Protein Engineering, Vol. 15, No. 5, 365-371,
May 2002
© 2002 Oxford University Press
Modelling the structure of the fusion protein from human respiratory syncytial virus
Biomolecular Research Institute, 343 Royal Parade, Parkville, Victoria 3052, Australia
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Abstract |
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The fusion protein of respiratory syncytial virus (RSV-F) is responsible for fusion of virion with host cells and infection of neighbouring cells through the formation of syncytia. A three-dimensional model structure of RSV-F was derived by homology modelling from the structure of the equivalent protein in Newcastle disease virus (NDV). Despite very low sequence homology between the two structures, most features of the model appear to have high credibility, although a few small regions in RSV-F whose secondary structure is predicted to be different to that in NDV are likely to be poorly modelled. The organization of individual residues identified in escape mutants against monoclonal antibodies correlates well with known antigenic sites. The location of residues involved in point mutations in several drug-resistant variants is also examined.
Keywords: fusion/homology modelling/Newcastle disease virus/respiratory syncytial virus
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Introduction |
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Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in infants and children, with almost all children becoming infected by the age of 2 years. Primary infection does not induce immunity and recurrent infection occurs throughout life. The virus is also an important pathogen in immunocompromised adults and the elderly (Collins et al., 1996
). RSV is a member of the Pneumovirus genus of the family of Paramyxoviridae. The Paramyxoviridae also include the human pathogens measles, mumps and parainfluenza virus types 1–4. Other members include Sendai virus, Newcastle disease virus (NDV), simian virus 5 (SV5), pneumonia virus of mice (PVM) and turkey rhinotracheitis virus (TRT). RSV has a single strand of negative-sense RNA, encoding for 10 viral proteins. Three of these are exposed on the surface of virion and infected cells; the F (fusion) protein, the G (attachment) protein and SH (small hydrophobic) integral membrane protein, the first two being the major immunogenic proteins.
The G protein is responsible for attachment of the viral particle to the host, a role adopted by the haemagglutinin of other members of the family (although G itself does not bind sialic acid). Following attachment, fusion of the viral particle with its host is achieved by F. While F is able to mediate fusion in recombinant viruses lacking G and SH, efficient fusion is obtained only when all three surface proteins are coexpressed (Karron et al., 1997). The F protein also promotes infection of neighbouring cells by the formation of syncytia.
The F protein is synthesized as a single N-glycosylated polypeptide precursor of 574 amino acids (F0), that is assembled in the rough endoplasmic reticulum into a homo-oligomer and cleaved by a cellular protease into two disulphide-linked chains, F2 and F1, before reaching the cell surface. The peptide is anchored in the viral membrane by a transmembrane segment found toward the C-terminus of F1. At the N-terminus of F1, 
20 residues compose a highly hydrophobic domain (fusion peptide) that is believed to insert into the target membrane during the fusion process. The F proteins of all Paramyxoviridae family display heptad repeat sequences. One of these (HR-A) extends from the C-terminus of the fusion peptide approximately 56 residues to a conserved cysteine residue, while the second is N-terminal to the transmembrane anchoring domain. The X-ray structure of NDV-F shows that the helix of HR-A extends a further 22 residues C-terminal to the conserved cysteine residue. A third heptad repeat region (residues 53–100) identified in the sequence of the F2 chain of RSV-F (Lambert et al., 1996) maps to the HR-C helix observed in the X-ray structure of NDV-F. The structures of a complex of regions of HR-A and HR-B from both RSV-F (Zhao et al., 2000) and SV5-F (Baker et al., 1999) show a trimeric coiled-coil core formed by HR-A, with three HR-B 
-helices packed anti-parallel within the grooves formed by adjacent HR-A segments. This hexameric motif has now been observed in a large number of viral fusion proteins and may reflect a common mechanistic element (Lentz et al., 2000).
The hexameric coiled-coil structure observed for the HR-A/HR-B complex probably corresponds to the stable post-fusion conformation. The three-dimensional atomic structure of the remainder of the RSV-F protein is at present unknown. Single-molecule electron microscopy images of RSV-F (Calder et al., 2000) suggest two morphologies, `cone' shape and `lollipop' shape. These probably relate to the pre-fusion metastable and post-fusion forms of the protein, respectively, analogous to those seen for haemagglutinin from influenza virus (Skehel and Wiley, 2000).
The structure of the F protein from NDV, however, has been determined recently (Chen et al., 2001). The molecule is trimeric and is organized into three regions, head, neck and stalk (Figure 1). In the head, each monomer comprises an immunoglobulin type ß-sandwich domain and a highly twisted ß-sheet domain. Residues 171–221 (NDV-F numbering), that include HR-A, form a central coiled-coil spanning both neck and stalk regions; residues 465–495 (HR-B) are disordered in the structure. The neck includes all of HR-C (77–105) that forms an -helix in which the first 15 residues (77–91) pack parallel against the central coiled-coil, a mixed four-stranded ß-sheet and an irregular bundle of four -helices. The structure is fenestrated by three radial channels between the head and neck regions. These connect to a wide central channel that extends 50 Å down through the head region.
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The sequence homology of F proteins within the Paramyxoviridae family is generally fairly low although, within any one subfamily, the level of homology can be high. The sequence homology between F proteins from NDV and RSV is particularly low, <15%, certainly well below the so-called twilight zone (Rost, 2000
) for homology modelling. There are, however, several features that allow the generation of a realistic model of RSV-F based on the structure of NDV-F; they are known to have the same function, their overall morphology is the same and key sequence/structural features are present in both in identical positions within their sequences. The presence of such features improves the chances of obtaining a successful model (Colman et al., 1993; Crennell et al., 2000). We report here our efforts in deriving a model of RSV-F and an appraisal of this model. |
Methods |
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Alignments of representative sequences from several members of the Paramyxoviridae family were performed using ClustalW (Thompson et al., 1994
) implemented in the BioEdit program (Hall, 1999). Minor manual changes were performed to ensure maximum overlap of predicted secondary structure with no insertions in these regions. These changes were restricted primarily to the N-terminal region of F2 and residues in the regions 329–346 and 385–391 (RSV-F numbering). Secondary structure prediction was performed through the PHD server (Rost and Sander, 1993, 1994).
The model of RSV-F was first generated by homology with NDV-F using the Modeller package (
ali and Blundell, 1993) within the InsightII program (Molecular Simulations). Compatibility of the model with the sequence was evaluated with Profiles-3D (Luthy et al., 1992). Three-fold symmetry was applied by superimposing the -carbon atoms of one RSV-F monomer on to the other two related monomers. The trimer resulting from this assembly was then energy minimized using the Discover program (Molecular Simulations) with all backbone atoms held fixed. The molecular mechanics minimization used the AMBER force field and an electrostatic cut-off of 18 Å. The method of steepest descents was applied until the gradient fell below 10.0 kcal/mol.Å, at which point the conjugate gradients method was applied until the gradient fell below 0.01 kcal/mol.Å. The final model was again assessed using Profiles-3D and Procheck (Laskowski et al., 1993).
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Results |
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The alignment of sequences from several members of the Paramyxoviridae family is presented in Table I
. Also indicated in Table I is the secondary structure observed in the three-dimensional X-ray crystal structure NDV-F. The sequence identity matrix based on this alignment is presented in Table II. Secondary structure was correctly predicted by PHD for 78% of the residues in NDV-F. Residues involved in the ß-bulges (Chan et al., 1993) in strands Ia and Ib were not predicted as strand although the flanking regions were. Residues 314–318 and 322–326 had secondary structure incorrectly assigned (i.e. assigned strand when the structure was helix and vice versa). Notably, the X-ray structure near residues 311–314 is poorly defined. The residues in strand Ig were predicted to be coil. Residues 371–377 were incorrectly predicted to be strand (although they do not form of a ß-sheet they do adopt an extended conformation).
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The success of PHD in predicting the secondary structure in NDV-F provides encouragement for the prediction in RSV-F. The predicted secondary structure for the F proteins from NDV, SV5, HPIV 1–4, mumps, Sendai and measles appears to be highly conserved, despite pairwise sequence identities being as low as 20%. The members of the Pneumovirinae subfamily (that includes TRT, PVM and RSV) also share a high degree of similarity in their predicted secondary structure, but there are clear differences between the F proteins from this genus and the members of the Paramyxovirinae subfamily. This dichotomy is also reflected in the level of sequence identity, ranging from as low as 12% to a maximum of only 18%. In particular, the sequence identity between NDV-F, a member of the rubulavirus family and RSV-F is only 13.5%.
The predicted secondary structure of RSV-F (Table I
) differs significantly from that predicted for NDV-F in several regions: (1) in HR-A, 13 residues (202–214) are not predicted to adopt a helical structure, although there is a cysteine present that is strictly conserved; (2) in the 4-helix bundle, where the helices N2–N4 were successfully predicted in NDV-F, only two helices are predicted for RSV; (3) in RSV, strand IIIb is predicted to be significantly shorter, with an intervening helical segment between strands IIIb and IIIc; (4) residues in strand Ic, incorrectly predicted by PHD as helix in NDV-F, are predicted to be strand in RSV-F; and (5) the residues in helix H2 of NDV-F are predicted to be random coil in RSV-F.
The homology model of RSV-F was based on the sequence alignment in Table I. The r.m.s. difference in -carbon positions between individual monomers of RSV-F and NDV-F is 0.9 Å. In the X-ray structure of NDV-F, five disulphide linkages are observed in each monomer. In the sequence alignment presented in Table I, eight of the disulphide-forming cysteine residues are aligned with cysteine residues in RSV-F. While the likelihood of disulphide formation in the model is reasonably unambiguous in a few cases, as judged by the close proximity of the constituent cysteine residues, the cysteine-rich nature of the head region makes unambiguous assignment in that region somewhat more difficult. In particular, a shift in the sequence alignment by just two residues of cysteines 322 and 393 would bring them within a distance capable of forming a disulphide link, at the loss of the NDV-F equivalent disulphide link between residues 382 and 393 (362 and 370, NDV-F numbering). Similar shifts in sequence alignment of cysteines 37, 416 and 439 could also change the disulphide pattern in RSV-F. Several alternative disulphide linkages were explored and their suitability was assessed by examining the Profiles-3D score in the region of the newly formed disulphide link. The Profiles-3D score was significantly poorer for all models in which an NDV-F equivalent disulphide link was removed. In the final model all NDV-F equivalent disulphide links were therefore maintained. Residues 37, 322 and 439 all lie in close proximity: the disulphide link between 37 and 322 provided the better Profiles-3D score.
Disulphide links were therefore built between the following pairs of cysteine residues in the RSV-F model, 37:322, 313:343, 69:212, 358:367, 382:393 and 416:422. The latter four links correspond to those observed in the structure of NDV-F; the link between cysteine residues 401 and 424 (NDV-F numbering) is not present in RSV-F. The former two links are additional to those observed in NDV-F; they connect, respectively, the loop between helix H1 and strand Ia with the C terminus of strand Ib and the loop between the central region of strand Ib with the C terminus of strand Ic. Two inter-chain disulphide links are predicted for RSV-F, between cysteine pairs 37:322 and 69:212; the former of these has no corresponding partner in NDV-F.
The residue compatibility score (S-score) from Profiles-3D for both the X-ray structure of NDV-F and the model structure of RSV-F proteins is presented in Figure 1 (only the score for one of the monomers is presented, the profile for the other monomers being very similar). The compatibility score for NDV-F falls below zero for residues Asp277 and Ser278, at the C-terminus of strand IIIb. The compatibility score for the RSV-F model falls below zero in five regions, four of which are where the secondary structure prediction by PHD differs for NDV-F and RSV-F (region 1, Val207–Ser215; region 2, Leu257–Met274; region 3, Tyr286–Val296; and region 5, Leu381–Asp392), while the fifth occurs at Lys470–Asp479 (region 4).
Several resides fall into disallowed regions of the Ramachandran plot (Cys37, Arg235, Thr324, Asp344, Cys416, Ser436). Residue Thr324 corresponds to Thr310 in NDV-F, which also lies in the disallowed region of the Ramachandran diagram in the X-ray structure of NDV-F. Both cysteine residues (37 and 416) have been modelled in disulphide links.
The proline residue involved in the wide ß-bulge, responsible for introducing a twist into the connection between strands IIIc and Ib (Pro290 NDV-F, Pro304 RSV-F), is strictly conserved in all sequences. Apart from cysteine residues, the only other residues that are strictly conserved in the alignment presented in Table I are Gly145, Ala147, Gly411 and Asp486. Only Gly411 is observed in the structure of NDV-F; this residue is in a type I' (inverse common) ß-turn (Chou and Fasman, 1977; Richardson and Richardson, 1989) connecting stands IIa and IIb, found at the outside opening of the radial channel. Asp486 is located on the surface of coiled-coil HR-A/HR-B complex (Zhao et al., 2000).
The sequence of RSV-F differs from all other sequences in that it has a large insertion prior to the fusion peptide cleavage site. This region contains three potential N-linked glycosylation sites (at Asn116, Asn120 and Asn126). Proteolytic removal of this region appears to be necessary for activation of the fusion protein (Zimmer et al., 2001). Three other potential N-linked glycosylation sites are at Asn27, Asn70 and Asn500. Only Asn70 has been included in the current model, located at the N-terminus of helix HR-C at the base of the neck region.
The heptad repeat pattern of hydrophobic amino acids in the a and d positions in HR-A observed in NDV-F is shown in Table III. Two stutters are found in this region (Brown et al., 1996). There is predicted to be a disruption of the helix in HR-A between residues 202 and 214 of RSV-F [also previously noted by Chambers et al. (Chambers et al., 1992)]. In the alignment with NDV-F this region contains various residues in positions that are likely to be unfavourable towards the formation of a coiled coil (Lupas et al., 1991), specifically Leu204 in a b position, Val207 in an e position and Arg213 in a d position. In addition, Pro205 (conserved between SV5-F and RSV-F) could introduce a kink into the helix (Chang et al., 1999) further disrupting the coiled-coil. In the sequence alignment presented in Table I, the observed structure of NDV-F and the HR-A/HR-B complex from RSV-F have 24 residues in common through HR-A (Gly184–Lys209). The HR-A helix in NDV-F contains a shorter (3–4–4–3) stutter than that observed (3–4–4–4–3) in the N-terminus of both RSV-F and SV5-F HR-A helices (Table III). The longer stutter, however, may be induced by the artificial nature of the constructs employed in the last two structures. The difference in heptad repeat pattern corresponds roughly with onset of poor Profiles-3D compatibility scores in region 1.
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A 21-residue peptide, corresponding to residues 255–275 of RSV-F, adopts a helix–loop–helix conformation in 30% trifluoroethanol (Toiron et al., 1996
). The secondary structure matches very well the prediction from PHD for this region, but as was noted above, this is different to that observed in the X-ray structure of NDV-F. It is likely, therefore, for the structures of RSV and NDV fusion proteins to differ in this region.
The spatial location of various antigenic sites on the surface of RSV-F has been provided through EM images of monoclonal antibodies (MAbs) of known specificity in complex with full length F (Calder et al., 2000). The angle at which each MAb binds F is compared with the position of the altered residue of the resistant mutants in the model of RSV-F in Table IV. Angles in the model were calculated from the -carbon of each residue to a point 30 Å from the top of the head of the trimeric assembly lying on the molecular symmetry axis. The agreement is very good, with most comparisons lying within a few degrees. All residues that confer antibody resistance through mutation are exposed on the surface of the protein in the model. Residues forming antigenic site II are found in helices N3 and N4, on the exterior of the neck and at the mouth of the radial channel. Site I is near the top of the head, while sites IV, V and VI lie at the apex of the trimeric structure of the head. These sites are indicated in Figure 2.
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A segment from the C-terminal region of HR-A from the Sendai virus fusion protein has been shown to be able to induce membrane fusion (Peisajovich et al., 2000
). This segment corresponds to residues 221–242 in RSV-F and encompasses the C-terminus of HR-A and all of helix N1. In the present model, several residues in N1 are surface exposed at the base of the axial channel. These residues could become exposed to the target membrane after opening and disassembly of the head (Chen et al., 2001).
Polypeptides from the HR-B domain of RSV demonstrate antiviral activity, inhibiting syncytia formation (Lambert et al., 1996). These are believed to function by interfering with the association of HR-B with HR-A. The HR-B region is not observed in the structure of NDV-F; however, the overlay of the HR-A/HR-B complex from RSV-F suggests an onward extension of the coiled-coil from HR-A with HR-B packed into the grooves. It is anticipated that the association of HR-B with HR-A, which brings together the transmembrane and fusion peptides, occurs only in the post-fusion form of the protein. The polypeptide that inhibits syncytia formation could associate with the extended -helical N-terminal region of HR-A, preventing it from adopting its correct post-fusogenic association with HR-B.
Several small-molecule inhibitors of RSV have been developed recently that function either by inhibiting fusion or by interfering with formation of the multimeric state or early processing of RSV-F (Aulabaugh, et al., 2000; Meanwell and Krystal, 2000). In a few cases, resistant viruses have led to an indication of the site of interaction. Resistance to the compound RD3-0028 was mapped to residue N276Y in RSV-F (Sudo et al., 1998). This compound presumably acts by interfering with the synthesis or intracellular processing of the F protein. Residue 276 lies in helix N4, at the interface between head and neck regions. Benzimidazole derivatives have demonstrated potent antiviral activity (Andries et al., 2000); resistant mutants to these compounds have single point mutations at residues S398L and D486N. Residue 486 has not been included in the present model, while residue 398 lies N-terminal to the immunoglobulin-like ß-sandwich domain and is found surface exposed on the rim of the axial channel at the junction between two monomers.
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Conclusions |
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The recently determined three-dimensional structure of NDV-F has permitted the construction of a reasonably reliable model of RSV-F. The model of RSV-F, however, is clearly inadequate in several regions. We have shown that the heptad repeat pattern in HR-A of NDV-F and RSV-F differs. The secondary structure prediction in RSV-F in this region is also not consistent with the structure observed in NDV-F. Similarly, the secondary structure prediction for RSV-F in the region of the 4-helix bundle differs from what is observed in the structure of NDV-F, but is consistent with the structure of a small polypeptide taken from this region. Other regions where the secondary structure prediction in RSV-F differs from the X-ray structure of NDV-F also have poor Profiles-3D structure compatibility scores and are likely to be poorly modelled. Despite these reservations, the model is consistent with the known three-dimensional arrangement of antigenic sites and provides an indication of the location of residues involved in drug-resistant variants and hence should provide a useful framework for future investigations in these areas.
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Notes |
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1 Present address, The Walter & Eliza Hall Institute of Medical Research, P.O. Royal Melbourne Hospital, Parkville, Victoria 3050, Australia
3 Present address, CSIRO, Division of Health Science and Nutrition, 343 Royal Parade, Parkville, Victoria 3052, Australia
2 To whom correspondence should be addressed
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Acknowledgments |
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The support by Biota Holdings (Melbourne, Australia) for some of the work described here is gratefully acknowledged.
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Received October 5, 2001; accepted January 4, 2002.
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J. L. Douglas, M. L. Panis, E. Ho, K.-Y. Lin, S. H. Krawczyk, D. M. Grant, R. Cai, S. Swaminathan, and T. Cihlar Inhibition of Respiratory Syncytial Virus Fusion by the Small Molecule VP-14637 via Specific Interactions with F Protein J. Virol., May 1, 2003; 77(9): 5054 - 5064. [Abstract] [Full Text] [PDF]
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