Background
Genome variation is very high in influenza A viruses. However, viral evolution and spreading is strongly influenced by immunogenic features and capacity to bind host cells, depending in turn on the two major capsidic proteins. Therefore, such viruses are classified based on haemagglutinin and neuraminidase types, e.g. H5N1. Current analyses of viral evolution are based on serological and primary sequence comparison; however, comparative structural analysis of capsidic proteins can provide functional insights on surface regions possibly crucial to antigenicity and cell binding.
Results
We performed extensive structural comparison of influenza virus haemagglutinins and of their domains and subregions to investigate type- and/or domain-specific variation. We found that structural closeness and primary sequence similarity are not always tightly related; moreover, type-specific features could be inferred when comparing surface properties of haemagglutinin subregions, monomers and trimers, in terms of electrostatics and hydropathy. Focusing on H5N1, we found that variation at the receptor binding domain surface intriguingly relates to branching of still circulating clades from those ones that are no longer circulating.
Conclusions
Evidence from this work suggests that integrating phylogenetic and serological analyses by extensive structural comparison can help in understanding the ‘functional evolution’ of viral surface determinants. In particular, variation in electrostatic and hydropathy patches can provide molecular evolution markers: intriguing surface charge redistribution characterizing the haemagglutinin receptor binding domains from circulating H5N1 clades 2 and 7 might have contributed to antigenic escape hence to their evolutionary success and spreading.
IMPORTANT
Variation of some protein properties sometimes may depend only on local and limited hanges, e.g. mutation of a few - or even only one - residue(s) within linear or conformational motifs. In fact, even when local variation in sequence is seemingly poorly evident, it may result in locally dramatic changes in accessible surface area, electrostatic potential, hydropathy or hydrophilicity features that can deeply change motif functionality. It is common knowledge that variation in surface features of a protein can modulate recognition interactions of the protein itself. Since variation often depends on mutation of a number of residues and changes in side chains can vary multiple biochemical features, it is difficult or even nonsense trying to establish a priori which specific property (among e.g. surface area and shape, electrostatics or hydrophobicity) should be more relevant than others in modulating recognition interactions. In fact, changes in each specific property can result in such modulation, and this can be independent on variation of other features, or modulation can result from the aggregate or synergistic effect of multiple feature changes. So far, several sequence-based studies on variation could provide valuable phylogenetic evidence; however, such studies are of minor help in inferring variation at protein regions including amino acids that are far each other in the primary sequence and quite close within the 3D protein structure (conformational epitopes). In practice, while sequence-based investigation can be good in highlighting very evident changes at individual positions of a protein chain, in general they fail in highlighting meaningful `group variation’, i.e. in identifying - especially when the overall variation is relevant and spread - relationship of specific multiple changes to variation in conformational epitopes hence in interactions they mediate.
The two track Rule
Stressing relevance of local surface variation is particularly important when considering special constraints addressing viruses evolution: keeping basic properties in simplified but complex pathogenic systems while simultaneously varying - as much as possible - all variable epitopes, in order to escape the immune responses of their hosts. Therefore, viral genome evolution runs along two parallel tracks, both of which, like in railways, must be followed: (i) mutations in sites crucial to protein machinery mediating basic functions (e.g. in motifs relevant to host recognition or cell entrance) are not allowed because they strongly impair viral fitness, and at the same time, (ii) hyper-variability is needed to escape recognition by neutralizing antibodies (`antigenic drift’, [7]). Given that surface viral proteins do not interact only with antibodies (as their original function is to contact the host), in addition to determining antigenic drift, variation can also influence pathogenicity (because e.g. of modified interaction with cell receptors in different tissues and organ districts) or host specificity. Influenza viruses do not escape such a two-tracks rule, hence while global structure conservation ensures basic functions, limited or even subtle changes in local structural features may modulate interactions of the viral proteins with the host molecules/cells and thus mechanisms underlying antigenic drift, pathogenicity shifts and host specificity change.
sequence based studies
Thanks to the availability of thousands of viral genomes/gene sequences and of several specific antibodies/vaccines, a large number of sequence-based/phylogenetic and serological analyses of avian flu viruses have been performed and published so far. This notwithstanding, mechanisms in viral evolution are still elusive, as genome/proteome-wide analyses on sequence variation or antigenic features are able to only partially unveil a number of relevant changes, because of the overall mutational noise. Therefore, structural zoom in is needed to integrate such analyses by identifying `meaningful’ variation. This prompted us to take advantage from availability of structural templates to perform structural comparison among different HA subtypes, in order to identify subtype- and subregion-specific feature variation suggestive for possible involvement in antigenic recognition, or pathogenicity and host specificity. Last but not least, evidence from structural comparison can check relationship among serological, phylogenetic and structural closeness.
comparison among solved HA structures: prior work
Preliminary analysis of the six available HA structures was performed in order to evaluate intra- and inter-group structural variation by superposition of all structure pairs and computation of their Root Mean Square Deviation (RMSD). Indeed, the RMSD of two superposed structures indicates their `structural divergence’ from one another. As both sequence mutation and conformational variation inflate the RMSD, values up to 2 Ångstrom indicate structural similarity [17].
%17
@article{carugo2001normalized,
title={A normalized root-mean-spuare distance for comparing protein three-dimensional structures},
author={Carugo, Oliviero and Pongor, S{\'a}ndor},
journal={Protein science},
volume={10},
number={7},
pages={1470--1473},
year={2001},
publisher={Wiley Online Library}
}