JOURNAL OF VIROLOGY, May 1997, p. 4055–4061 0022-538X/97/$04.0010 Copyright q 1997, American Society for Microbiology
Vol. 71, No. 5
Antigenic Structure of the Central Conserved Region of Protein G of Bovine Respiratory Syncytial Virus JOHANNES P. M. LANGEDIJK,1* ROB H. MELOEN,2 GERALDINE TAYLOR,3 JULIE M. FURZE,3 AND JAN T. VAN OIRSCHOT1 Departments of Mammalian Virology1 and Molecular Recognition,2 The Institute for Animal Science and Health (ID-DLO), 8200 AB Lelystad, The Netherlands, and The Institute for Animal Health, Compton, Newbury RG20 7NN, United Kingdom3 Received 20 August 1996/Accepted 17 January 1997
Epitopes were resolved at the amino acid level for nine monoclonal antibodies (MAbs) directed against the central conserved region of protein G of bovine respiratory syncytial virus (BRSV-G). Peptide binding studies showed which amino acids in the epitope contributed to antibody binding. The details of the epitopes were compared with the high-resolution structure of a synthetic peptide corresponding to the central conserved region of BRSV-G, and this indicated which face of the central conserved region is the antigenic structure. The major linear epitope of the central conserved region of BRSV-G is located at the tip of the loop, overlapping a relatively flat surface formed by a double disulfide-bonded cystine noose. At least one, but possibly two sulfur atoms of a disulfide bridge that line the conserved pocket at the center of the flat surface, is a major contributor to antibody binding. Some of the residue positions in the epitope have mutated during the evolution of RSV-G, which suggests that the virus escaped antibody recognition with these mutations. Mutations that occur at positions 177 and 180 may have only a local effect on the antigenic surface, without influencing the structure of the backbone, whereas mutations at positions 183 and 184 will probably have major structural consequences. The study explains the antigenic, structural, and functional importance of each residue in the cystine noose which provides information for peptide vaccine design. Additionally, analysis of the epitopes demonstrated that two point mutations at positions 180 and 205 define the preliminary classification of BRSV subgroups. lar (10). All of these features apply to the top surface of the cystine noose corresponding to the C-terminal half of the central conserved region of BRSV-G. The flat surface and the conserved pocket suggest that this site may bind a receptor with a complementary flat counterstructure with a small protrusion which docks in the pocket. The identity of the receptor for RSV-G is not yet known. However, we obtained much detailed information on antibodies that bind to the cystine noose. We describe the results of a study of an important functional epitope with a known high-resolution structure. The epitopes of several different MAbs, of which most bind to this antigenic site, were resolved at the amino acid level by peptide binding studies. The results explain the antigenic, structural, and functional importance of each residue in the cystine noose.
The attachment protein G of respiratory syncytial virus (RSV-G) is an envelope glycoprotein which is structurally very different from its counterparts (hemagglutinin-neuraminidase and hemagglutinin) in other paramyxoviruses. It has been proposed that the ectodomain of RSV-G has a modular architecture with a central conserved folded region bounded by two highly glycosylated, polymeric mucin-like regions (9, 12, 14). The C-terminal part of a synthetic peptide corresponding to the central conserved region (residues 171 to 186) forms a very rigid structure with unique structural features (2). The C-terminal half of the central conserved region is composed of two helices, connected by a type I9 turn and linked by two disulfide bridges. This double-linked cystine noose folds into a relatively flat disc with a hydrophobic pocket lined by highly conserved residues (2) and is probably located at the distal tip of the protein. Previous studies showed that this structured region corresponds exactly to the immunodominant site of bovine RSV-G (BRSV-G) (12, 13) and human RSV-G (HRSV-G) subtype A (1, 15). A synthetic peptide spanning this region induced protection against HRSV infection in mice, and an antibody directed against this peptide conferred passive protection against challenge (21). The BRSV-G-specific monoclonal antibody MAb 20, directed to the C-terminal side of the central conserved region (12), showed low neutralizing activity in a plaque reduction assay and gave protection after passive immunization of mice (13a). On average, interfaces in protein-protein heterocomplexes tend to be relatively rigid, planar, and circular and the residues contained in the interface are preferably hydrophobic and po-
MATERIALS AND METHODS Pepscan analysis. Peptides were previously synthesized on polyethylene rods (12). The peptides were tested for their reactivities with MAbs in an enzymelinked immunosorbent assay according to established procedures (7, 8). The following peptides were synthesized: 186 overlapping 12-residue peptides of the ectodomain of BRSV-G strain 391-2 (14) and a set of 240 analogs for the 12-residue peptide STCEGNLACLSL in which each amino acid was consecutively replaced by the other 19 amino acids. For this study, the following additional sets of overlapping peptides were synthesized: overlapping 8- to 16-residue peptides from the region between residues 169 to 187 and 18 overlapping 12residue peptides spanning residues 182 to 210 of BRSV-G strain Lelystad (accession no. U33539). Most pepscan data were obtained in one single measurement. The reactivities of the peptides with one MAb on different days were very reproducible. Exactly similar reactivity patterns were obtained. MAbs. MAbs 48, 49, 50, 53, 57, 61, 62, and 66 were raised in mice vaccinated with vaccinia virus expressing the G protein of BRSV strain 391-2 (4, 14). All MAbs reacted with a 32-residue peptide corresponding to residues 158 to 189 of BRSV-G strain 391-2 (14). MAb 70 was raised in mice immunized with serumfree supernatant from cells infected with BRSV strain 127 (4).
* Corresponding author. Mailing address: Institute for Animal Science and Health (ID-DLO), P.O. Box 65, 8200 AB Lelystad, The Netherlands. Phone: 31-320-238216. Fax: 31-320-238050. E-mail:
[email protected]. 4055
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RESULTS Mapping of MAbs with overlapping 12-residue peptides. MAbs 48, 49, 50, 53, 57, 61, 62, 66, and 70 were tested for their reactivities with all 186 overlapping 12-residue peptides of the ectodomain of BRSV-G (Fig. 1a). MAbs 49, 50, and 66 showed similar reactivities; therefore, only the reactivity of the representative MAb 66 is shown. All MAbs, except for MAb 70, reacted with peptides corresponding to the central conserved region of BRSV-G. The results (Fig. 1a) showed that most MAbs bound the same successive overlapping peptides. For MAbs 48, 49, 50, 53, 62, and 66, the highest reactivity was directed against the peptide with the amino acid sequence STCEGNLACLSL. This region corresponds to the C-terminal half of the central conserved region for which the atomic structure has been resolved (2). MAb 70 did not react with any peptide of BRSV-G strain 391-2. This is in accordance with previous BRSV subgroup classifications (4, 16, 20). These studies demonstrated that MAb 70 was directed against another subgroup (e.g., BRSV-G strains 127 and Lelystad). Therefore, an extra set of peptides was synthesized which spanned the variable part of BRSV-G strain Lelystad, C-terminal of the central conserved region, up to the highly basic region (residues 182 to 210). MAb 70 reacted with peptides 198 and 199 which share the amino acid sequence 199TITLKKAPKPK209 (Fig. 1b). Mapping of MAbs with peptides in which each position in the epitope is systematically replaced. For MAbs that reacted with peptide 174STCEGNLACLSL185, the contribution of each amino acid side chain of the epitope to antibody binding was determined by testing the reactivity of the MAbs to peptide analogs in which each amino acid position of the epitope was replaced by all 19 naturally occurring amino acids (Fig. 2). If replacement of an amino acid by most other amino acids results in loss of antibody binding, that specific amino acid is important for antibody binding. Figure 2 shows that some key residues (C176, C182, and L180, in order of importance) are essential for binding many different MAbs. Other residues are also quite important for antibody binding of most MAbs (E177, N179, A181, L183, S184, G178, and L185, in order of importance). For each MAb that bound peptide STCEGNLACLSL (Fig. 1), an arbitrary ranking was made which indicates the contribution of the residue to antibody binding. The binding contribution of each residue is color coded in Fig. 3. Identification of the length of the reactive peptides. All MAbs that reacted with peptides corresponding to the central conserved region were tested for reactivity to all overlapping peptides with multiple lengths. With this approach the minimal reactive peptide and the additional contributions of residues N or C terminal to this epitope core could be obtained. The reactivities with the set of peptides with increasing length were almost identical for many different MAbs. Therefore, only some representative examples are shown in Fig. 4. The shortest reactive peptide for MAbs 48, 49, 50, 61, and 66 was CEGN LACLS; that for MAb 62 (and MAb 20) (12) was CEGN LACL; that for MAb 53 was EGNLACLSL; and that for MAb 57 was PYVPCSTC (Fig. 4). A major contribution to this core of the epitope was mostly the C-terminal addition of L185, and no additional binding contributions were found outside this decapeptide. Many additions of residues N and C terminal of the epitope disrupt binding with the MAbs. This phenomenon is illustrated for the reactivities of the MAbs for the 16-residue peptides. Although all 16-residue peptides contain the residues important for antibody binding, peptide VPCSTCEGNLACLSLC is the highest, or in some cases, the only reactive 16-residue peptide (Fig. 4).
FIG. 1. (a) Reactivities of overlapping 12-residue peptides of BRSV-G strain 391-2 with MAbs 48 (diluted 1:10,000) and 62, 53, 66, 61, and 57 (all diluted 1:75). Only the reactivities of peptides 155 to 187 are shown, because the other peptides did not show any reactivity with the MAbs. (b) Reactivities of 18 overlapping 12-residue peptides of BRSV-G strain Lelystad with MAb 70 (diluted 1:175). The peptide sequences represent the amino acids common to each of the peptides that is recognized by the MAbs, as illustrated for MAb 48. The N-terminal residue of each 12-residue peptide runs along the x axis. Absorbances at 405 nm were plotted vertically.
DISCUSSION In this study we showed that all tested MAbs except for MAb 70 mapped to the same region at the C-terminal half of the central conserved region of BRSV-G. Most of these (MAbs 48,
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FIG. 2. Reactivities of MAbs 48 (diluted 1:60,000), 66 (diluted 1:2,000), 53 (diluted 1:1,000), and 62 (diluted 1:2,000) with peptide analogs in which each position of the reactive peptide 174STCEGNLACLSL185 is replaced by all other 19 residues. The binding activity of each antibody with a peptide is shown as a vertical line proportional to the absorbance at 405 nm. Each group of 20 lines corresponds to the complete set of substitutions at a particular position of the original peptide (STCEGNLACLSL). The sets are arranged in alphabetical order according to the one-letter code of residues (ACDEFGHIKLMNPQRSTVWY). The reactivity with the original peptide is shown as a thick line. The original residue is shown at the bottom for each set. The reactivities of MAbs 49 and 50 are not shown because the pattern of reactivity was very similar to that of MAb 66.
These substitutions cluster C or N terminal of Cys176 and Cys182. Therefore, it is likely that MAbs 48, 49, 50, 62, and 66 all interact directly with a sulfur atom(s) of the disulfide bridge between Cys176 and Cys182. In a previous study we showed that a covalent bond between both Cys residues was not essential for antibody binding but that substitution of Cys or chemical modification of Cys impaired binding (12). According to the results shown in Fig. 2, Cys176 is the most important residue in the epitopes. This critical residue has a relatively low surface accessibility, and the sulfur atom of Cys176 makes up part of the wall of the conserved pocket (Fig. 3). This suggests that the sulfur atoms in the cystine bridge between Cys176 and Cys182 at the wall and the rim of the pocket, respectively, are part of the energetic core of the epitope. Another essential residue in the epitope, Leu180, lies at the tip of the loop, at the side of the disc. Because the antibody paratopes should span the distance
49, 50, 53, 62, and 66) showed the strongest reactivity to the same peptide (STCEGNLACLSL). This reactive peptide is diagrammed in Fig. 5. The epitope is located at the rim of the disc, half at the tip of the loop, half at the flat top surface. In Fig. 3, the residues important for antibody binding according to Fig. 2 and 4 are color coded. For many residues, the possible substitutions are conservative and therefore easy to comprehend. For instance, Leu180 in the epitope of MAb 48 can be replaced by Ile or Val which are hydrophobic amino acids. However, some nonconservative substitutions (Fig. 2) are unexpected; therefore, these are discussed below. In agreement with results for MAb 20 in a previous study (12), unusual substitutions for several amino acid positions are the high number of favorable substitutions to Met at positions Glu177, Gly178, Ala181 (e.g., MAbs 48 and 66), and Leu183.
TABLE 1. Alignment of residues 171 to 208 of RSV-G, including linear epitopesa Strain
BRSV BRSV BRSV BRSV
391-2 (A) LELY (AB) 271SW BOV-X (B)
HRSV-B HRSV-A
Sequence
V – – –
P – – –
£ C – – –
£ helix S – – –
T – – –
C – – –
turn ¢ E – K –
G – – –
helix
¢ £
N – – –
L – – P E
A – – –
C – – –
L S S S
¢
S – – P
L – – –
C – – –
H Q Q Q
I – – –
E G G G
T L L L
E – – –
R – – –
A – – –
P – – –
S – – –
R – – –
A – – –
P – – –
T – – –
I – – –
T L K K T P K P K – – – –E A – – – – – – – – –E A – – – –
V P C S I C G N N Q L C K S I C K T I P S N K P K K K P T I K P T N K P T T K – – – – – – S – – P T – W A – – – R – – N K – – G – – T – T – – – K – – F –
a The sequences of strains were taken from the following sources (reference): strain 391-2 (14), strains Lelystad and BOV-X (16), strain 271SW (3), HRSV-B (9), and HRSV-A (22). –, identical residue in subgroups of RSV-G; boxes, conserved residues; underlines, linear epitopes; circles, mutations that determine antigenic subgrouping.
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FIG. 3. Structure of the cystine noose of BRSV-G (residues 171 to 189) in which the side chains are colored based on contribution to antibody binding. MAbs 49, 50, and 66 are placed in one group because of the nearly identical pattern of reactivity. The binding contribution ranking is based on the replaceability of the residues in the peptide binding study (Fig. 2). Residues which could not be replaced by any of the other 19 residues are considered very important for binding, followed by residues that can only be replaced by residues with similar physiochemical properties, residues that can be replaced by unrelated residues, and finally residues that can be replaced by many other residues. The residue side chains are arbitrarily ranked in order of decreasing contribution to antibody binding: white, essential for binding; yellow, pink, orange, and red, less important for binding; blue, not important for binding. Backbone atoms of residues present in the core of the epitope, according to the results shown in Fig. 4, are colored red. (A) MAbs 49, 50, and 66. (B) MAb 48. (C) MAb 53. (D) MAb 62. In panel C, the side chain of Cys176 is colored turquoise because it is important for binding to MAb 53 according to the results shown in Fig. 2 but not according to those of Fig. 4. Orientation of the cystine noose is viewed perpendicular to the plane of the antibody and the epitope, showing the epitope at the rim of the disc formed by the tip of the loop and the relatively flat surface. The same angle is used for all panels, except for panel D in which the orientation of the antibody with the cystine noose is slightly different. The orientation of panels A, B, and C is similar to that shown in Fig. 5C. P, the pocket in the surface.
between the essential disulfide at the pocket and the essential Leu180 at the rim of the disc (Fig. 5), the antibody paratopes must have a concave shape. This agrees with the general three-dimensional (3-D) structures of protein-protein interfaces in heterocomplexes which are in general concave for antibody-antigen and planar for other protein-protein complexes (9). Besides the direct contribution of a residue to antibody binding, the pepscan data also indicate which residue in the epitope has an important structural role. A nonconservative mutation is the allowed substitution of Ser at the position of Asn179 (Fig. 2). Asn179 has a very important structural role in the peptide, because it is involved in an H-bond network (2). Asn179 functions as an N-cap residue to stabilize the second helix and is involved in stabilization of the type I9 turn. This structural role can also be performed by Ser, and according to statistical analysis, Ser is the only residue that occurs as frequently as Asn at the N-cap position of a helix (17). The strict occupation of this position for residues that allow N-capping of
a helix suggests that the reactive peptides in the pepscan have an ordered helical structure in the C-terminal region. The lack of reactivity to peptides with a typical helix-breaking residue like Pro at positions 182 to 185 also suggests that this part of the peptide adopts a helical structure (Fig. 2). In most epitope mapping studies with peptides of increasing lengths, MAbs bind with all peptides which contain the essential residues in the core of the epitope. For example, if a MAb binds a pentapeptide, it should bind six overlapping decapeptides of the corresponding antigenic region (11). However, this is not the case for all MAbs in this study. The addition of residues N or C terminal to the core of the epitope reduces or abrogates antibody binding. Therefore, elongation of the reactive sequence may have structural consequences for the antigenic site. The 3-D structure of the antigenic site is probably very important. For the longest peptides of 16 residues, the only (or strongest) binding was directed against peptide VPCSTCEGNLACLSLC. The reactivities of the 16-residue peptides suggest that synthetic peptides may not always adopt the native fold. In
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FIG. 4. Reactivities of overlapping 8, 9, 10, 11, 12, 13, 14, 15, and 16-residue peptides of BRSV-G strain 391-2 (residues, PYVPCSTCEGNLACLSLCH) with MAbs 48 (diluted 1:150,000), 66 (diluted 1:6,000), 53 (diluted 1:500), 62 (diluted 1:6,000), and 57 (diluted 1:200). Reactivities of the other MAbs are not shown because the pattern of reactivity was very similar to that of MAb 48 or 66.
this study we used the 3-D structure obtained from a 32residue synthetic peptide (2) which showed high affinity with MAb 20 (12). The solution structure of the peptide agrees with the pepscan data because the most reactive 16-residue peptide is the only peptide of the 16-residue set which can mimic the complete top surface of the cystine noose of BRSV-G. This finding suggests that the energetic epitope may be the rim of the disc, but that the complete epitope spans the complete top surface of the disc. Comparison of the 3-D structure with the detailed analysis of the antigenic structure explains the known mutations that have accumulated during evolution within the cystine noose of G proteins of RSV types and subtypes. The mutations in the cystine noose of BRSV-G listed in Table 1 (E1773K177, L1803P180, L1833S183, and S1843P184, arbitrarily from the perspective of the BRSV-G 391-2 strain) are all mutations with major consequences for antibody binding (Fig. 2). These mutations are located at the rim or at the bottom of the disc,
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leaving the top surface unchanged (Fig. 6). Remarkably, mutations have occurred at the same position (and one additional position, 181) of HRSV-G when the sequences of HRSV-A and HRSV-B are compared (Table 1). This suggests a similar 3-D structure and antigenic structure for the central conserved region of HRSV-G. The side chain of E177 is well exposed, and therefore the mutations at that position in BRSV and HRSV, as shown in Table 1, will change the local antigenic surface of the protein. Similarly, mutation of the exposed side chain of L180 changes the local antigenic surface of the protein. Remarkably, in both BRSV and HRSV the antigenic variants have P180 at the N-cap 1 1 position of the helix. Pro is the only residue with a high preference for the N-cap 1 1 position of a helix (17). Therefore, a mutation to (or from) Pro at position 180 seems an ideal solution for antigenic escape, because the mutation will result in a local change of the antigenic surface of the protein but leaves the 3-D structure of the backbone intact. The large side chain of L183 occupies the crevice between the two helices on the back of the disc. Therefore mutations of this residue (Table 1) may have structural consequences. Because S184 forms helix-stabilizing H bonds with Leu180 via its backbone amide, mutation to a helix-breaking Pro (Table 1) which is not able to form this H bond may have an impact on the 3-D structure. Interestingly, two in vitro-selected virus escape mutants resistant to an antibody directed against the central conserved region of protein G of HRSV-A contained unusual substitutions of Cys182 or Cys186 to Arg (19). Both mutations would have a drastic impact on the antigenic surface and the structure of the protein. Furthermore, both mutations would destroy the integrity of the conserved pocket. Although both mutants could be propagated in vitro, it seems unlikely that these mutations caused by a rare hypermutation event (19) would occur in vivo. Furthermore, other dramatic genetic changes in G which are possible in vitro (5, 18) suggest that the in vivo role of protein G might be different compared with the in vitro role. Of all the MAbs that were used in this study, only MAb 70 mapped outside the central conserved region of BRSV-G. MAb 70 was mapped to residues 199TITLKKAPKPK209 of BRSV-G strain Lelystad. This epitope corresponds exactly to a linear epitope on protein G of HRSV-A (204KKPTFK209) (Table 1) (6). Several MAbs directed to the cystine noose and MAb 70 have been used for the antigenic subgrouping of BRSV isolates (4, 16, 20). Because MAb 70 did not bind peptide 199TITLKKTPKPK209, corresponding to BRSV-G strain 391-2 (data not shown), the Ala2053Thr205 mutation determines the subgroup classification based on MAb 70 (Tables 1 and 2). Only the Thr205 substitution is sufficient to escape antibody binding. Because Thr is a potential O-glycosylation site, a possible carbohydrate at that position would result in a very different antigenic variant. According to epitope mapping of MAb 48 (Fig. 2) and MAb 20 (12), the classification based on these MAbs (4, 16, 20) directed to the cystine
TABLE 2. Point mutations that determine BRSV subgrouping based on linear epitopes Subgroup
A AB B
Mutation(s) in BRSV-Ga
L180 L180 P180 (S183, P184)
T205 A205 A205
a Residues in parentheses are not strictly necessary for classification in subgroup B.
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FIG. 5. Molecular surface (white) of the top view (A), oriented as shown in Fig. 3A, B, and C, and side view, rotated 908 over the x axis (B) of the cystine noose of BRSV-G. Residues conserved in RSV-G are indicated with a blue dot surface. Residues in epitope STCEGNLACLSL are indicated with a red line surface. P, the pocket which is visible in panel A and indicated with an arrow in panel B. (C) Secondary structure (blue ribbon for helices) in the transparent surface, which is oriented the same as panel A.
noose is also determined by a single mutation of Leu1803 Pro180 and in the case of strain BOV-X it may be determined by an extra mutation of Ser1843Pro184. Figure 2 shows that binding of MAb 48 is lost when L183 is replaced by S183. However, MAb 48 still recognizes BRSV strains with L183 to S183 substitutions (4). This discrepancy may be explained by differences in affinity for both strains, because higher reactivity was found with peptide STCEGNLACSSL when a higher concentration of MAb 48 was used (data not shown). Alternatively, the reactivities to small peptides may have different structural constraints for binding compared to those of complete virus. It is possible that the solution structure of a synthetic peptide is different than that of the authentic protein. Therefore, results obtained from a comparison of the peptide
structure with peptide binding studies may not automatically apply for the authentic protein. In conclusion, the 3-D structure of the cystine noose of BRSV-G and the detailed antigenic structure of the noose explain the occurrence of a particular residue at a specific location in the primary sequence. Several conserved and nonconserved residues are essential for antibody binding. The conserved residues have an apparent structural role or probably form a conserved receptor binding site. The nonconserved residues in the epitope have a major effect on the antigenic structure. Mutations at these latter positions can be divided into mutations with and without major structural changes and explain the way a virus can escape antibody recognition.
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FIG. 6. Molecular surface of cystine noose of BRSV-G, as shown in Fig. 5A. The epitope is indicated with a red line surface. Mutations in BRSV-G are indicated with black dots.
Because both BRSV-G and HRSV-G have similar mutations in the cystine noose, their overall structures are probably similar. The limited number of actual escape mutations compared with the large number of possible mutations determined by pepscan analysis suggests a strong structural constraint for the cystine noose. ACKNOWLEDGMENTS We thank Wouter Puijk and Drohpati Parohi for technical assistance and Jurgen Doreleijers for helpful discussions. REFERENCES ¨ rvell, R. A. Lerner, and 1. Åkerlind-Stopner, B., G. Utter, M. A. Mufson, C. O E. Norrby. 1990. Subgroup-specific antigenic site in the G protein of respiratory syncytial virus forms a disulfide-bonded loop. J. Virol. 64:5143–5148. 2. Doreleijers, J. F., J. P. M. Langedijk, K. Hård, R. Boelens, J. A. C. Rullmann, W. M. M. Schaaper, J. T. van Oirschot, and R. Kaptein. 1996. Solution structure of the immuno dominant region of protein G of bovine respiratory syncytial virus. Biochemistry 35:14684–14688. 3. Elvander, M., S. Vilcek, A. Uttenthal, A. Ballagi-Pordany, and S. Belak. 1996. Genetic heterogeneity of G attachment protein of bovine respiratory syncytial virus (BRSV) strains, p. 1–15. Ph. D. thesis, University of Uppsala, Uppsala, Sweden. 4. Furze, J., G. Wertz, R. Lerch, and G. Taylor. 1994. Antigenic heterogeneity of the attachment protein of bovine respiratory syncytial virus. J. Gen. Virol. 75:363–370. 5. Garcia-Barreno, B., A. Portela, T. Delgado, J. A. Lopez, and J. A. Melero. 1990. Frame shift mutations as a novel mechanism for the generation of neutralization resistant mutants of human respiratory syncytial virus. EMBO J. 12:4181–4187.
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6. Garcia-Barreno, B., T. Delgado, B. Åkerlind-Stopner, E. Norrby, and J. A. Melero. 1992. Location of the epitope recognized by monoclonal antibody 63G on the primary structure of human respiratory syncytial virus G glycoprotein and the ability of synthetic peptides containing this epitope to induce neutralizing antibodies. J. Gen. Virol. 73:2625–2630. 7. Geysen, H. M., R. H. Meloen, and S. J. Barteling. 1984. Use of synthetic peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc. Natl. Acad. Sci. USA 81:3998–4002. 8. Geysen, H. M., S. J. Barteling, and R. H. Meloen. 1985. Small peptides induce antibodies with a sequence and structural requirements for binding antigen comparable to antibodies raised against the native protein. Proc. Natl. Acad. Sci. USA 82:178–182. 9. Johnson, P. R., M. K. Spriggs, R. A. Olmsted, and P. L. Collins. 1987. The G glycoprotein of human respiratory syncytial viruses of subgroups A and B: extensive sequence divergence between antigenically related proteins. Proc. Natl. Acad. Sci. USA 84:5625–5629. 10. Jones, S., and J. M. Thornton. 1996. Principles of protein-protein interactions. Proc. Natl. Acad. Sci. USA 93:13–20. 11. Langedijk, J. P. M., N. K. T. Back, E. Kinney-Thomas, C. Bruck, M. Francotte, J. Goudsmit, and R. H. Meloen. 1992. Comparison and fine mapping of both high and low neutralizing monoclonal antibodies against the principal neutralization domain of HIV-1. Arch. Virol. 126:129–146. 12. Langedijk, J. P. M., W. M. M. Schaaper, R. H. Meloen, and J. T. van Oirschot. 1996. Proposed three-dimensional model for the attachment protein G of respiratory syncytial virus. J. Gen. Virol. 77:1249–1257. 13. Langedijk, J. P. M., W. G. J. Middel, W. M. M. Schaaper, R. H. Meloen, J. A. Kramps, A. H. Brandenburg, and J. T. van Oirschot. 1996. Type-specific serologic diagnosis of respiratory syncytial virus infection, based on a synthetic peptide of the attachment protein G. J. Immunol. Meth. 193:157–166. 13a.Langedijk, J. P. M. Unpublished data. 14. Lerch, R. A., K. Anderson, and W. G. Wertz. 1990. Nucleotide sequence analysis and expression from recombinant vectors demonstrate that the attachment protein G of bovine respiratory syncytial virus is distinct from that of human respiratory syncytial virus. J. Virol. 64:5559–5569. 15. Norrby, E., M. A. Mufson, H. Alexander, R. A. Houghten, and R. A. Lerner. 1987. Site-directed serology with synthetic peptides representing the large glycoprotein G of respiratory syncytial virus. Proc. Natl. Acad. Sci. USA 84:6572–6576. 16. Prozzi, D., K. Walravens, J. P. M. Langedijk, F. Daus, J. A. Kramps, and J. J. Letesson. Antigenic and molecular analyses of the variability of bovine respiratory syncytial virus G protein. J. Gen. Virol., in press. 17. Richardson, J. S., and D. C. Richardson. 1988. Amino acid preferences for specific locations at the ends of a helices. Science 240:1648–1652. 18. Rueda, P., T. Delgado, A. Portela, J. A. Melero, and B. Garcia-Barreno. 1991. Premature stop codons in the G glycoprotein of human respiratory syncytial viruses resistant to neutralization by monoclonal antibodies. J. Virol. 65:3374–3378. 19. Rueda, P., B. Garcia-Barreno, and J. A. Melero. 1994. Loss of conserved cysteine residues in the attachment (G) glycoprotein of two human respiratory syncytial virus escape mutants that contain multiple A-G substitutions (hypermutations). Virology 198:653–662. 20. Schrijver, R. S., F. Daus, J. A. Kramps, J. P. M. Langedijk, R. Buijs, W. G. J. Middel, G. Taylor, J. Furze, M. W. C. Huyben, and J. T. van Oirschot. 1996. Subgrouping of bovine respiratory syncytial virus strains detected in lung tissue. Vet. Microbiol. 53:253–260. 21. Trudel, M., F. Nadon, C. Se´guin, and H. Binz. 1991. Protection of BALB/c mice from respiratory syncytial virus infection by immunization with a synthetic peptide derived from the G glycoprotein. Virology 185:749–757. 22. Wertz, G. W., P. L. Collins, Y. Huang, C. Gruber, S. Levine, and L. A. Ball. 1985. Nucleotide sequence of the G protein of human respiratory syncytial virus reveals an unusual type of viral membrane protein. Proc. Natl. Acad. Sci. USA 82:4075–4079.
