Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, NM 87545, USA
Correspondence
Catherine A. Macken
cmacken{at}lanl.gov
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ABSTRACT |
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Present address: Department of Biology, Indiana University, 1001 E. Third Street, Bloomington, IN 47403, USA.
Present address: 454 Life Science Corporation, 20 Commercial Street, Branford, CT 06405, USA.
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INTRODUCTION |
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Solution of the structure of the HA of influenza A virus (subtype H3) strain A/Aichi/2/68 (Wilson et al., 1981) and studies of variant viruses enabled mapping of the antigenic sites on this protein (reviewed by Wilson & Cox, 1990
). Similar mapping of the antigenic sites on the influenza B virus HA has been hampered by the lack of a known crystal structure. Studies of variants of influenza B viruses have allowed inference of some of the antigenic nature of the HA (Berton et al., 1984
; Hovanec & Air, 1984
; Berton & Webster, 1985
; Rivera et al., 1995
). These authors inferred the structure of antigenic sites on influenza B virus HA based on the assumption, proposed by Krystal et al. (1982)
, that the three-dimensional structures of the HAs of influenza A (H3) and B viruses are very similar. Qualitatively, this assumption is reasonable because of the functional similarity of these proteins; in reality, it leaves out details in structural differences that may occur due to mutations, insertions and deletions.
More recently, the structure of the HA esterase (HEF) protein from C/Johannesburg/1/66, an influenza C virus (the third type of influenza virus that infects humans), was solved (Rosenthal et al., 1998), followed by structures of the influenza A virus HA for three additional subtypes: H1 (A/South Carolina/1/18 and others; Gamblin et al., 2004
; Stevens et al., 2004
), H5 (A/duck/Singapore/3/97; Ha et al., 2001
) and H9 (A/swine/Hong Kong/9/98; Ha et al., 2001
). Whilst the functions of the HEF of influenza C virus are not equivalent to those of the HAs of influenza A and B viruses (the HEF has a combined HA and receptor-destroying esterase activity), nevertheless, the structures of the HEF and HA of A/Aichi/2/68 (H3) are remarkably similar (Rosenthal et al., 1998
). We decided to utilize this structural and functional knowledge to model the structure of the HA of influenza virus B/Lee/40, the oldest influenza B virus with an HA sequence in public databases.
Two classes of structure-prediction methods, ab initio and knowledge-based, have attracted great interest in recent decades and objective evaluation of their performance has received public scrutiny (http://PredictionCenter.llnl.gov). Ab initio methods have made notable progress toward predicting structures of small (<60 aa) proteins (e.g. Huang et al., 1999; Moult, 1999
; Bonneau & Baker, 2001
), but have not yet been successful on proteins as large as HA. The knowledge-based class of methods uses available information from protein-structure databases to develop models of proteins of unknown structure. Within this class, comparative modelling (or homology modelling) works at its best when the protein with unknown structure shares substantial sequence homology with a protein of known structure. Under these circumstances, the known structural features of one protein (the template) can be used to model the unknown structural features of the other (the target).
The quality of a homology model depends critically on the extent of identity between the template and target sequences. A rule of thumb is that one needs 35 % sequence identity between the template and target proteins in order for homology modelling to be successful. An identity of >50 % ensures that most of the features of a homology model will be accurate and that the root mean square error will be <2 Å, regardless of the size of the protein (Sánchez &
ali, 1997
), an accuracy equivalent to that of a medium-resolution crystal structure. Low sequence identity (<25 %) leads to much less accurate predictions. Still, low sequence identity can lead to a useful model, as described by Tramontano (1998)
.
As ab initio predictions of the structure of the influenza B virus HA are not yet possible, we chose a homology modelling approach. We selected the HA1 polypeptide chain, one of two chains into which HA is cleaved during virus replication, as our target of interest, as it shows most of the variation of the HA.
Identity between the HA sequence of B/Lee/40 and the HA sequence from any of the influenza A viruses with known HA structure is substantially below the desired minimum of 35 %. However, the HA of A/Aichi/2/68 (H3) and the HEF of C/Johannesburg/1/66 have lower sequence identity than any of the B/A comparisons, yet their structures are similar (Rosenthal et al., 1998). Hence, we were optimistic about the prospects for modelling the structure of the HA1 of B/Lee/40 successfully. To improve our chances of success, we included knowledge of the structural similarity between the HEF of C/Johannesburg/1/66 and the known HA structures of the influenza A viruses in our analysis. By the criteria described below, our modelling of the B-HA1 structure produced results of high quality that are consistent with the available sequence/function information on the HA of B/Lee/40, thereby providing a basis for investigating the evolution of this protein.
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METHODS |
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Choice of template.
The function of the HA of B/Lee/40 is closer to that of the HA of influenza A viruses than to that of the HEF of influenza C viruses. Hence, for comparative purposes, we selected two templates from known A-HA structures by assessing pairwise distances among A-HA structures and pairwise identity between B/Lee/40 and A-HA sequences in the multiple alignment of A-HA, B-HA and C-HEF sequences that we derived by using the procedure described below.
Sequence alignments.
We chose the HA1 sequence of B/Lee/40 (GenBank accession no. NP_056660) as the representative of influenza B virus HA, for which we inferred structural models. The profile alignment option of CLUSTAL_W (Thompson et al., 1994) with default parameters was used for initial alignments.
To generate an alignment between the HA1 sequences of B/Lee/40 and influenza A viruses, we began with a structure-informed alignment of the HEF sequence from C/Johannesburg/1/66 with one sequence from each of the 15 HA subtypes of influenza A virus, provided to the Influenza Sequence Database (ISD) (http://www.flu.lanl.gov; Macken et al., 2001) by Dr J. Skehel, and available at http://www.flu.lanl.gov/review/review.html. To this profile, we added sequences manually, while maintaining the alignment, to capture the variation among types and subtypes by year and host species. We aligned this augmented A/C profile to an alignment of influenza B HA sequences that was used as a profile, thus generating a multiple alignment of A, B and C sequences. Insertions in the HA sequence of B/Lee/40 were then adjusted manually to preserve secondary-structure features in the A-HA sequences. Elements of secondary structure were assigned according to the crystal structure of the HA from A/Aichi/2/68.
Confirmation of alignment of HA/HEF sequences from influenza A, B and C viruses.
For each site that was conserved in the A/B/C alignment, we counted the sequences in which that site was conserved within an alignment of sequences from each single type/subtype.
Homology modelling.
We used the homology modelling techniques of Tung (1999). Briefly, we first matched the main-chain structures of the target to those of the template in the aligned regions. Insertions in the target relative to the template were treated as loops with known end-structure. Stretching the predicted structure accommodated insertions in the template relative to the target. The main-chain structures of the loops were modelled by using an efficient Monte Carlo loop-sampling method (Tung, 1997
; Ryu et al., 1998
). Once the main-chain structure was modelled, the side-chain atoms were attached. As the head of the HA molecule is compact, limited space is available to place the side-chain atoms. Hence, in this analysis, side-chain torsional angles were initialized to equal or be close to those in the template structure. This consideration is particularly useful in avoiding clashes between side chains in the modelled structure. Finally, the all-atom models were subjected to a short run of energy minimization (1000 cycles) by using AMBER (Weiner et al., 1986
) to relieve unfavourable steric interactions and to optimize the stereochemistry.
Quality of homology model.
We used PROCHECK (Laskowski et al., 1993) to calculate the main-chain torsional angles, i.e. the Ramachandran plot (Ramachandran et al., 1963
), for our predicted structures.
Predicted model functionality.
We asked whether our model structures supported three important functionalities of the HA1 molecule: binding sialic acid on the surface of the host cell (receptor binding), evolution to escape immune pressure and attachment of carbohydrates. To examine receptor-binding potential, we used the known structure of sialic acid as it binds to the HA of A/Aichi/2/68 (PDB accession code 1HGG) and tested the fit of this sialic acid structure to the predicted B-HA receptor-binding site. To check the structural plausibility of escape mutants, we used NACCESS (Hubbard & Thornton, 1993) to calculate the proportion of the surface areas of mutating residues that is solvent-accessible in our models. The same approach was used to check the plausibility of carbohydrate binding at potential glycosylation sites.
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RESULTS |
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Sequence alignments
Our alignment of HA sequences from A/Aichi/2/68, A/swine/Hong Kong/9/98 and B/Lee/40, extracted from our A/B/C multiple alignment, is shown in Fig. 1. Twenty-one sites were conserved across these sequences and the HEF sequence of C/Johannesburg/1/66.
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Confirmation of the alignment
The majority of the 21 conserved residues in our A/B/C alignment were highly or completely conserved in single-type alignments of influenza A and B virus HA1 sequences (Table 1). Positions 83 and 269 (B/Lee/40 numbering) were interesting exceptions; variation at these positions will be discussed later in the context of model structure.
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Homology models
Fig. 2 shows the energy-minimized predicted structure of the HA1 of B/Lee/40 based on the A-H9 template, together with the crystal structures of the influenza A (H9) virus HA1 and influenza C virus HEF1 (PDB accession code 1FLC). All of the secondary-structural elements, as well as the overall shape, of the influenza A virus HA1 were preserved in the model structure of influenza B virus HA. The comparisons are essentially the same when the crystal structure and template refer to the A-H3 subtype (data not shown).
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All five conserved Cys residues in the A/B/C alignment (Fig. 1) are involved in disulphide bridges of the influenza A virus (H9) HA and influenza C virus HEF structures (Rosenthal et al., 1998
; Ha et al., 2001
), as well as those of the modelled influenza B virus HA1 structure. Four of the Cys residues are involved in disulphide bridges within the HA1 or HEF1 polypeptide chain, whereas the fifth forms a bridge with the other polypeptide chain of HA. An additional two Cys residues were conserved between the A and B sequences of this alignment (Fig. 1
). They form a disulphide bridge between Cys-80 and Cys-92 in the HA1 of A/swine/Hong Kong/9/98 (A/swine/Hong Kong/9/98 numbering) (Ha et al., 2001
) and aligned with Cys-75 and Cys-87 (B/Lee/40 HA1 numbering) to form a disulphide bridge in our structural model. A matching bridge is not observed in the crystal structure of the HEF1 of C/Johannesburg/1/66. The observed disulphide bridge between Cys-68 and Cys-293 in the HA1 of A/swine/Hong Kong/9/98 (A/swine/Hong Kong/9/98 numbering) did not have analogues in either the modelled influenza B virus HA1 structure or the crystal structure of influenza C virus HEF1. It is well-known that disulphide bridges serve the critical function of stabilizing protein folds and, hence, the predominance of conserved Cys residues in these functionally related proteins is not surprising.
The high rate of conservation of Gly and Pro residues was interesting. The small side chains of Gly and the main-chain ring structures of Pro enable these two residue types to take specialized roles in protein folding. Gly and Pro are special amino acids (Tramontano, 1998) that play important roles in the protein fold. Indeed, in both the crystal structure of A (H9)-HA1 and the modelled structure of B-HA1, three out of 11 of these conserved Gly and Pro residues are involved in forming
-turns (i.e. four consecutive residues that are not involved in a helical conformation and C
(i) to C
(i+3) distance no larger than 7 Å; Cai et al., 1999
). Three additional conserved Pro and Gly residues are involved in both the crystal structure of A-HA1 and the B-HA1 model in borderline
-turns [i.e. C
(i) to C
(i+3) distance <8 Å]. The formation of a
-turn is a crucial step in protein folding and an important motif in protein stability (Zimmerman & Scheraga, 1977
; Fasman, 1989
; Fetrow et al., 1998
).
Taken together, the prevalence of Cys, Pro and Gly residues among the conserved residues of our A/B/C alignment showed that conserved sites were typically involved in the folding stability of the HA protein.
Predicted model functionality
The three-dimensional structure of the HA of A/Aichi/2/68 complexed with the natural receptor analogue sialyllactose has been solved (Weis et al., 1988). Matrosovich et al. (1993)
inferred the receptor-binding site of B/Lee/40 from an alignment of influenza A (H1 and H3) virus and B virus HA1 proteins. Their alignment required two 2 aa insertions into the left-hand edge and a 4 aa insertion adjacent to the right-hand edge of the receptor-binding site of A/Aichi/2/68 HA. In contrast, our A/B/C alignment required only one 2 aa insertion into the left-hand edge.
To study the receptor binding of our model, we used the structure of sialyl-2-3-lactose in its complex with the HA1 of A/Aichi/2/68 (PDB accession code 1HGG) and examined the contacts that this sialic acid structure would make with our modelled influenza B virus HA1. The results are shown in Fig. 4 for the model based on the A-H9 template. Both the crystal structure of HA1 from A/Aichi/2/68 and the modelled structure of HA1 from B/Lee/40 contain a deep binding pocket (see the top panel of Fig. 4
). Whilst the shapes of these two pockets are not identical, they each show a number of points of contact with the target molecule (sialic acid) and, hence, we predict that the modelled HA1 of B/Lee/40 would bind with an affinity approximating that of the binding of A-HA1 to its target molecule. Free-energy calculations can provide an estimate of binding energy between two interacting molecules, but such calculations are beyond the scope of this work.
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The amino acid at position 269 is a signature of the sublineages Yamagata and Victoria, into which the influenza B virus HA bifurcated in 1976 (Hay et al., 2001). Residue 269 is Pro in the Yamagata sublineage, unchanged since B/Lee/40, but Ser in all viruses of the Victoria sublineage. The change from Pro to Ser is a non-conservative change, involving charge distributions (neutral to polar) and shapes of the amino acids. We asked why the amino acid at position 269 showed little variation, as a single nucleotide mutation in Pro can lead to seven different amino acids, namely Ala, Ser, Thr, Leu, His, Gln and Arg. Fig. 5
shows residue 269 together with residues in close proximity to 269 in our model. Thr is significantly larger than Pro and would interfere with Gly-198 and Glu-199 unless the local structure is disturbed. Hence, mutations from Pro to Thr or to the larger Leu, His, Gln or Arg were not favourable. In contrast, both Ser and Ala are somewhat smaller than Pro. Therefore, Pro-269-Ser and Pro-269-Gly mutations will not cause any significant alterations in the general fold of the protein. Both Pro-269-Ser and Pro-269-Ala mutations resulted in the loss of some favourable contacts, due to their smaller sizes. However, the Pro-269-Ser mutation enabled a hydrogen bond with Glu-197 to stabilize the local fold; this bond was not possible with the Pro-269-Ala mutation. Hence, our model provides simple reasons, based on considerations of contacts and hydrogen bonds, for only two amino acid types at the variable position 269.
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Positions 71 and 144 were selected for additional study as examples of sites at which changes occurred that appeared to confer a selective advantage, as the new amino acid quickly predominated in the majority of viruses of a sublineage in which the change occurred. Both of these positions had residues in close proximity (within 9 Å) and were not in a loop. Our model structure showed clear evidence of size-compensating changes (i.e. covariation) between position 71 and a neighbouring site; an Asn to Thr change (decrease in size) at position 71 is compensated by a Thr to Ile change (increase in size) at position 90, 7·8 Å away.
Position 144 interacted with the residues in the loop starting at position 177 that contained the pattern of insertions and deletions that was noted by McCullers et al. (1999). The C
C
distance between residues 144 and 177 is 5·0 Å in our model. The residue types at position 144 can be non-polar (Ala, Met, Trp), polar (Gly, Thr, Tyr), acidic (Glu) or basic (Lys, Arg). Whilst the changes in the loop involved insertions, mutations and deletions, an overwhelming 85 % of 500 sequences that are currently in the ISD had an overall neutral charge when the charge at positions 144 and the charge in the loop were summed. It seems likely that charge compensation is an important factor in the variations observed in the loop at position 177.
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DISCUSSION |
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We used the known structures of H3 and H9 subtypes of influenza A virus HA to predict, by using homology modelling, two structures of influenza B virus HA1. These homology models depend critically on our alignment of highly diverged HA1 and HEF1 sequences from influenza A, B and C viruses. We assessed the quality of our three-way alignment in two ways: statistically, by looking at conservation of aligned sites, and structurally, by observing the effects of insertions and deletions on secondary structure.
We examined our models to ensure that they supported an observed, persistent pattern of insertions and deletions and several essential functionalities of the HA molecule. Differences between the two predicted structures were insignificant for the purposes of this study, although by the criteria described here, the structure predicted from the H9 subtype of A-HA was slightly better.
Whilst the accuracy of a structure predicted by homology modelling can approach that of a medium-resolution structure determined experimentally (Martí-Renom et al., 2000) and our B/Lee/40 HA1 models are consistent with the functionality of the molecule, some of the structural details (e.g. regions involving deletions/insertions) remain to be tested experimentally. Although supported strongly for the uses described here, the predicted structures are not sufficiently accurate to study specific molecular interactions (i.e. number of hydrogen bonds, hydrophobic patches etc.). Nevertheless, our models are likely to be a valuable tool for studying many functional aspects of the molecule, based on its structure.
In influenza A virus, amino acid variation at hypervariable sites in the HA molecule is attributed largely to evolution under pressure to escape the immune system. However, analysis of varying sites in the head region of our models of influenza B virus HA1 and the crystal structures of influenza A virus HA1 suggests that excess amino acid variation in these molecules is by no means restricted to surface positions. Whilst it was largely true that buried (<20 % surface-accessible) positions in the head region of influenza B virus HA1 varied little, above this level of accessibility, there was no apparent relationship between degree of exposure and variability.
Our homology models allowed us to examine structural details at two of the more variable sites of influenza B virus HA1. By looking in the immediate neighbourhood of each of these sites, we found clear evidence of size- and charge-compensating co-variation. The case of charge compensation involved a loop in which a recurring pattern of insertions, mutations and deletions has been observed over the years. Knowledge of the compensatory changes to maintain neutral charge may lead to insight into possible functions of this region of the influenza B virus HA molecule.
The presence of co-varying sites has implications for statistical analysis of sequence data. Inferences of antigenicity based on variability alone may lead to erroneous interpretation of the antigenic sites of the influenza B virus HA. Careful examination of a crystal or model structure can help in deeper understanding of the capacity for variation in the influenza virus HA.
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ACKNOWLEDGEMENTS |
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Received 5 February 2004;
accepted 19 July 2004.