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J Virol, August 1998, p. 6922-6928, Vol. 72, No. 8
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Antigenic Structure of Human Respiratory Syncytial
Virus Fusion Glycoprotein
Juan A.
López,1
Regla
Bustos,1
Claes
Örvell,2
Mabel
Berois,3
Juan
Arbiza,3
Blanca
García-Barreno,1 and
José A.
Melero1,*
Centro Nacional de Biología
Fundamental, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid,
Spain1;
Laboratory of Clinical Virology,
Huddinge Hospital, S-141 86 Huddinge, Sweden2;
and
Sección de Virología, Facultad de
Ciencias, Universidad de la República, Montevideo,
Uruguay3
Received 21 January 1998/Accepted 1 May 1998
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ABSTRACT |
New series of escape mutants of human respiratory syncytial virus
were prepared with monoclonal antibodies specific for the fusion (F)
protein. Sequence changes selected in the escape mutants identified two
new antigenic sites (V and VI) recognized by neutralizing antibodies
and a group-specific site (I) in the F1 chain of the F molecule. The
new epitopes, and previously identified antigenic sites, were
incorporated into a refined prediction of secondary-structure motifs to
generate a detailed antigenic map of the F glycoprotein.
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TEXT |
Human respiratory syncytial virus
(HRSV) is the major cause of severe respiratory infections in very
young children (reviewed in reference 10). Viral
isolates have been classified into two antigenic groups (A and B) on
the basis of differences in reactivity with panels of monoclonal
antibodies (1, 27). Like other paramyxoviruses, HRSV encodes
two major surface glycoproteins (G and F), which are incorporated in
the virus particle. The attachment protein (G) mediates virus binding
to the cell receptor (21), and the fusion protein (F)
promotes fusion of the viral and cell membranes, allowing penetration
of the viral ribonucleoprotein into the cell cytoplasm (43).
The F protein also promotes fusion of the membranes of infected cells
with those of adjacent cells, leading to the formation of syncytia.
Antibodies directed against either G or F neutralize virus infectivity.
Furthermore, experimental animals immunized with vaccinia virus
recombinants expressing either antigen are protected against challenge
with live HRSV (29, 37). However, whereas the immune response against the F protein protects the animals against viruses of
both antigenic groups, the G protein induces a homotypic response protective only against viruses of the same antigenic group. These results reflect the extensive antigenic and genetic divergence in the G
protein between group-A and group-B viruses (16), in contrast to the high degree of conservation of the F glycoprotein (17).
The F glycoprotein is synthesized as an inactive precursor
(F0) (14) that is cotranslationally modified by
the addition of N-linked carbohydrates in the endoplasmic reticulum,
where it assembles into a homooligomer (probably a tetramer)
(9). The F0 precursor is cleaved by trypsin-like
proteases into two chains (F2 N-terminal to F1) before reaching the
cell surface. The two chains remain disulfide linked. The F protein
also contains palmitate (9).
Several laboratories have reported the isolation of monoclonal
antibodies directed against the F protein that neutralize virus infectivity and/or inhibit membrane fusion. Virus-binding competition between antibodies identified three to four antigenic sites in the F
molecule (4, 12). Some epitopes have been mapped by testing
the reactivities of antibodies with synthetic peptides (5,
41) or protein fragments expressed in bacteria (26). This approach, however, is not applicable to epitopes that require the
native conformation of the protein for antibody binding. As an
alternative, we have isolated and sequenced HRSV escape mutants, resistant to certain anti-F antibodies, in order to identify amino acid
residues that are essential for epitope integrity (2, 23).
In this way, two major antigenic sites (II and IV) were located in the
F protein primary structure (2, 23), and some of their
epitopes were further characterized with synthetic peptides (24).
Identification of new antigenic sites recognized by neutralizing
anti-F antibodies.
To expand our view of the antigenic sites in
the F molecule, 12 neutralizing anti-F monoclonal antibodies,
previously isolated against the WV4843 strain (antigenic group B)
(30), were used to select escape mutants. Those antibodies
cross-reacted and neutralized the Long strain (antigenic group A).
Since the Long strain had been used in our previous studies of epitope
mapping, we decided to use this virus for selecting new mutants.
The selection procedure involved passaging the virus in the presence of
antibodies, as was done previously (2, 12). Although escape
mutants could be selected after 4 to 5 passages with antibody 47F
(which was done as a control), as previously reported (2), only four mutants resistant to antibody 7.936 and three mutants resistant to antibody 9.432 were selected after 12 to 20 passages. This
might reflect more stringent structural or functional constraints in
the new epitopes than in previously identified antigenic sites.
The reactivities of the new escape mutants with anti-F specific
monoclonal antibodies are shown in Fig.
1. For comparison,
previously described
mutants and antibodies were included in the
same assay. Antibody 7.957 and those below it on Fig.
1 reacted
with mutants resistant to
antibodies 47F, AK13A2, 7C2, and B4
of antigenic site II, indicating
that their epitopes lie outside
this region of the F molecule.
Antibodies 7.936, 8.858, 8.075,
8.138, and 8.139 did not react with
previously described mutants
resistant to antibodies 19 and 20. These
mutants had a single
amino acid change (R429S) (
2) that
ablated reactivity with
antibodies recognizing epitopes of antigenic
site IV (see Table
1). In contrast, three mutants selected with
antibody 7.936 (R7.936/1,
R7.936/2, and R7.936/6) reacted
normally with antibodies 56F,
19, and 20, and a fourth mutant
(R7.936/4) reacted partially with
these antibodies. These results
indicated that epitopes 7.957,
7.936, 8.858, 8.075, 8.138, and 8.139, which were lost in some
or all of the mutants resistant to antibody
7.936, identified
a new antigenic site (V) that overlapped partially
with site IV.
At least three different classes of epitopes could be
distinguished
in antigenic site V by the reactivity of antibodies with
escape
mutants (Fig.
1): (i) epitope 7.957, (ii) epitopes 7.936 and
8.858,
and (iii) epitopes 8.075, 8.138, and 8.139.
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FIG. 1.
Reactivities of anti-F monoclonal antibodies with escape
mutants of the Long strain. Each monoclonal antibody (MAb) was tested
with an enzyme-linked immunosorbent assay (ELISA) using as the antigen
extracts of HEp-2 cells (12) infected with the different
escape mutants listed at the top. Antibodies included in antigenic
areas I, II, and IV were classified previously by their competition for
antigen binding (12) and reactivities with escape mutants
(2). Mutants resistant to antibodies 47F, AK13A2, 7C2, B4,
19, and 20 have been described previously (2). Antibody
7.957 and those below it on the chart have also been described
elsewhere (30). Viruses resistant to antibodies 7.936 and
9.432 are new to this study. Absorbance results:  , >75% of the
value obtained with Long-infected cell extracts;  , between 25 and
75%;  , <25%.
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The three escape mutants selected with antibody 9.432 (R9.432/1,
R9.432/2, and R9.432/4) lost reactivity only with this antibody
and
with antibody 7.916. Thus, epitopes 7.916 and 9.432 were included
in a
new antigenic site (VI). However, these epitopes were also
lost in
mutant R.7936/4, indicating partial overlapping of the
newly identified
antigenic sites V and VI of the F molecule. Finally,
four antibodies
(7.901, 9.137, 9.226, and 9.596) reacted with
all the escape mutants in
Fig.
1 and thus recognized epitopes
of the F molecule as yet
unidentified.
To locate the amino acid changes that were selected in the new escape
mutants, poly(A) RNA extracted from cells infected with
the different
mutants was used to amplify the F gene by reverse
transcription-PCR
(RT-PCR). The amplified DNAs were cloned into
pGEM-4 and sequenced by
the dideoxy method with F-specific primers.
Regions including the
changes described below were confirmed by
direct sequencing of the PCR
product.
The sequence changes detected in the different mutants compared with
the parental Long virus are listed in Table
1. Mutants
R7.936/1 and R7.932/2 each had
a single-nucleotide change at position
1353 (T to C) that led to the
amino acid substitution V447A. The
other two mutants (R7.936/4 and
R7.936/6) resistant to antibody
7.936 had single-nucleotide
substitutions at positions 1311 (A
to C) and 1308 (T to C) that were
translated into amino acid changes
K433T and I432T, respectively. All
these changes placed residues
essential for epitopes of antigenic site
V in a segment of 16
amino acids (residues 432 to 447) of the F1 chain.
This segment
is near residue 429, which was changed (R429S) in mutants
resistant
to antibodies 19 and 20 of site IV, and overlaps partially
with
a peptide (amino acids 422 to 438) that reacted to high titers
of
antibody 19 (
2). These results are in agreement with the
partial overlapping of profiles of reactivity with antibodies
of
antigenic sites IV and V observed in Fig.
1.
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TABLE 1.
Sequence changes in the F proteins of escape mutants
resistant to antibodies from antigenic sites IV, V,
and VIa
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It is interesting that the drastic change (K433T) selected in mutant
R7.936/4 ablated reactivity with all the antibodies from
sites V and VI
and partially inhibited reactivity with antibodies
from site IV (Fig.
1). In contrast, a more conservative change
at the adjacent position
432 (I432T), selected in mutant R7.936/6,
influenced epitopes of site V
only. Finally, the conservative
change V447A (selected in mutants
R7.936/1 and R7.936/2) eliminated
only two of the epitopes included in
antigenic site V.
Sequencing of the cDNA clones derived from escape mutants R9.432/1,
R9.432/2, and R9.432/4 revealed the same nucleotide substitution
at
position 1320 (C to T), which resulted in the amino acid change
S436F
(Table
1). Mutant R9.432/2, however, had a second nucleotide
substitution at position 323 (A to G), which resulted in the amino
acid
change N104D in the F2 chain. Although this change was also
present in
the RT-PCR product used for cDNA cloning, chimeric
F proteins
expressing only the N104D change reacted normally with
antibody 9.432 (data not shown). Thus, it is likely that the substitution
N104D
represents an adventitious mutation incorporated during
the selection
process of mutant R9.432/2. It is worth emphasizing
that residue 436 is
included in the F gene segment (encoding residues
432 to 447) where
mutations in mutants selected with antibodies
from site V were located.
Nonetheless, the three escape mutants
selected with antibody 9.432 reacted normally with antibodies
from site V (Fig.
1). In contrast,
antibodies 7.916 and 9.432,
included in site VI, did not react with
mutant R7.936/4, which
had a drastic change (K433T) only 3 residues
away from amino acid
436.
Location of a group-specific antigenic site in the F protein
primary structure.
In contrast to antibodies from other antigenic
sites, some of the antibodies included previously in antigenic area I
reacted exclusively with viruses from antigenic group A
(12). Therefore, it was of interest to identify the residues
of the F protein required for the integrity of these group-specific
epitopes. Although antibodies from area I reduced virus infectivity by
less than 1 log unit, two escape mutants could be obtained after the
Long strain was passaged five to six times in the presence of antibody
55F (R55F/9 and R55F/11). The reactivities of these mutants with
antibodies representative of previously identified antigenic sites are
shown in Fig. 2.
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FIG. 2.
Characterization of escape mutants selected with
antibody 55F. Extracts of HEp-2 cells infected with either Long,
CH18537, R55F/9, or R55F/11 were used in an ELISA. The results are
presented as in Fig. 1. The amino acid at position 389 is indicated at
the bottom for each virus.
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Mutant R55F/9 lost reactivity with all the antibodies from site I but
retained reactivity with antibodies from sites II and
IV. Mutant
R55F/11 showed a similar profile of reactivity, except
that it reacted
with antibody 44F (Fig.
2). Thus, at least two
different epitopes could
be distinguished in antigenic area I.
The reactivity of virus CH18537
(a representative strain of antigenic
group B) with antibodies
resembled that of mutant R55F/9 (Fig.
2). cDNA copies of the F gene
from viruses R55F/9 and R55F/11
were cloned in pGEM-4 and sequenced.
Single-nucleotide substitutions
were found at position 1179 in both
viruses, C to T in mutant
R55F/9 and C to A in mutant R55F/11. Those
substitutions were
translated into amino acid changes P389L and P389H,
respectively
(Fig.
2).
Amino acid 389 (P) is unchanged in the F sequence available for a
second HRSV strain (A2) from antigenic group A, but it is
changed in
other pneumoviruses. Thus, it is S in an HRSV strain
from antigenic
group B (
17), it is T or A in bovine respiratory
syncytial
virus (
31), it is T in pneumonia virus of mice
(
8),
and it is S in turkey rhinotracheitis virus
(
45). It is worth
noting that amino acid changes in residue
389 can have different
effects on antibody binding. Thus, the
replacement P389H did not
eliminate reactivity with antibody 44F,
whereas the replacement
P389S (in strain CH18537) or P389L (in the
mutant R55F/9) ablated
that epitope (Fig.
2). Residue 389 is located in
the middle of
the cysteine-rich region of the F1 chain (see Fig.
4).
Escape mutants doubly resistant to antibodies from antigenic sites
II and IV.
Until now only mutants resistant to individual anti-F
antibodies have been reported in the literature (2, 4, 12). These viruses frequently displayed miniplaque phenotypes, indicative of
functional alterations in the F molecule (4). This may be related to the unusual amino acid changes selected in the escape mutants at positions which are unchanged in HRSV isolates. Thus, it was
of interest to know whether or not viruses doubly resistant to
antibodies recognizing nonoverlapping antigenic sites could be
isolated, and the effect of the accumulated mutations on F antigenicity.
The mutant C4848f, which is resistant to antibody 19 (
2)
(antigenic site IV), was chosen for selecting viruses also resistant
to
antibody 47F (antigenic site II). Four mutants (RRA3, RRA6,
RRA9, and
RRA10) were obtained after four passages in the presence
of antibody
47F. The double mutants were impaired in cell membrane
fusion, as
denoted by a very small syncytium phenotype (data not
shown). Those
viruses lack reactivity with antibodies from sites
IV and V, as does
the parental C4848f virus, and in addition they
did not react with most
antibodies from antigenic site II (Fig.
3). Mutants RRA3 and RRA9 could be
distinguished from RRA6 and
RRA10 by their reactivity with antibodies
7C2 and B4. The reactivity
profiles of the double mutants were those
expected from the reactivities
of mutants resistant to individual
antibodies, and no major antigenic
reorganization of the F protein was
observed.
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FIG. 3.
Characterization of double escape mutants. Extracts of
HEp-2 cells infected with the viruses listed at the top were used in an
ELISA. The results are presented as in Fig. 1. Amino acids at positions
262, 275, and 429 are indicated at the bottom for each virus.
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The F genes of viruses doubly resistant to antibodies 19 and 47F were
amplified by RT-PCR and sequenced. The four mutants
had the original
substitution at nucleotide 1298 (C to A) that
resulted in the amino
acid change R429S and distinguished the
parental C4848f virus from the
Long strain (Table
1). Mutants
RRA6 and RRA10 had, in addition, a
transversion at nucleotide
797 (A to T) that resulted in the amino acid
change N262Y (Fig.
3). Single mutants with the change N262Y had been
selected previously
with antibody 47F or AK13A2, and they showed a
reactivity profile
with antibodies from site II identical to that of
RRA6 and RRA10
(
2). The other two double mutants shown in
Fig.
3 (RRA3 and
RRA9) had a transition at position 837 (C to T) that
resulted
in the amino acid change S275F. This change had not been
observed
previously among escape mutants of the Long strain, but it had
been found in mutants of the A2 strain (
10). It is
interesting
that the change S275F did not affect epitope B4, which is
eliminated
by an amino acid change at residue 262, 268, or 272 (
2). B4
is the only epitope, among those included in site
II, which is
efficiently reproduced in a synthetic peptide spanning
residues
255 to 275 (
2,
24).
In conclusion, the changes selected in the double mutants were also
found among those selected in single mutants, indicating
that amino
acid changes in individual antigenic sites of the F
protein did not
influence the changes selected in a separate site.
Nevertheless,
viruses with changes in sites II and IV had a clear
impairment for
syncytium formation (data not shown). This may
reflect structural
and/or functional constraints of the F protein
that prevent
accumulation of sequence changes and may explain
the high degree of
sequence conservation among the F protein genes
of HRSV strains
(
17).
Structural considerations about the antigenic organization of the F
molecule.
Figure 4A is a diagram of
the primary structure of the F protein of the Long strain, showing the
locations of amino acid changes selected in escape mutants described in
this work and in previous publications (2, 23). To gain
further insight into the structural requirements of the F protein
epitopes, a model of secondary-structure motifs was built by using
published sequences of different pneumoviruses (Fig. 4). This
prediction is in good agreement with, and refines the structural
elements proposed by Chambers et al. (8) on the basis of the
comparison of 5 sequences of paramyxovirus fusion proteins. The F
protein spikes of HRSV particles represent, at least, F1-F2 homodimers
that are observed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis without heating and reduction (3, 9, 25).
However, homotetramers have been observed after chemical cross-linking
(9); presumably, these represent less-stable association
between two dimers. Intermonomer associations appear to involve the F1
chain, which would form the tetramer core. By analogy with Sendai virus
(15), a single disulfide bridge between cysteines 69 and 212 was proposed to link the F1 and F2 chains.
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FIG. 4.
Prediction of secondary-structure motifs of the F
protein. (A) Linear diagram of the F protein polypeptide of the Long
strain (22), showing cysteine residues ( ), potential
N-glycosylation sites ( ), site of
proteolytic processing ( ), amino acid changes in escape mutants
reported here and in previous publications ( ) and hydrophobic
regions
().
S-S, disulfide bridge. (B) Predicted secondary-structure elements of
the F protein:  -helices (cylinders),  -sheets (rectangles), loops
(turns). Other symbols are as explained for panel A. The segment
between residues 255 and 275 (F255-275), predicted to fold in a
helix-loop-helix structure, is boxed. F1h and F2h, amphipathic
-helices in the F1 and F2 chains, respectively. The diagram is not
drawn to scale. The predictions were made with published sequences of
pneumovirus F proteins and by using the following methods available on
Worldwide Web servers: the PHD method (33, 34)
(http://www.embl-heidelberg.de/predictprotein/predictprotei.html), the
DSC method (18)
(http://bonsai.lif.icnt.uk/bmm/dsc_form_align.html), and the PREDATOR
method (11)
(http://embl-heidelberg.de/predator/predator_info.html), which use
multiple aligned protein sequences as input to estimate the probability
of secondary-structure motifs and solvent accessibility; and the SOPMA
method (13) (http://www.ibcp.fr/serv_pred.html), the
nnPREDICT method (19)
(http://www.cmpharm.ucsf.edu/~nomi/nnpredict.html), and the PSA
method (38) (http://bmerc-www-bu.edu/psa/), which use single
protein sequences as input. The final secondary-structure model was a
consensus of the information obtained by the different methods. Details
of the prediction studies are available from the authors upon
request.
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The F2 chain of 139 amino acids is the most divergent segment of the F
molecule both between and within the antigenic groups
of HRSV (
17,
22). It is likely to be located outside the internal
core, as
indicated by high scores of solvent accessibility throughout
its
sequence (data not shown). F2 has the majority of potential
sites for N
glycosylation. Estimates of carbohydrate content suggest
that most, if
not all, of these sites are actually used. A region
of turns and
-sheets is predicted at the N-terminal end before
the amphipathic
-helix, which precedes a stretch of 28 hydrophilic
amino acids that
is present in HRSV but absent in other pneumoviruses
(Fig.
4B)
(
8). The C-terminal end of F2 has the sequence KKRKRR,
which
is presumably the site of proteolytic processing of the
F
0
precursor.
The N-terminal end of the F1 chain has 19 contiguous hydrophobic amino
acids that form the fusion peptide (Fig.
4). This is
probably buried in
the F-protein tetramer. Not only the hydrophobic
character of this
peptide but also certain amino acids are conserved
among pneumoviruses,
suggesting that they are required for fusion
activity. The N-terminal
one-third of F1 is rich in
-helices,
including a long heptad repeat,
which might be involved in coiled-coil
interactions, and an amphipathic
-helix (Fig.
4B). The relevance
of both the fusion peptide and the
heptad repeat region for fusion
activity has been demonstrated in
Newcastle disease virus (NDV)
by site-directed mutagenesis
(
35). The segment between residues
255 and 275 is predicted
to fold in a helix-loop-helix structure,
which is precisely the
conformation adopted by a 21-residue peptide
in 30% trifluoroethanol,
as determined by two-dimensional nuclear
magnetic resonance
(
39).
The cluster of cysteines in the middle of the F1 chain is larger in
pneumoviruses than in other paramyxoviruses, which lack
the last 4 cysteines. Nevertheless, the overall structure of this
region is likely
to be similar in all those viruses. Multiple
intrachain cysteine loops
have been found in the Sendai virus
F protein (
15), and
several
-sheets are predicted in HRSV (Fig.
4B). Thus, a set of
alternating loops and
-sheets should confer
a globular conformation
on this region of the F molecule, which
might have an external location
in the tetramer, as indicated
by high hydrophilicity values (data not
shown). Following the
cysteine cluster there is a second heptad repeat,
in close proximity
to the membrane, which may participate in intra- or
intermonomer
coiled-coils.
The N-terminal segment of the F1 chain, up to the cysteine-rich region,
has structural similarities with the HA2 subunit of
influenza virus
hemagglutinin (HA), another viral glycoprotein
with membrane fusion
activity and a known three-dimensional structure
(
44). This
resemblance is reflected in a high
-helix content,
the presence of
fusion peptides, and resistance to trypsin digestion
(
23,
36). The relevance of this region for F protein assembly
is
inferred from the phenotype of mutants with mutations at positions
236 to 237 that are retained in the endoplasmic reticulum as a
result of
protein misfolding (
25). The epitopes of antigenic
site II
are located in this protease-resistant region of F1. Previous
studies
indicated that a peptide spanning residues 215 to 275
could reproduce
the bizarre local conformation of that region
of the F molecule. Most
antibodies included in site II (
24)
recognized that peptide.
The other region that included epitopes recognized by neutralizing
antibodies (sites IV, V, and VI) is located near the C-terminal
end of
the cysteine-rich region and not far from the heptad repeat
adjacent to
the membrane. Thus, neutralizing antibodies bind to
both sides of the
cysteine cluster. It is likely that those antibodies
would inhibit
conformational changes of the F1 chain associated
with the fusion
process (
20). These changes would be analogous
to the
structural reorganization of the HA2 subunit of influenza
virus HA
during fusion of the virus and cell membranes (
6,
7). In
this case, the HA2 chain folds back at an acid pH (
6),
bringing in close proximity the fusion peptide, inserted in the
cell
membrane, and the transmembrane anchor, inserted in the viral
membrane.
This notion of direct apposition of the two membranes
before fusion
would require that the cysteine-rich region of the
F1 chain have
flexible behavior. This is in agreement with the
marginal effect that
antibodies binding to site I (located in
the middle of the cysteine
cluster) have in virus neutralization.
Antigenic sites equivalent to
those of HRSV F protein have been
described in Sendai virus
(
32), NDV (
28,
40), and parainfluenza
virus type
3 (
42). Thus, virus neutralization afforded by anti-F
antibodies may take place via a general mechanism in paramyxoviruses.
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ACKNOWLEDGMENTS |
J.A.L. and R.B. contributed equally to this work.
We thank P. Coppe for antibody AK13A2, M. Trudel for antibody 7C2, and
G. Taylor for antibodies B5, B4, 11, 13, 19, and 20.
This work was partially funded by grant PM96-0025 from the
Dirección General de Enseñanza Superior (to J.A.M.) and by
a grant from the Comisión Sectorial de Investigación
Científica (to J.A.). A grant from the Agencia Española
de Colaboración Internacional funded travel expenses between
Madrid and Montevideo. J.A.L. was the recipient of a postdoctoral
fellowship from the Instituto de Salud Carlos III.
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FOOTNOTES |
*
Corresponding author. Mailing address: Centro Nacional
de Biología Fundamental, Instituto de Salud Carlos III,
Majadahonda, 28220 Madrid, Spain. Phone: 34-91-509 7941. Fax:
34-91-5097919. E-mail: jmelero{at}isciii.es.
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J Virol, August 1998, p. 6922-6928, Vol. 72, No. 8
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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