Different rates of (non-)synonymous mutations in astrovirus genes; correlation with gene function

Human astrovirus has been recognized as the second most common cause of diarrhoea among children under 5 years old [1]. In animal and bird farms, an astrovirus infection is fatal for a considerable part of the livestock [2, 3]. The family of Astroviridae is divided in two genera: Mamastrovirus (mammalian astroviruses) and Avastrovirus (avian astroviruses). The pathogen is a non-enveloped virion with a single-stranded, positive-sense RNA of approximately 6.8 kb in size [4]. The virus genome contains three open reading frames designated ORF1a (2.8 kb), ORF1b (1.6 kb) and ORF2 (2.4 kb). Translation of ORF1b depends on translation of ORF1a by a ribosomal frame-shift mechanism [5]. The primary translation products are processed into the virus protease (ORF1a) and the virus polymerase (ORF1b). ORF2 encodes a structural protein that is intracellularly processed at the C-terminal part by caspase protease, by which genome packaging and virus particle release is promoted [6]. As part of the released virus particle, ORF2 protein is processed further by trypsin to acquire the mature capsid proteins VP34 and VP27/25, a process accompanied by a considerable increase of virus infectivity [7]. Consensus on the post-translational processing routes of ORF1a, ORF1b and ORF2 polyproteins has not yet been attained [8, 9].

An inventory of evolutionary relationships among astroviruses has been published confirming the topology that is generally accepted for the astroviruses and pointing to a strong selection against non-synonymous substitutions [10]. Phylogenetic analyses based solely on the synonymous substitutions resulted in loss of tree structure due to the shortening of specific ancestral branches in trees of all ORFs. Avian, sheep, and human virus species appeared to be virtually equidistant in these trees. Such a loss of tree structure or tree compression may be interpreted either as the result of saturation of synonymous substitutions or to a peculiar pattern of synonymous substitutions that is typical for specific members of the astrovirus family. To address this issue, we analyze the astrovirus genes by means of nucleotide substitution models based on maximum likelihood approximation because these models are better suited for the estimation of mutational rates in highly divergent genes than models relying on the Jukes-Cantor correction for multiple hits at the same site.

Phylogenetic analysis by maximum likelihood (PAML3.14) [11] offers a set of sophisticated models to assess the extent of (non-) synonymous substitutions in genes. The current status of the PAML programs displays a profound documentation for the inference of sites and branches prone to molecular adaptation and is supported by validated statistics [12‐14]. For instance, adaptive evolution is observed in the hemagglutinin gene of human influenza virus type A [15].

Applying PAML to the genes of the complete astrovirus family, we show that tree compression (shortening of ancestral branches) can be ascribed to deviant rates of synonymous mutation at discrete regions of ORF2 in certain astrovirus species. Tree compression is absent in ORF1a and ORF1b as revealed by PAML due to its ability to include more evolutionary history. Sites that tend to escape from purifying selection correlate to protein domains dedicated to environmental communication rather than to replication and assembly of the virus. Finally, we propose that integrin recognition of ORF2 domains plays a key role in the process of cell binding by astrovirus.

Previous research showed that avian astrovirus species displayed different topologies in trees based on either synonymous or non-synonymous substitutions suggesting a deviant pattern of synonymous substitutions specifically in these species [10]. More specifically, tree compression due to shortening of ancestral branches caused loss of resolution among the non-human mammalian and the avian species in all ORFs leaving solely the human serotypes properly resolved. Recently, we demonstrated a switch in the recent evolution of Astroviridae driving the synonymous codon usage in genes of specifically the non-human mammalian viruses towards the mean codon usage in genes of their hosts [21]. The present study employs phylogenetic analysis by maximum likelihood approximation to assess the extent of (non-) synonymous mutational rates at the expense of tree-building capacity. Fortunately, there is consensus in literature on the phylogenetic topology of astrovirus species derived from amino acid replacements or non-synonymous nucleotide substitutions [10, 16‐21]. This provides the opportunity to decorate a tree carrying this consensus topology with the branch lengths estimated by PAML for synonymous or non-synonymous substitutions in astrovirus ORFs. By these means, we obtained standard-like trees without significant compression for astrovirus ORF1a and ORF1b, despite the large extent of synonymous substitutions in the non-human species. Apparently, substitution models subjected to maximum likelihood approximation tolerate considerably higher levels of mutational saturation than "classic" substitution models relying on the Jukes-Cantor correction for multiple hits at the same site and hence allow the inclusion of significantly more evolutionary history during phylogenetic analysis. Improvement of dN and dS estimation is the result.

With respect to astrovirus ORF2, the tree based on non-synonymous substitutions is very much standard-like, whereas the tree based on synonymous substitutions clearly suffers from compression due to the extended branch lengths of pig, sheep, mink and turkey astrovirus species. A bipartition of ORF2 into the two regions encoding the VP34 and VP27 capsid proteins shows that the branch extension of these species is observed in the VP34 domain, but confined to sheep and the turkeys in VP27. In mink and particularly pig, enhanced rates of synonymous mutation are present in the VP34 domain, but absent in the VP27 domain of ORF2. At present, we cannot offer a proper explanation for this species- and domain-specific enhancement in the rates of synonymous mutation. The consistency of dN trees with data in literature [10, 16‐20] argues against an improper alignment of the astrovirus sequences. It is conceivable that the substitution model applied reaches its limit at a certain level in the rate of mutation. However, VP34 carries the majority of elongated branch lengths, but is slightly better conserved than its ORF2 colleague VP27. The avian clade may pose a biological argument relevant to the problem. As shown above, turkey astroviruses consistently do and avian nephritis viruses do not display branch length extension indicating a possibly relevant difference between these species. Investigation at the source of the sequences involved has pointed out that the sequences of turkey species have been determined by RT-PCR of virus RNA extracted directly from stools and organs [18, 29], whereas RNAs of avian nephritis virus have been prepared from cell-culture supernatants after three consecutive rounds of plaque purification before being subjected to RT-PCR amplification [30]. It has already been mentioned that adaptation of human astroviruses to grow in continuous cell lines induces a 45-nucleotide deletion near the 3'-end of ORF1a [25]. In coronavirus, mutations have been associated with isolation and passage in primate cell lines [31, 32]. In conclusion, selection at the level of isolation and purification as well as mutation at the level of propagation may affect the difference between the rates of synonymous and non-synonymous substitution in astrovirus genes. Future research may address this issue.

The relationship between negative and positive selection carries an antagonistic character. Random mutations that may be a menace to intrinsic virus functionality are meshed during cycles of virus replication and propagation and are subsequently removed from the virus population leading to conservation of the sequences involved. Astrovirus protease, polymerase and VP34 are exemplary for this process of negative or rather purifying selection. Positive selection of substitutions on the other hand occurs in response to environmental changes and hence is also designated as molecular adaptation or adaptive molecular evolution. Obviously, sites prone to positive selection may be expected at domains in charge of communicative functionality like host range and immune response. It is therefore not surprising that sites belonging to the predicted serotypic epitope and to the putative RGD site for cell attachment in astrovirus ORF2 as well as to the two variable regions in astrovirus ORF1a display dN/dS values indicative for weak positive selection. The two clusters with dN/dS values >1 at the borders of VP25 may mark adaptive responses to maintain the structure of the virus spike protein allowing variability in the central part of VP25 that carries the serotypic epitope. The range of neutral evolution (0.3 < dN/dS < 1) is not very popular in astrovirus ORFs indicating that during evolution astrovirus has reached equilibrium between purifying selection and molecular adaptation.

Our attempts to correlate positively selected sites with virus functions also resulted in the identification of an RGD recognition motif for cellular attachment present in ORF2. In all astrovirus species (except Turkey1 isolate), an integrin recognition motif is found in ORF2. One of these (NGR) is located near the N-terminus. Bass and Qui [7] have shown that the aminoterminal 70 amino acids of the astrovirus ORF2 polyprotein can be deleted without consequences for virus assembly. Remarkably, they demonstrated this property for human astrovirus serotype 1 being the only species with all three integrin-binding motifs present in the sequence. Data on the process of cell entry by astrovirus have not been reported since 1992 [33]. Although experimental support is lacking, we tend to propose that integrin recognition plays a key role in astrovirus to target cell attachment.