The shift in the receptor-binding specificity of influenza A viruses is mostly determined by mutations in viral haemagglutinin. In this Review, Gao and colleagues discuss recent crystallographic studies that provide molecular insights into haemagglutinin–host receptor interactions that have enabled several influenza A virus subtypes to ‘jump’ from avian to human hosts.
Abstract
The recent emergence of the H7N9 avian influenza A virus and its ability to infect humans emphasize the epidemic and pandemic potential of these viruses. Interspecies transmission is the result of many factors, which ultimately lead to a change in the host tropism of the virus. One of the key factors involved is a shift in the receptor-binding specificity of the virus, which is mostly determined by mutations in the viral haemagglutinin (HA). In this Review, we discuss recent crystallographic studies that provide molecular insights into HA–host receptor interactions that have enabled several influenza A virus subtypes to 'jump' from avian to human hosts.
Key points
- Interspecies transmission of influenza A viruses is the result of many factors. One of the key factors involved is a shift in the receptor-binding specificity of the virus, which is mostly determined by mutations in viral haemagglutinin (HA).
- Recent structural studies have provided molecular insights into the HA–host receptor interactions that have enabled several influenza A virus subtypes to jump from avian to human hosts.
- The combination of distinct amino acids at positions 225 and 190 of HA is important for determining the receptor-binding specificity of the H1 subtype.
- The Q226L and G228S substitutions in the HA glycoproteins of the H2 and H3 subtypes are sufficient to change the binding preference from the avian receptor to the human receptor.
- In an experimentally adapted H5 subtype, the Q226L substitution and loss of a glycosylation site near the receptor-binding site contribute to the shift in binding preference from the avian receptor to the human receptor.
- In the H7 subtype, amino acid substitutions at positions 186 and 226 of HA increase the preference for binding to the human receptor.
Introduction
Influenza outbreaks have occurred since at least the Middle Ages, if not since ancient times1. In the past century, there were four severe influenza pandemics, in 1918 (Spanish flu), in 1957 (Asian flu), in 1968 (Hong Kong flu) and in 2009 (swine flu), as well as a moderate pandemic in 1977 (Russian flu)1. There is a high probability that we will face another influenza pandemic, but it is impossible to predict when it will happen, where it will originate, what virus subtype will cause the pandemic and the severity of such an outbreak. However, it is likely that new animal-derived influenza strains, particularly avian strains, will contribute to new pandemics2, and an increased understanding of the molecular mechanisms involved in determining influenza host tropism should facilitate such predictions in the future.
Influenza A virus is a zoonotic pathogen that can infect a broad range of species, including birds, pigs, dogs, horses, tigers and humans, causing annual epidemics (known as seasonal flu) and, at irregular intervals, pandemics (Box 1). The virus is an enveloped, single-stranded, negative-sense RNA virus with a segmented genome comprising eight gene segments3 that encode 16 proteins4, 5 (Fig. 1a), although not all viruses express all 16 proteins. Haemagglutinin (HA) and neuraminidase (NA) are the two major viral envelope glycoproteins that recognize sialic acid (SA) on host cells. HA binds to sialylated host cell receptors and mediates membrane fusion, whereas NA removes sialyl residues from the membrane of infected cells and from viral membranes to enable budding and release of newly synthesized virus particles6. In the infected host, both HA and NA are targeted by neutralizing antibodies, and based on their antigenic properties, influenza type A viruses are classified into 18 HA subtypes (H1–H16 in wild waterfowl, and H17 and H18 in bats; note that the functions of the bat HA subtypes are currently unknown) and into 11 NA subtypes (N1–N9 in wild waterfowl, and N10 and N11 in bats)6, 7, 8, 9, 10.
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| Figure 1: Structure and life cycle of influenza A viruses.
a | Influenza A viruses are enveloped, single-stranded, negative-sense RNA viruses that contain eight gene segments that encode 16 proteins (although not all influenza viruses express all 16 proteins). The non-structural segment encodes the nuclear export protein NS2 and the host antiviral response antagonist NS1; the matrix segment encodes the matrix protein M1, the ion channel protein M2 and the M2-related protein M42 (which can functionally replace M2); the haemagglutinin (HA) segment encodes the receptor-binding glycoprotein HA; and the neuraminidase (NA) segment encodes NA (which cleaves sialic acid from cell surfaces). In addition, nucleoprotein (NP) and the components of the RNA-dependent RNA polymerase complex (PB1, PB2 and PA) are expressed from their respective genome segments. The two newly identified proteins N40 (the function of which is unknown93) and PA-X94, which represses cellular gene expression, are encoded by the PB1 and PA segments, respectively. Another two forms of PA (with amino-terminal truncations) have been found recently, named PA-N155 and PA-N182, which are likely to have important functions in the replication cycle of influenza A viruses5. In addition, some viruses express the pro-apoptotic protein PB1-F2, which is encoded by a second ORF in the PB1 segment. b | Virus infection is initiated by binding of the virus to sialylated host cell-surface receptors, and entry is mediated by endocytosis. In the host cell, fusion of viral and endosomal membranes occurs at low pH, which enables the release of the segmented viral genome into the cytoplasm. The viral genome is subsequently translocated to the nucleus, where it is transcribed and replicated. Following synthesis in the cytoplasm, viral proteins are assembled into viral ribonucleoproteins (vRNPs) in the nucleus. Export of vRNPs to the cytoplasm is mediated by M1 and NS2. Virus particles are assembled at the cell membrane, and the newly generated progeny virus buds into extracellular fluid.
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To achieve interspecies transmission (known as a 'host jump'), influenza A virus must change its tropism to preferentially target new host species, and both viral and host factors have been implicated in this event11, 12. The high mutation rate of the virus enables it to evolve rapidly and thereby overcome host barriers. All eight gene segments evolve continuously, but this evolution is most pronounced for the HA and NA glycoproteins. Evolution is achieved by two main mechanisms: genetic reassortment between different subtypes (known as antigenic shift if it occurs in either the HA or NA segments) and point mutations owing to antibody-mediated immune pressure (known as antigenic drift), including substitutions, deletions and insertions within the antibody-binding sites. This results in the generation of modified influenza virus genomes, which facilitates virus evasion of the host immune response.
HA proteins exhibit specific binding affinities for the different SA-linked glycoproteins that are expressed on cell-surface receptors. Avian viruses preferentially bind to SA linked to the terminal oligosaccharide by an α2,3 bond (which is referred to as the avian receptor), whereas human strains favour the α2,6-linked SA receptor (which is referred to as the human receptor). Specific amino acid mutations in HA lead to a change in receptor-binding preference and thus to altered host specificity and tropism. In addition to the structural determinants of viral HA and their corresponding receptors, other viral determinants contribute to host-specific adaptation, such as the balance between HA receptor-binding activity and NA-mediated release from infected cells, and amino acid substitutions in viral RNA polymerase (reviewed in Refs 11,12).
In this Review, we describe recent crystallographic studies that have identified the structural determinants of viral HA that enable interspecies transmission, and we also briefly consider corresponding changes in the host receptor. First, we provide a brief overview of the host cell receptors and viral HA proteins that are involved in the initial stages of influenza A virus infection. We then discuss recent crystallographic structures of the H1, H2, H3, H5 and H7 HA subtypes in complex with both avian and human receptors, with a focus on the amino acid substitutions in the receptor-binding site of HA that enable the host jump. Please note, all amino acid residues throughout the paper are numbered according to the H3 subtype (as is convention in the field), which enables the different virus subtypes to be compared.
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| Figure 2: The haemagglutinin binding site and host receptors.
a | The crystal structure of the ectodomain of haemagglutinin (HA) reveals two distinct domains: the globular domain and the stem domain. The receptor-binding site is located on the membrane-distal globular domain and forms a shallow pocket comprising three secondary elements: the 130-loop, 190-helix and 220-loop (the numbers correspond to the amino acids in the H3 subtype; see inset). Four highly conserved residues (Y98, W153, H183 and Y195) form the base element of the receptor-binding site (indicated in orange). The fusion peptide, which inserts into the host membrane during membrane fusion, is indicated. The structural figures were created using Protein Data Bank (PDB) accession 4JUG. b,c | The HA receptor analogues can be categorized into two types: the avian receptor analogue (α2,3-linked sialylated glycan receptor) (part b) and the human receptor analogue (α2,6-linked sialylated glycan receptor) (part c). Sialylated glycans of the host receptors that influenza viruses bind to contain the three terminal saccharides: the terminal sialic acid SA1, the galactose ring Gal2 (at position two relative to SA) and N-acetylglucosamine GlcNAc3 (at position three relative to SA). When bound by HA, the α2,3-linked SA receptor adopts a trans conformation and the hydrophilic glycosidic oxygen atom faces the 220-loop in the receptor-binding site (part b), whereas the α2,6-linked SA receptor adopts a cis conformation and the hydrophobic C6 atom points towards the 220-loop (part c). The structural figures are created using the PDB accessions 4JUH and 4JUJ.
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| Figure 3: Haemagglutinin proteins from the H1, H2 and H3 subtypes in complex with the avian and human receptor analogues.
Crystal structures of H1, H2 and H3 haemagglutinin (HA)–receptor complexes have revealed the structural basis for the switch in binding specificity. a | H1 HA from the avian subtype contains the residues E190 and G225 in the receptor-binding site, and can bind to both α2,3-linked (avian; shown in cyan) and α2,6-linked (human; shown in magenta) sialic acid (SA) receptors. The structural figures were created using Protein Data Bank (PDB) accessions 1RVX and 1RVZ. b | H1 HA proteins from two human isolates from the 1918 and 2009 pandemics contain the residues D190 and D225 and specifically bind to the human receptor. The crystal structure revealed that D190 interacts with N-acetylgalactosamine GlcNAc3, whereas D225 contacts the galactose ring Gal2.These interactions are absent in the avian HA–receptor complex owing to the different configurations of the human receptor (folded) compared with the avian receptor (extended)29, 30. The structural figure was created using the PDB accession 4JTV. c | H1 HA mutants from later isolates from the 1918 and 2009 pandemics contain the residues D190 and G225 and can bind to both avian and human receptors. It is likely that G225 increases the flexibility of the 220-loop, which provides a suitable microenvironment to accommodate the extended configuration of the avian α2,3-linked SA receptor. The structural figures were created using PDB accessions 4JUH and 4JUJ. d | Avian HA proteins from the H2 and H3 subtypes contain the residues Q226 and G228, and can bind to both avian and human receptors. The structural figures were created using PDB accessions 2WR3 and 2WR4. e | HA proteins from human-adapted H2 and H3 subtypes contain the residues L226 and S228, and preferentially bind to the human receptor. L226 creates a hydrophobic environment that is favourable for the orientation of the hydrophobic C6 atom of the α2,6-linked SA receptor but is incompatible with the orientation of the hydrophilic glycosidic oxygen of the α2,3-linked SA receptor. The residue S228 forms a hydrogen bond with SA1, which increases the binding affinity of HA for the human receptor. Moreover, the avian H2 HA binds more efficiently to the human receptor than the avian H3 HA owing to hydrogen-bond interactions between N186 in H2 HA and Gal2, which are absent in H3 HA owing to the short side chain of S186. The structural figure was created using PDB accession 2WR7.
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| Figure 4: Haemagglutinin proteins from the H5 and H7 subtypes in complex with the avian and human receptor analogues.
Crystal structures of H5 and H7 haemagglutinin (HA) proteins in complex with avian and human receptor analogues. a | Wild-type H5 HA contains the residue Q226 and is glycosylated at residue N158. It has the capacity to bind preferentially to the avian receptor (α2,3-linked sialic acid (SA) receptor; shown in cyan) in a favourable trans conformation, but it binds weakly to the human receptor (α2,6-linked SA receptor; shown in magenta) in an unfavourable trans conformation. The hydrophilic residue Q226 provides a hydrophilic environment, which is compatible with the hydrophilic glycosidic oxygen atom of the avian receptor and incompatible with the hydrophobic C6 atom of the human receptor. Thus, the H5 HA preferentially binds the avian receptor. The structural figures were created using the Protein Data Bank (PDB) accessions 4K63 and 4K64. b | The ferret-transmissible mutant H5 HA (H5mut) has the residue L226 and residue 158 is deglycosylated. It binds preferentially to human receptor in a favourable cis conformation, whereas it binds weakly to the avian receptor in an unfavourable cis conformation. The hydrophobic residue L226 creates a hydrophobic environment, which is compatible with the hydrophobic C6 atom and incompatible with the hydrophilic glycosidic oxygen atom. Thus, H5mut preferentially binds to the human receptor. The structural figures were created using PDB accessions 4K66 and 4K67. c,d | The crystal structures of Anhui-H7N9 HA (AHH7, carrying the four amino acid substitutions S138A, G186V, T221P and Q226L) and an Anhui-H7N9 HA mutant (AHH7mut), in which L226 has been mutated to Q226, in complex with the avian or human receptor analogues provide insights into the structural basis of the shift in receptor binding. AHH7 and AHH7mut bind to both avian and human receptor analogues. The avian receptor analogues bind in different conformations to AHH7 (cis) and the AHH7mut carrying Q226 (trans), whereas the human receptor analogue binds to both proteins in a cis conformation. The four amino acid substitutions in AHH7 create a more hydrophobic environment that is favourable for binding to the human receptor. Furthermore, mutagenesis assays showed that the amino acid substitution Q226L is not solely responsible for the shift in receptor-binding preference of AHH7, and other substitutions also have a role. The structural figures were created using the PDB accessions 4KOM, 4KON, 4LKJ and 4LKK.
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Summary and conclusions
The determinants that contribute to the host jump of avian influenza A viruses are complex and involve several viral and host factors. Recent crystallographic studies have provided molecular insights into the shift in receptor binding caused by mutations in the viral envelope protein HA, which is a major (but not the only) determinant of the host switch. To efficiently switch, viruses must acquire a preference for the human receptor, or at least the ability to bind weakly to the human receptor in addition to the avian receptor to successfully infect and replicate in human epithelial cells in the URT, which predominantly express the α2,6-linked SA receptor. It is likely that decreased binding to avian receptors is required for human-to-human transmission. This is because the human URT is covered with secreted mucin molecules that contain the α2,3-linked SA receptor, so viruses could get trapped in the URT as these mucin molecules (containing attached virus) are tightly bound to the respiratory epithelium and are unlikely to be transmitted in droplets generated by coughing or sneezing91.
Amino acid substitutions in HA have been identified as major determinants for preferential targeting of human, rather than avian, receptors. The H1, H2 and H3 subtypes of influenza A viruses have naturally adapted to humans, causing worldwide pandemics and epidemics. In the H1 subtype, the amino acids at positions 190 and 225 in the receptor-binding sites seem to be important for the shift in receptor-binding specificity and, in addition, different combinations of mutations result in altered receptor-binding specificity: H1 HA proteins that contain E190/G225, E190/D225 or D190/G225 in their receptor-binding site have dual receptor-binding specificity, whereas those that contain the D190/D225 and D190/E225 substitutions specifically bind to the human receptor. The Q226L and G228S substitutions in the HA glycoproteins of H2 and H3 subtypes are sufficient to change the receptor-binding preference from the avian receptor to the human receptor. Moreover, experimental adaptation of the H5 subtype showed that the Q226L substitution and loss of a glycosylation site near the receptor-binding site contribute to the shift in receptor-binding preference from avian to human. Finally, in the H7 subtype, amino acid substitutions at positions 186 and 226 increase binding to the human receptor; however, H7 HA still preferentially binds to the avian receptor, and the amino acid substitutions that are responsible for the shift in receptor-binding specificity remain to be determined.
Although the molecular bases of the receptor-binding preference shifts for H1, H2, H3 and H5 HA proteins have been established, the mechanism for other HA subtypes remains unknown, particularly for H7 and H9. In the future, more efforts are needed to elucidate the molecular basis of the host jump of the other HA subtypes, which should aid the rapid identification of newly emerging epidemic and pandemic strains. Furthermore, owing to technical limitations, we can currently only solve the structure of HA in complex with simple receptor analogues with pentasaccharide or trisaccharide. However, the sialylated glycan receptors in different hosts and tissues are far more complex, with different oligosaccharide chains in the receptor, and the molecular interaction between HA and complex sialylated glycan receptors should be studied in the future.
In conclusion, as single amino acid changes seem to be sufficient to alter receptor-binding preference, and as natural selection is unpredictable, extensive surveillance of influenza viruses will be crucial for the prevention and control of future pandemics.
Nature Reviews Microbiology | Review
Nature Reviews Microbiology (20140 Volume: 12, Pages: 822–831
DOI: doi:10.1038/nrmicro3362
Published online
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