 17B1). Pin peptides were
precoated with 1 % gelatin and 1 % Tween 20 in 0±01 M PBS pH 7±2 for
1 h at room temperature to block non-specific absorption of antibodies.
This was followed by a wash in 0±05 % Tween 20–PBS. The 2 h
incubation at room temperature in a dilution of antiserum in pre-coat
buffer was followed by four washes (10 min each on a shaker platform)
with 0±05 % Tween 20–PBS. Reaction for 1 h at room temperature with
a 1 : 3000 dilution of goat anti-mouse IgG coupled to horseradish
peroxidase (Dakopatts) and 1 % sheep serum in precoat buffer was again
followed by extensive washing to remove excess conjugate. The presence
of antibody was detected by reaction with a freshly prepared substrate
solution (0±5 mg}ml diammonium 2,2’-azino-bis[3-ethylbenzthiazoline 6sulphonate] or ABTS, 0±01 M PBS and 0±01 % hydrogen peroxide) and the
colour produced was read in a Titertek Multiskan Plus MKII plate reader
at 405 nm. Prior to retesting, bound antibody was removed from pin
peptides by sonicating in 0±1 M PBS–1 % SDS and 0±1 % 2-mercaptoethanol at 60 °C. The sample was then washed several times with
distilled water (60 °C) before immersing in boiling methanol and air
drying.
Results
Characterization of escape mutants
The pattern of cross-neutralization of BEFV escape mutants
by the G protein MAbs was reported previously (Cybinski et
al., 1992). For clarity, these results have been reproduced in Fig.
1 and expanded to indicate the pattern of reactivities of the
individual mutants. The mutants have been assigned to groups
corresponding to four distinct antigenic sites that were defined
by competitive binding assays (Cybinski et al., 1990, 1992).
Mutants selected by site G1 or site G2 MAbs were neutralized
by all MAbs assigned to other sites, but were not neutralized
by the selecting MAb, and often were totally or partially
resistant to neutralization by other MAbs assigned to the same
site. Antigenic site G3 was defined by competitive binding
assays (Cybinski et al., 1990) as a complex site comprising two
elements (G3a and G3b). Site G3a MAbs 8B6 and 3D6
competed for antigen binding and selected mutants that were
resistant to cross-neutralization. Site G3b MAbs 16A6 and
8D3 also competed in antigen binding assays but selected
escape mutants that failed to be cross-neutralized. MAb 8D3
also competed with both 8B6 and 3D6 for antigen binding,
establishing the relationship between sites G3a and G3b.
Antigenic site G4 was defined by a single MAb (5A5) which
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BEFV neutralization epitopes
Fig. 1. Neutralization of BEFV escape mutants by G protein neutralizing MAbs. Individual plaque clones selected with each MAb
were designated as resistance mutants (R) and grouped according to their patterns of reactivity. Mutant susceptible to
neutralization (*) ; mutant resistant to neutralization by selecting MAb (E) ; mutant resistant to neutralization by non-selecting
MAb ( ★ ) ; mutant partially resistant to neutralization (D).
failed to compete with other MAbs for antigen binding and
selected a unique escape mutant (Cybinski et al., 1990, 1992).
Thirty-three neutralization escape mutants representing
sites G1, G2, G3a and G3b were examined by sequence
analysis of the 1527 nt region encoding the entire ectodomain
of the G protein. The region encoded 509 aa between the
signal peptide and the transmembrane domain (aa 18–521) of
the G protein. The nucleotide and deduced amino acid
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K. Kongsuwan and others
Table 1. Nucleotide and amino acid sequence changes found in escape mutants of BEFV
strain BB7721 selected with anti-G neutralizing MAbs
Antigenic
site
G1
Selecting
MAb
DB5
13A3
17B1
9C5
13C6
G2
12A5
15B5
1C6
G3a
8B6
3D6
G3B
16A6
8D3
Escape
mutant
DB5R1
DB5R2
DB5R3
13A3R1
13A3R2
13A3R3
13A3R4
17B1R3
17B1R4
17B1R5
17B1R6
9C5R3
9C5R4
13C6R5
13C6R10
13C6R12
12A5R2
12A5R4
15B5R5
1C6R1
1C6R5
8B6R6
8B6R8
3D6R3
3D6R5
3D6R6
16A6R4
16A6R5
16A6R6
16A6R7
8D3R1
8D3R7
8D3R9
Codon
change
TTT to CTT
CCA to CTA
CCA to CTA
AAA to ACA
AAA to ACA
AAA to AAC
AAT to ACT
TAT to TTT
TAT to TTT
TAT to GAT
AAA to AAC
GAG to GAT
GAG to AAG
GAT to TAT
CCA to CTA
CCA to CAA
ATA to AGA
ATA to ATG
TTA to TCA
ATA to AGA
TTA to TCA
GAT to GAG
AGG to AGT
GAT to GAG
CAA to AAA
AGG to GGG
CA to CGA
CAA to AAA
CAA to AAA
CAA to AAA
GAA to GGA
GAA to GGA
GAA to GGA
sequences of the mutants were compared with those of the
parental BEFV isolate BB7721 (Walker et al., 1992). Point
mutations resulting in a single amino acid substitution were
detected in each mutant (Table 1). For 16 site G1 mutants
selected with MAbs DB5, 13A3, 17B1, 9C5 and 13C6, 13
different base substitutions resulted in 10 changes in a sequence
of 17 aa (487–503) in the stem of the G protein just upstream
of the transmembrane domain. Only three of the amino acid
substitutions were conservative (Y to F in 17B1R3 and
17B1R4 ; E to D in 9C5R3). In accordance with crossneutralization results (Fig. 1), the mutants clustered into two
overlapping subsets. Substitutions in mutants selected with
MAbs DB5 and 13A3 were located between aa 493–503.
MAbs 17B1, 9C5 and 13C6 selected substitutions between aa
487–495. The only two mutants which cross-neutralized
CFHG
Amino
acid
change
F to L
P to L
P to L
K to T
K to T
K to N
N to T
Y to F
Y to F
Y to D
K to C
E to D
E to K
D to Y
P to L
P to Q
I to R
I to M
L to S
I to R
L to S
D to E
R to S
D to E
Q to K
R to G
Q to R
Q to K
Q to K
Q to K
E to G
E to G
E to G
Amino
acid
493
500
500
503
503
503
499
487
487
487
494
489
491
490
495
495
187
187
169
187
169
57
218
57
265
218
49
49
49
49
229
229
229
between the subsets contained substitutions located in the
overlapping sequence (DB5R1 at aa 493 ; 13C6R10 at aa 495).
In the five site G2 mutants selected with MAbs 12A5, 15B5
and 1C6, there were only two substitutions at aa 169 and 187.
The only mutant in this group which was not cross-neutralized
by other site G2 MAbs contained the only conservative amino
acid substitution (I to M in 12A5R4). MAb 1C6 selected
mutations at aa 169 or 187. Twelve mutants selected using site
G3 MAbs mapped to widely spaced locations in the cysteinerich N-terminal half of the G protein. Site G3a MAbs 8B6 and
3D6 selected substitutions at aa 57, 218 and 265. Both MAbs
selected for a conservative substitution at aa 57 (D to E) and a
non-conservative substitution at aa 218 (R to S}G). Site G3b
MAb 16A6 selected substitutions at aa 49 and MAb 8D3
selected substitutions at aa 229. The wide separation of these
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BEFV neutralization epitopes
Fig. 2. Alignment of deduced amino acid sequences of the G proteins of six Australian BEFV isolates. Residues differing from
the sequence of isolate BB7721 are indicated. Amino acid substitutions detected in MAb-selected neutralization escape
mutants of BB7721 are also indicated in bold and underlined.
sites is in accordance with the resistance of site G3b mutants to
cross-neutralization (Fig. 1).
Natural variants
An analysis of BEFV isolates recovered in Australia from
naturally infected cattle and insects between 1956 and 1989
has indicated that 13 of 18 isolates failed to react with MAb
3D6. However, all isolates were neutralized by BEFV polyclonal mouse antiserum and MAbs 17B1 and 12A5 (Cybinski
et al., 1992). Five isolates were selected for sequence analysis of
the 1527 nt region encoding the ectodomain of the G protein.
Two isolates (CS42}Beatrice Hill}1975 ; CS1818}Upper
Barron}1970) and the reference strain (BB7721}Charters
Towers}1968) were sensitive to neutralization by MAb 3D6.
Three
isolates
(CS1180}Peachester}1982,
CS1619}
Peachester}1984 ; CS1647}Peachester}1984) were 3D6-resistant. The deduced amino acid sequence of the isolates is
compared with that of BB7721 in Fig. 2. All changes in the
deduced protein sequence resulted from single nucleotide
substitutions except for a complete codon deletion in isolate
CS1619 at aa N'". No insertions or frameshifts were observed.
Among the six isolates studied, there was 98–99 % nucleotide
sequence identity and 96–98 % amino acid sequence identity.
The only amino acid substitution peculiar to all three 3D6resistant viruses was a conservative change at aa 218 (R to K).
Substitutions at R#") were also found in experimentally derived
3D6-resistant mutants selected with both MAbs 3D6 and 8B6
(Table 1), confirming the essential contribution of this arginine
residue to antigenic site G3a.
No other substitutions in the isolates were detected at
residues identified in escape mutants as critical to antigenic
sites G1, G2 or G3b. However, several substitutions were
detected in residues located at sites adjacent to critical residues.
In particular, substitutions at aa 215 (K to T) and aa 263 (E to
K) were adjacent to critical site G3a aa R#") and Q#'&
respectively, and substitutions at aa 488 (R to K) immediately
preceded the linear antigenic site G1. Despite their proximity
to critical sites, none of these substitutions changed the
neutralization characteristics of the isolates.
Location of B cell epitopes in the G1 antigenic site
Fourteen overlapping, polypropylene pin-bound octapeptides covering antigenic site G1 were assayed for reactivity
with MAbs 13A3, DB5, 9C5, 17B1 and 13C6 by ELISA (Fig.
3). The peptides, offset by one residue, spanned aa 485–505 of
the G protein. Minimal B cell epitopes recognized by the
MAbs were determined from the overlap of ELISA-positive
peptide sequences. In the case of 13A3 and 9C5, the minimal
epitope may include one extra residue not positively identified
with the available synthetic peptides. The MAbs clustered into
two subsets which recognized the N terminus (9C5, 17B1,
13C6) or the C terminus (13A3, DB5) of the antigenic site. The
subsets corresponded to those identified by neutralization
cross-reactions (Fig. 1) and sequence analysis (Table 1) of site
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K. Kongsuwan and others
comprises two distinct epitopes, local conformation determined by the central region affects antibody binding to the
terminal sites.
Alignment of rhabdovirus antigenic sites
Fig. 3. Pepscan analysis of the binding of BEFV neutralizing MAbs to
peptides encompassing the sequence of antigenic site G1
(IRYEEDEKFKPVNLNPHEKSQ) identified by escape mutants. Each
octapeptide (numbered 1 to 14) overlapped the preceding and following
peptides by one residue. (A) MAbs DB5 and 13A3 ; (B) MAbs 17B5, 9C5
and 13C6.
G1 escape mutants. The extent of each B cell epitope
determined from peptide binding, and neutralization and
sequence analysis of escape mutants are summarized in Fig. 4.
The analysis indicated that critical residues required for
generation of escape mutants and virus neutralization are not
confined to the minimal epitope recognized in peptide ELISA.
This indicates that, although the antigenic site is linear and
A CLUSTAL W alignment of the deduced amino acid
sequences of the G proteins of BEFV, VSV Indiana, VSV New
Jersey, IHNV and rabies virus is shown in Fig. 5. As described
previously, the alignment has been adjusted to align putative
signal peptides, transmembrane domains and common glycosylation sites, and illustrates the extensive conservation of
cysteine residues which stabilize the folded structure of the
glycoproteins (Walker et al., 1992). Also indicated for each
virus are amino acids which have been identified using escape
mutants as influencing antibody binding to neutralization sites.
As observed previously for a smaller set of viruses (Huang et
al., 1996), the critical residues are mostly clustered into a small
number of aligned sites. In particular, residues contributing to
the major discontinuous neutralization site of rabies virus (site
II), IHNV (site II) and BEFV (site G3) align in three small
regions that are distantly located in the amino acid chain.
These three regions also encompass identified neutralization
epitopes of VSV New Jersey (epitopes VI, VII and VIII) and
VSV Indiana (epitopes A and A ). The remaining neutral"
#
ization sites of VSV Indiana (epitope V) and VSV New Jersey
(epitope B) appear to correspond to rabies virus antigenic site
III (Vandepol et al., 1986 ; Luo et al., 1988 ; Seif et al., 1985 ;
Prehaud et al., 1988). Linear antigenic site G1 and discontinuous
site G2 of BEFV do not appear to have been identified in other
rhabdoviruses.
Discussion
The BEFV G protein, like those of other rhabdoviruses, is
the major protective antigen and the site for binding of
neutralizing antibody. This study defines amino acid residues
which are important in determining the integrity of epitopes in
the three major neutralization sites (G1, G2 and G3), allowing
localization of the sites in the G protein sequence. The G1
Fig. 4. Analysis of antigenic site G1 B cell
epitopes. The sequence of 21 aa
corresponding to antigenic site G1 of
BEFV strain BB7721 and residues
associated with neutralization or peptide
binding of each MAb are shown. Square
box, residue selected in escape mutant
by neutralizing MAb ; circle, residue
selected in escape mutant crossneutralized by MAb ; dashed circle,
residue selected in escape mutant
partially cross-neutralized by MAb ;
underlined, residue assigned to minimal
epitope from overlap of positive reactions
with synthetic peptides ; dashed
underline, residue tentatively assigned to
minimal epitope from positive reaction
with terminal synthetic peptide.
CFHI
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BEFV neutralization epitopes
Fig. 5. Alignment of the deduced amino acid sequences of the G proteins of BEFV strain BB7721, rabies virus strain CVS
(RABV), VSV Indiana (VSIV), VSV New Jersey (VSNV) and IHNV WRAC strain (IHNV). Signal sequences, transmembrane
domains, BEFV antigenic sites G1, G2 and G3, and rabies antigenic site III are identified with a line above the sequence. The
antigenic sites are not precisely defined but indicative of the general location in the sequence. Putative glycosylation sites are
underlined. Amino acids that have been identified in escape mutants as influencing the structure of a neutralizing epitope are
shown in bold and underlined. Positions of universally conserved amino acids are shown by the single-letter code below the
aligned residue. Highly conserved cysteine residues are also indicated (*).
antigenic site is located in a linear stretch of 17 aa (Y%)(to K&!$)
of the ectodomain adjacent to the transmembrane anchor.
Corresponding sites have not been identified in other rhabdoviruses. However, the BEFV G protein is significantly larger
than those of other known rhabdoviruses and sequence
alignments indicate that this is due to an extension of the G
protein stem between the transmembrane domain and the
cysteine-rich head region (Walker et al., 1992 ; Gaudin et al.,
1992). The BEFV G1 antigenic site lies within this domain at a
locus that does not align with other G protein sequences and
appears to be one of two sites of stem elongation.
The linear conformation of the G1 site was suggested
previously by Western blotting studies with G1-specific MAbs
which reacted with the G protein following denaturation under
reducing conditions. However, pepscan analysis, as well as
neutralization cross-reactions and sequence analysis of escape
mutants, indicate that G1 is more complex, comprising two
overlapping domains. MAbs 17B1, 9C5 and 13C6 recognize
epitopes in the N-terminal region (aa 487–495) which features
the highly acidic sequence EEDE. Epitopes recognized by
MAbs DB5 and 13A3 mapped to aa 493–503 and recognized
a common minimal epitope consisting of the sequence NPHE.
Furthermore, the size of minimal epitopes varied and those
recognized by MAbs DB5, 17B1 and 13C6 were longer than
those commonly found for linear B cell epitopes, suggesting
some tertiary structure in this region of the molecule. The DB5
epitope mapped to 11 residues (Fig. 5) and contained two
proline residues (P%*& and P&!!) which are a common feature in
antigenic β-turns. Epitope 17B1 comprised 8 residues and
13A6 comprised 9 residues, again suggesting conformation.
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K. Kongsuwan and others
Epitope 13A6 overlapped the DB5 epitope and also included
P%*&. Although this sequence does not contain any statistically
determined motifs characteristic of β-turns (Hutchinson &
Thornton, 1994), the G1 site would not be considered linear
and must involve some local conformation. Secondary structure and solvent accessibility predictions indicate that the G1
sequence is likely to be a loop (data not shown). This would
account for the high proportion of mutants with amino acid
substitutions in this region exposed to immune selection. The
observations also support previous studies which suggest that
far fewer residues are necessary for specific recognition than
the total number of residues in the contact region of the
antibody (Geysen et al., 1984 ; Getzoff et al., 1987 ; Jin et al.,
1992 ; Speller et al., 1993).
The G2 and G3 neutralization sites are located in the
cysteine-rich head portion of the G protein ectodomain (Fig. 5).
The non-linear conformation of these sites was suggested
previously by Western blotting studies (Cybinski et al., 1990).
Site G2 encompasses residues L"'* and I")(, intersected by two
cysteine residues (C"(# and C")#) which are conserved in all
animal rhabdoviruses other than sigma virus. Modelling studies
suggest that they are likely to form a disulphide bridge which
would align the components of the G2 site at the base of an Nglycosylated loop (P. J. Walker & K. Kongsuwan, unpublished).
A conservative substitution (I to M) at aa 187 produced
resistance only to the selecting MAb (12A5), but a nonconservative substitution (I to R) selected by the same antibody
produced a mutant that was resistant to neutralization by all
G2 MAbs (Fig. 1 ; Table 1). It has been reported previously for
rabies virus that the nature of amino acid substitutions at the
same site is important in determining their accessibility to
neutralizing antibodies (Prehaud et al., 1988).
Antigenic site G3 comprises amino acid residues which are
distributed at distant loci in the cysteine-rich head domain and
appears to correspond to the major conformational site
identified in the G proteins of other animal rhabdoviruses. Site
G3a MAbs 3D6 and 8B6, which compete for antigen binding,
each selected neutralization escape mutations at residues D&(
and R#"). The critical role of R#") in antibody binding to the
site was supported by the study of BEFV field isolates. MAb
3D6 also selected a mutation at Q#'& which remained
susceptible to neutralization by MAb 8B6. Site G3b MAbs
16A6 and 8D3, which also compete for antigen binding,
selected respective mutations at Q%* and E##* that were not
cross-resistant to neutralization. MAbs representing sites G3a
and G3b also competed for antigen binding and selected
escape mutants in similar regions but there was no crossresistance between the mutants. This suggests that elements of
the G3 site are derived from three distant regions of the
polypeptide chain represented by residues Q%* and D&( ; R#")
and E##* ; and Q#'&. Competitive binding and cross-neutralization data indicate that these regions are closely associated in
the folded protein to form a complex neutralization site
comprising several distinct epitopes. Similar complex non-
CFIA
linear neutralization sites have been recognized in the G
proteins of other rhabdoviruses. Alignment of the G protein
sequences indicates that there is a remarkable correspondence
between the locations of site G3 mutations and those of site II
in rabies (Prehaud et al., 1988), epitope A in VSV Indiana
(Vandepol et al., 1986), epitope VII of VSV New Jersey (Luo et
al., 1988 ; 1990) and sites I and II in IHNV (Huang et al., 1996)
(Fig. 5). As reported previously, there is also a remarkable
degree of conservation of cysteine residues in these proteins
and, despite the low level of amino acid sequence identity,
basic elements of the folded structure of animal rhabdovirus G
proteins appear to be preserved (Walker et al., 1992 ; Huang et
al., 1996).
Potential sites for N-linked glycosylation lie adjacent to all
three BEFV neutralization sites. Indeed, aa Q#'& in site G3
forms part of the N-glycosylation recognition sequence (NQT).
The proximity of glycosylation and antigenic sites has been
observed for other rhabdoviruses and this is not surprising as
each is likely to be located on the external surface of the
protein. However, there is evidence that some potential sites
may not be glycosylated (Benmansour et al., 1991) and that
glycosylation may influence the folded conformation of the G
protein (Grigera et al., 1991). The role of glycosylation in
neutralization site recognition or in protection from protease
digestion has yet to be established.
Neutralizing MAbs have previously been shown to induce
a significant protective response in mice against lethal BEFV
challenge (Cybinski et al., 1990). Purified BEFV G protein and
G protein expressed from recombinant vaccinia virus have also
been shown to protect cattle from experimental challenge and
there was a correlation between protection and the presence of
neutralizing antibody (Uren et al., 1994 ; Hertig et al., 1996).
The G1 neutralization site is located in a linear sequence close
to the transmembrane domain. Although the site may involve
some local conformation, G1 epitopes remain accessible in
reduced G protein and in synthetic peptides used for pepscan
analysis. We have found that G1 epitopes are preserved in 70
Australian BEFV isolates obtained from different geographical
locations during the period 1956–1992 and sequence analysis
of representative isolates indicates that site is highly conserved
(unpublished data). There appears potential to use the G1 site
as the basis of a synthetic peptide vaccine.
We thank Helen Zakrzewski, David Waltisbuhl and Roger Pearson for
assistance with ELISA and pepscan analysis.
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Received 22 April 1998 ; Accepted 15 July 1998
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CFIB