Bioinformatics Advance Access originally published online on November 7, 2006
Bioinformatics 2007 23(3):272-276; doi:10.1093/bioinformatics/btl559
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An RNA conformational shift in recent H5N1 influenza A viruses
Leiden Institute of Biology, Leiden University P.O.Box 9516, 2300 RA Leiden
1 Department of Biophysical Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen P.O.Box 9010, 6500 GL Nijmegen
2 Leiden Institute of Chemistry, Leiden University P.O.Box 9502, 2300 RA Leiden, The Netherlands
*To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTS AND DISCUSSIONREFERENCES |
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Recent outbreaks of avian influenza are being caused by unusually virulent H5N1 strains. It is unknown what makes these recent H5N1 strains more aggressive than previously circulating strains. Here, we have compared more than 3000 RNA sequences of segment 8 of type A influenza viruses and found a unique single nucleotide substitution typically associated with recent H5N1 strains. By phylogenetic analysis, biochemical and biophysical experiments, we demonstrate that this substitution dramatically affects the equilibrium between a hairpin and a pseudoknot conformation near the 3' splice-site of the NS gene. This conformational shift may have consequences for splicing regulation of segment 8 mRNA. Our data suggest that besides changes at the protein level, changes in RNA secondary structure should be seriously considered when attempting to explain influenza virus evolution.
Contact: olsthoor{at}chem.leidenuniv.nl
Supplementary information: Supplementary data are available at Bioinformatics online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONREFERENCES |
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Since the first documented transmission of influenza virus from birds to humans in 1997 Hong Kong outbreak, H5N1 strains of avian influenza A are the focus of the studies with the major goal to identify the molecular determinants of their virulence and host adaptation (for recent reviews, see Noah and Krug, 2005; Horimoto and Kawaoka, 2005). These studies show that the pathogenicity of influenza viruses is multifactorial and depends on various virus-encoded proteins. In addition to surface glycoproteins hemagglutinin and neuraminidase that determine recognition of host cell receptors and are the main targets of host immune response, other proteins have been shown to contribute to the virulence of highly pathogenic H5N1 strains. For instance, specific mutations in polymerase subunit PB2 protein (Hatta et al., 2001) and non-structural protein NS1 (Seo et al., 2002; Obenauer et al., 2006) were identified as potential virulence determinants of H5N1 viruses. However, some highly pathogenic H5N1 strains do not have these mutations, again emphasizing complex mechanisms of influenza virulence (Salomon et al., 2006; Krug, 2006).
In contrast to multiple studies with comparative analysis of proteins from various influenza strains, higher-order structure of influenza RNA remains mostly uninvestigated. On the other hand, RNA structure plays an important role in the life cycle of RNA viruses. Many functional viral RNA structures are known and evolution of virus RNA genomes is subject to various structural constraints (e.g. Simmonds et al., 2004). In the influenza virus genome, consisting of eight separate negative-sense RNAs (segments), highly conserved structures, located at both the 5' and 3' ends of each segment, have been shown to be important for RNA replication and packaging (Hsu et al., 1987; Fodor et al., 1994). However, nothing is known about the folding of other regions of influenza genomic RNAs or complementary positive-sense cRNAs and mRNAs. Here we describe the analysis of a structure in the coding region of segment 8 mRNA that is conserved in both influenza A and B viruses. This segment, usually consisting of 890 and 1096 nt in A and B viruses, respectively, encodes two proteins: NS1, synthesized on unspliced mRNA, and NEP (formerly called NS2), produced from spliced transcripts (Lamb and Horvath, 1991). We proposed that there exists equilibrium between two alternative structures in this mRNA that has significantly shifted in recent H5N1 strains.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONREFERENCES |
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We identified several conserved hairpin structures within the plus or coding strand of influenza A segment 8 (data not shown). Interestingly, despite rather low sequence similarity between influenza A and B segments, two mutually exclusive structures near the 3' splice-site (3'-ss) turned out to be remarkably conserved in both types. One of the structures is a hairpin with some mismatches at the top and the bottom and a conserved sequence (5'-GAGGAU-3'/5'-A(G)UCCUC-3') in the middle of the stem (Figure 1A). The existence of this hairpin is supported by nucleotide covariations at the bottom of the stem. A BLAST analysis of all available sequences (3017 influenza A and 162 influenza B strains at the moment of writing) showed that, although a minor fraction of influenza A isolates may have additional mismatches, the hairpin is thermodynamically stable in all viruses and supported by nucleotide covariations. The hairpin appears to be remarkably stabilized in recent H5N1 strains: whereas in the majority of the viruses the stem contains one or more mismatches, in recent H5N1 strains, for instance A/Vietnam/1194/04 (VT04), a perfect duplex of 16 bp is formed (Figure 1A).
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An alternative structure, consisting of a pseudoknot, can be formed by refolding of nucleotides at the top of the hairpin and pairing them with an upstream sequence (Figure 1B). Formation of this pseudoknot is also supported by covariations between A and B type influenza viruses in the pseudoknotted stem. Furthermore, a covariation between two main influenza A segment 8 clades (sometimes called ‘alleles’) A and B (Kawaoka et al., 1998; Basler et al., 2001) at the pseudoknot stem junction, with putative non-canonical base pair G524–A563 in clade B structure, lends further support to the pseudoknot folding (Figure 1B).
The structure of this pseudoknot is unusual. In principle, it corresponds to one of the three stem stacking topologies proposed for the so-called H-type (hairpin) pseudoknots (Westhof and Jaeger, 1992; Mans and Pleij, 1993). In contrast to the widespread classical H-type pseudoknot topology (Figure 1, inset), the inverted stacking configuration of the proposed pseudoknot we find is very rare and has been observed only in the structural context of long-range tertiary interactions in various ribozymes (Jaeger et al., 1991; Michel et al., 1989; Bergman et al., 2004; Soukup, 2006). However, in these cases the loop spanning both stems contains one or more structured domains. To our knowledge, the influenza ‘inverted’ pseudoknot is the first example with two relatively short single-stranded loops.
The suggested pseudoknots in influenza A and B viruses are remarkably similar: identical lower stems, similar sizes of upper stems and of both loops (Figure 1). Determined by the polarity in RNA helix, the loop spanning the upper stem has to cross its deep groove (Westhof and Jaeger, 1992; Mans and Pleij, 1993). Interestingly, in both influenza A and B viruses this loop is significantly longer (12–13 nt) than the typical length 1–3 nt of its topological homologues in long-range pseudoknots (Jaeger et al., 1991; Michel et al., 1989). In both A and B viruses this loop is A-rich. Another loop, spanning both stems in the proposed influenza pseudoknots, does not have, to our knowledge, structural analogues, because comparably oriented loops in long-range pseudoknots are much larger and are folded themselves into separate domains (Jaeger et al., 1991; Michel et al., 1989; Bergman et al., 2004; Soukup, 2006). The 3D-modeling of pseudoknot topologies shows that such loops do not cross any groove (Jaeger et al., 1991). In both influenza A and B pseudoknots (Figure 1B), this loop of 11 nt has to span a putative quasicontinuous helix of two stems comprising 11–12 bp, i.e. approximately one turn of A-form helix. Thus, this loop may be located at one side of RNA duplex. Additional base pairs can be formed at the end of the lower stem, but in the absence of a 3D-model for this type of pseudoknot it is difficult to estimate the allowed size of the loop spanning a larger helix.
Free energy estimates (Figure 1D) suggest a significant stabilization of the hairpin and destabilization of the pseudoknot in recent H5N1 strains, whereas in other viruses the alternative conformers have comparable stabilities. It should be noted, however, that such estimates do not take into account possible adiitional secondary and tertiary structure interactions involving the pseudoknot loops.
To investigate possible structural differences between recent H5N1 and other strains, we synthesized transcripts of segment 8 covering the region around the 3'-ss (see Materials and Methods in Supplementary material). These 95 nt transcripts differ only by the nucleotide changes shown in Figure 1B. Native gel-electrophoresis of these RNAs showed the existence of two species of RNA for one of the older H5N1 strains, A/duck/Shanghai/13/01 (DKSH01) and predominantly only one species for H1N1 (A/PR/8/34) and recent H5N1 (VT04) transcripts (Figure 1C). Occasionally, we observed only the slower migrating band for the DKSH01 transcript suggesting a relatively slow exchange between the two conformations (data not shown). The faster migrating RNA of VT04 was confirmed by enzymatic probing to consist of the hairpin conformation whereas the slower migrating RNA of A/PR/8/34 was more in agreement with a pseudoknot conformation (Supplementary Figure 1S). Enzymatic probing of the DKSH01 transcript showed features of both pseudoknot and hairpin structures (Supplementary Figure 1S).
To further investigate the equilibrium between the two conformations, we designed a set of variants based on the DKSH01 sequence in which we introduced base changes that were expected to either stabilize the pseudoknot or the hairpin conformation. Native gel-electrophoresis of these variants clearly showed the appearance of the hairpin and the concomitant disappearance of the pseudoknot conformation as a result of the introduced base changes (Supplementary Figure 2S). Structure probing supported the proposed conformations for these variants (Supplementary Figure 3S).
The existence of the hairpin in VT04 RNA was also confirmed by 2D-NOESY NMR experiments with a synthetic RNA corresponding to 523–571 nt of VT04 mRNA. In this 49 nt RNA the hairpin can form at most 13 bp. In the 2D spectrum all imino resonances could be assigned (Supplementary Figure 4S). Substituting C563 in this RNA by a G yields the ‘minimal’ pseudoknot construct depicted in Figure 2. The NMR spectrum of this RNA was more complex, possibly due to the presence of alternative conformations, i.e. a mixture of the hairpin and pseudoknot conformation (Supplementary Figure 5S). The observation of sequential NOEs between the two U•G base pairs at the junction, however, is consistent with stacking of the upper and lower stem as would be expected for the pseudoknot (Figure 2).
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The above data are consistent with equilibrium between a pseudoknot and a hairpin, which is strongly shifted to the hairpin conformation by the G563
C substitution in recent H5N1 viruses. The earliest strain with C563 in the database is A/duck/Guangxi/50/2001 (Dk/Gx/01, accession no. AY585453
[GenBank]
), isolated in 2001 (Figure 3). Since 2002, this mutation is present in a number of strains, belonging to various clades A genotypes, which acquired their NS genes from a common ancestor having a 15 nt deletion (positions 264–278). In particular, C563 is abundant in strains of genotype Z that has become dominant in South East Asia since 2002, causing severe outbreaks in poultry and infecting a number of humans (Li et al., 2004; Chen et al., 2006; Smith et al., 2006). The mutation is found exclusively in H5N1 strains, and only after 2001. Interestingly, apart from the 335 H5N1 strains with C at position 563, there are only few strains with other mutations that destabilize the base pair 524/563 at the junction of the suggested pseudoknot (Figure 1B). While disruption of Watson–Crick pairing was never observed in other clade A sequences (2268 U-G, 45 U-A and 2 C-G combinations), only 20 out of 367 clade B sequences could not form the suggested non-canonical pair G-A. The 17 out of these 20 sequences, with A.A or A.G combinations, are found in segments 8 from the H5N1 strain A/Goose/Guandong/1/96 (Gs/Gd/96, accession no. AF144307
[GenBank]
) and related viruses, considered to be predecessors of the viruses that caused the 1997 Hong Kong outbreak (Xu et al., 1999). The recent H5N1 strains have inherited their hemagglutinin genes from Gs/Gd/96-like strains as well (Guan et al., 2002; Li et al., 2004), so a tendency to evolve a pseudoknot-hairpin conformational shift in both independent clades of NS genes looks like a unique property of the current H5N1 lineage. Intriguingly, both these shifts have occurred about a year before major outbreaks of H5N1 influenza with infections of humans (Figure 3).
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The G563
C substitution is remarkably stable in current H5N1 genotype Z viruses, although it is silent at the level of NS1 protein and leads to a substitution at a non-conserved position in the NEP protein (Ludwig et al., 1991). The segments 8 of all strains isolated from wild birds and poultry outside of China and South East Asia in very distant geographic regions, such as Mongolia, Russia, Italy and Nigeria contain the G563C substitution. The same is true for all sequences of strains isolated from humans after 2003. Bearing in mind the location of the alternative structures, it is very likely that the equilibrium between them is implicated in the regulation of splicing of NS mRNA. However, other mechanisms or functions of the proposed alternative RNA structures of segment 8 of influenza A and B viruses cannot be excluded. For instance, they may be involved in modulating the activity of certain host antiviral factors, such as PKR kinase, which is antagonized by NS1 (Krug et al., 2003) and can be regulated by complex pseudoknotted structures (Ma and Mathews, 1996; Ben-Asouli et al., 2002).
Whatever the function of these structures, our results suggest that the equilibrium between them is shifted in recent H5N1 strains. Although the virulence of influenza strains is mainly determined by properties of viral proteins, the changes of virus fitness caused by RNA structure shifts can also modulate virus infectivity. Similar to a punctuated character of antigenic evolution of influenza (Smith et al., 2004), the evolution of the virus RNA structure may be punctuated as well, with some drastic conformational changes caused by just a single mutation.
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Acknowledgments |
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The authors thank C. Pleij, P. Haccou, J. van Duin and R. Fouchier for useful comments on the manuscript. The authors are grateful to C. Pleij for stimulating and continuing interest, R. Fouchier for fruitful discussions and cDNA clones, O. Reshetnikova for assistance in the analysis of early structure predictions, C. Erkelens and F. Lefeber for initial NMR measurements. This research was supported by The Netherlands Organization for Scientific Research (NWO) and by the Beijerinck Premium of the Beijerinck Virology Fund and VIDI grant awarded to R.C.L.O. Funding to pay the Open Access publication charges was provided by the M.W. Beijerinck Virology Fund, Royal Netherlands Academy of Arts and Sciences.
Conflict of Interest: none declared.
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FOOTNOTES |
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Associate Editor: Martin Bishop
Received on August 19, 2006; revised on October 11, 2006; accepted on October 31, 2006
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