Introduction
Influenza A virus (IAV) is an enveloped, segmented, negative-sense RNA virus that poses a major threat to human and animal health (Wille and Holmes, Reference Wille and Holmes2020). Its evolution and transmission are shaped in part by the functional interplay between its two principal surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) (Le Sage et al., Reference Le Sage, Rockey, French, McBride, McCarthy, Rigatti, Shephard, Jones, Walter, Doyle, Xu, Barbeau, Wang, Frizzell, Myerburg, Paulson, McElroy, Anderson, Vincent Baker and Lakdawala2024; Myers et al., Reference Myers, Wetzel, Linderman, Li, Sullivan and Hensley2013; Xu et al., Reference Xu, Zhu, McBride, Nycholat, Yu, Paulson and Wilson2012). HA recognizes and binds sialic acid (SIA)-containing receptors, whereas NA cleaves terminal SIA residues to facilitate virion movement through mucus and across cell surfaces and to promote the release of progeny virions. Although HA and NA have opposing receptor-binding and receptor-cleaving activities, their functions are interdependent, and their relative activities must remain compatible with the receptor landscape of a given host environment (Guo et al., Reference Guo, Rabouw, Slomp, Dai, van der Vegt, van Lent, McBride, Paulson, de Groot, van Kuppeveld, De Vries and de Haan2018; Huang et al., Reference Huang, Kim, Song, Grigorieff, Munro, Schiffer and Somasundaran2025; Vahey and Fletcher, Reference Vahey and Fletcher2019; Wallace et al., Reference Wallace, Liu, van Kuppeveld, de Vries and de Haan2021). Increasing evidence indicates that HA/NA functional coordination contributes not only to viral attachment, release, and transmission but also to cross-species adaptation, pathogenicity, immune escape, and antiviral resistance (Le Sage et al., Reference Le Sage, Rockey, French, McBride, McCarthy, Rigatti, Shephard, Jones, Walter, Doyle, Xu, Barbeau, Wang, Frizzell, Myerburg, Paulson, McElroy, Anderson, Vincent Baker and Lakdawala2024; Myers et al., Reference Myers, Wetzel, Linderman, Li, Sullivan and Hensley2013; Xu et al., Reference Xu, Zhu, McBride, Nycholat, Yu, Paulson and Wilson2012). This coordination is not static; rather, it can be reshaped by changes in the functional properties of either glycoprotein. For example, changes in HA receptor-binding avidity may be accompanied by corresponding changes in NA activity, whereas impaired NA function may be partly compensated for by adaptive changes in HA (Du et al., Reference Du, Wolfert, Peeters, van Kuppeveld, Boons, De Vries and de Haan2020; Lai et al., Reference Lai, Karunarathna, Wong, Peiris and Nicholls2019; Liu et al., Reference Liu, Wang, Tan, Wu and Brooke2022). The functions of HA and NA therefore cannot be fully understood in isolation.
Studies of HA/NA functional balance have elucidated the molecular basis of this interdependence at multiple stages of the IAV life cycle. As reviewed by de Vries et al. (Reference de Vries, Du, Guo and de Haan2020), Du et al. (Reference Du, Cui and Rong2019a), and Gaymard et al. (Reference Gaymard, Le Briand, Frobert, Lina and Escuret2016), studies of HA/NA functional balance have elucidated the molecular basis of this interdependence at multiple stages of the IAV life cycle and substantially advanced our understanding of its adaptive significance. However, previous reviews have generally emphasized individual biological processes or specific adaptive contexts and have not fully integrated how HA/NA functional balance is reshaped by distinct selective pressures or how these adaptive processes are interconnected. A functional change in either HA or NA can disrupt a pre-existing functional match and alter the selective constraints acting on its counterpart, thereby favouring subsequent adaptive or compensatory changes (Diederich et al., Reference Diederich, Berhane, Embury-hyatt, Hisanaga, Handel, Cottam-birt, Ranadheera, Kobasa and Pasick2015; Hensley et al., Reference Hensley, Das, Gibbs, Bailey, Schmidt, Bennink and Yewdell2011; Myers et al., Reference Myers, Wetzel, Linderman, Li, Sullivan and Hensley2013). HA/NA functional balance is therefore better viewed as a dynamic relationship that is continually adjusted in response to changes in the host environment and selective pressures. Selective forces may disrupt an established balance, whereas coordinated or compensatory changes may restore functional compatibility and establish a new equilibrium. This dynamic cycle of ‘balance–imbalance–rebalance’ may influence the adaptation of IAV to different hosts and environmental conditions.
Against this background, this review focuses on the dynamic reshaping of HA/NA functional balance and systematically examines its roles in viral infection and transmission, cross-species adaptation, pathogenicity, immune escape, and antiviral resistance. Defining how functional constraints and compensatory mechanisms interact to reshape this balance is important for understanding the evolutionary trajectories and adaptive potential of IAV.
The structure and function of HA
HA is the most abundant glycoprotein on the IAV surface and is anchored in the viral envelope as a homotrimer. Structurally, HA comprises a globular head and a membrane-proximal stalk (Harris et al., Reference Harris, Cardone, Winkler, Heymann, Brecher, White and Steven2006). The head contains the receptor-binding site (RBS), a shallow pocket formed primarily by the 130-loop, 150-loop, 190-helix, and 220-loop. The RBS recognizes SIA-containing glycans, thereby mediating initial viral attachment and contributing to host-cell entry (Huang et al., Reference Huang, Kim, Song, Grigorieff, Munro, Schiffer and Somasundaran2025; Weis et al., Reference Weis, Brown, Cusack, Paulson, Skehel and Wiley1988). Several hypervariable antigenic sites surround the RBS and frequently acquire substitutions under immune pressure. These regions are major targets of neutralizing antibodies and are important molecular determinants of antigenic drift and immune escape during IAV evolution (Hensley et al., Reference Hensley, Das, Bailey, Schmidt, Hickman, Jayaraman, Viswanathan, Raman, Sasisekharan, Bennink and Yewdell2009; Maurer et al., Reference Maurer, Vu and Schmidt2025).
Eighteen established HA subtypes (H1–H18) and the recently proposed H19 subtype exhibit marked differences in receptor usage and binding specificity. With the exception of the bat-derived H17 and H18 subtypes and the recently reported H19 subtype, which use noncanonical entry pathways rather than SIA receptors, other HA subtypes typically mediate host-cell attachment by recognizing sialylated glycans (Benton et al., Reference Benton, Gamblin, Rosenthal and Skehel2020; Fereidouni et al., Reference Fereidouni, Starick, Karamendin, Genova, Scott, Khan, Harder and Kydyrmanov2023; Karakus et al., Reference Karakus, Mena, Kottur, El Zahed, Seoane, Yildiz, Chen, Plancarte, Lindsay, Halpin, Stockwell, Wentworth, Boons, Krammer, Stertz, Boyce, De Vries, Aggarwal and García-sastre2024; Weis et al., Reference Weis, Brown, Cusack, Paulson, Skehel and Wiley1988). In general, avian influenza viruses (AIVs) preferentially bind glycans containing α2,3-linked SIA (α2,3-SIA), whereas human-adapted IAVs preferentially bind glycans containing α2,6-linked SIA (α2,6-SIA). These receptor types differ in their abundance and spatial distribution across host species and tissues; notably, α2,6-SIA predominates in the human upper respiratory tract, whereas α2,3-SIA is more abundant in the human lower respiratory tract and at many sites in avian respiratory and intestinal tissues. This mismatch between host receptor distribution and viral HA-binding preference constitutes an important barrier to cross-species transmission (de Graaf and Fouchier, Reference de Graaf and Fouchier2014; Long et al., Reference Long, Mistry, Haslam and Barclay2019). Substitutions at key residues within or adjacent to the HA RBS can alter receptor-binding specificity or avidity. Examples include E190D/G225D in H1 subtypes (Glaser et al., Reference Glaser, Stevens, Zamarin, Wilson, Garcia-Sastre, Tumpey, Basler, Taubenberger and Palese2005), Q226L/G228S in H2 and H3 subtypes (Connor et al., Reference Connor, Kawaoka, Webster and Paulson1994), G225D in H6 subtypes (de Vries et al., Reference de Vries, Peng, Grant, Thompson, Zhu, Bouwman, de la Pena, Van Breemen, Ambepitiya Wickramasinghe, de Haan, Yu, McBride, Sanders, Woods, Verheije, Wilson and Paulson2017), V186G/K193T/G228S, V186K/K193T/G228S, or V186N/N224K/G228S in H7 subtypes (De Vries et al., Reference de Vries, Peng, Grant, Thompson, Zhu, Bouwman, de la Pena, Van Breemen, Ambepitiya Wickramasinghe, de Haan, Yu, McBride, Sanders, Woods, Verheije, Wilson and Paulson2017), Q226L in H9 subtypes (Wan and Perez, Reference Wan and Perez2007), and K158A/D193T/Q226L/G228S in H10 subtypes (Tzarum et al., Reference Tzarum, De Vries, Peng, Thompson, Bouwman, McBride, Yu, Zhu, Verheije, Paulson and Wilson2017). Additional substitutions at positions 145, 186, 189, 219, and 227 in H1 (Chutinimitkul et al., Reference Chutinimitkul, Herfst, Steel, Lowen, Ye, van Riel, Schrauwen, Bestebroer, Koel, Burke, Sutherland-cash, Whittleston, Russell, Wales, Smith, Jonges, Meijer, Koopmans, Rimmelzwaan, Kuiken, Osterhaus, García-Sastre, Perez and Fouchier2010; Maines et al., Reference Maines, Jayaraman, Belser, Wadford, Pappas, Zeng, Gustin, Pearce, Viswanathan, Shriver, Raman, Cox, Sasisekharan, Katz and Tumpey2009) and positions 186, 193, 194, 195, 198, 219, 223, and 225 in H3 (Stevens et al., Reference Stevens, Chen, Carney, Garten, Foust, Le, Pokorny, Manojkumar, Silverman, Devis, Rhea, Xu, Bucher, Paulson, Cox, Klimov and Donis2010) can further modulate receptor-binding avidity and specificity by altering RBS conformation. Such changes may facilitate adaptation to new hosts, although receptor switching alone is generally insufficient to confer efficient transmission. After establishment in humans, HA continues to evolve under immune pressure, accumulating substitutions within or adjacent to the RBS that must balance antigenic escape against the preservation of receptor-binding function. This continuing process contributes to the sustained circulation and recurrent epidemics of human IAVs.
The structure and function of NA
NA is a homotetrameric glycoprotein anchored in the IAV envelope and occurs as isolated or locally clustered spikes. Its protrusion from the virion surface is determined largely by the length of the stalk region (Harris et al., Reference Harris, Cardone, Winkler, Heymann, Brecher, White and Steven2006). Eleven NA subtypes have been reported. Each NA monomer comprises four structural regions: a cytoplasmic tail, a transmembrane domain, a stalk, and a catalytic head (Tong et al., Reference Tong, Zhu, Li, Shi, Zhang, Bourgeois, Yang, Chen, Recuenco, Gomez, Chen, Johnson, Tao, Dreyfus, Yu, McBride, Carney, Gilbert, Chang, Guo, Davis, Paulson, Stevens, Rupprecht, Holmes, Wilson and Donis2013). The catalytic head adopts a six-bladed β-propeller structure, with each blade comprising four antiparallel β-strands stabilized by disulfide bonds. The catalytic site is a charged cavity formed by eight highly conserved residues – Arg118, Asp151, Arg152, Arg224, Glu276, Arg292, Arg371, and Tyr406 (N2 numbering) – that catalyse cleavage of the glycosidic linkage between terminal SIA and the adjacent sugar residue, thereby facilitating progeny virion release. The catalytic site is surrounded by a scaffold of framework residues, including Glu119, Arg156, Trp178, Ser179, Asp198, Ile222, Glu227, Glu277, Asn294, and Glu425, which help maintain the conformation of the catalytic pocket (Colman et al., Reference Colman, Varghese and Laver1983).
NA catalytic activity is influenced by multiple factors, including local pH, divalent cations such as Ca2⁺, stalk length, and the spatial and functional relationship between NA and HA. Many influenza NAs exhibit optimal enzymatic activity at mildly acidic pH, and both NA stability and catalytic function depend on Ca2⁺ binding. Each NA tetramer can bind several Ca2⁺ ions at sites that stabilize the quaternary structure or support the conformation of individual catalytic heads (Wang et al., Reference Wang, Dou, Östbye, Revol and Daniels2019). Additional Ca2⁺-binding sites have been identified in N1 proteins from the 1918 H1N1 virus and the 2009 pandemic H1N1 virus (Xu et al., Reference Xu, Zhu, Dwek, Stevens and Wilson2008). NA stalk length varies substantially among subtypes and lineages. These differences can influence enzymatic activity by altering the accessibility and mobility of the catalytic heads relative to sialylated substrates and neighbouring HA molecules. Stalk truncation can restrict head-domain mobility and modify substrate access, catalytic activity, host adaptation, and pathogenicity (Blumenkrantz et al., Reference Blumenkrantz, Roberts, Shelton, Lycett and Barclay2013; Hoffmann et al., Reference Hoffmann, Munier, Larcher, Soubieux, Ledevin, Esnault, Tourdes, Croville, Guérin, Quéré, Volmer, Naffakh and Marc2012; Sorrell et al., Reference Sorrell, Song, Pena and Perez2010). Many avian NAs also contain a second sialic acid-binding site (2SBS), or hemadsorption site, adjacent to the catalytic site. This shallow pocket is formed by three surface loops and can bind SIA through residues including Ser367, Ser370, Ser372, Asn400, Trp403, and Lys432 without catalysing its cleavage (Varghese et al., Reference Varghese, Colman, Van Donkelaar, Blick, Sahasrabudhe and McKimm-Breschkin1997). The 2SBS is highly conserved in many AIVs but is frequently disrupted in human H1N1 and H3N2 viruses and in some mammalian-adapted strains. It preferentially engages α2,3-sialylated glycans and can enhance catalytic efficiency by recruiting and positioning multivalent substrates near the catalytic site (Dai et al., Reference Dai, Du, Martínez-romero, Leenders, Wennekes, Rimmelzwaan, van Kuppeveld, Fouchier, Garcia-sastre, de Vries and de Haan2021, Reference Dai, McBride, Dortmans, Peng, Bakkers, de Groot, Van Kuppeveld, Paulson, de Vries and De Haan2017; Du et al., Reference Du, Dai, Li, Boons, Peeters, van Kuppeveld, de Vries and de Haan2018). Changes in 2SBS function may therefore contribute to the adaptation of NA activity to the receptor environment encountered during cross-species transmission (Dai et al., Reference Dai, McBride, Dortmans, Peng, Bakkers, de Groot, Van Kuppeveld, Paulson, de Vries and De Haan2017; Du et al., Reference Du, Dai, Li, Boons, Peeters, van Kuppeveld, de Vries and de Haan2018).
Molecular basis of HA/NA functional balance
HA and NA perform interconnected functions throughout IAV infection. This interplay is important for virion attachment, motility, entry, and release. Upon entering the respiratory tract, IAV first encounters the overlying mucus layer rather than the epithelial surface itself. This barrier contains mucins and other glycoconjugates bearing abundant terminal SIAs that can engage the HA RBS (Delaveris et al., Reference Delaveris, Webster, Banik, Boxer and Bertozzi2020; Pan et al., Reference Pan, Duggal, Lakdawala, Rockey and Marr2025; Wallace et al., Reference Wallace, Liu, van Kuppeveld, de Vries and de Haan2021; Wallert et al., Reference Wallert, Nie, Anilkumar, Abbina, Bhatia, Ludwig, Kizhakkedathu, Haag and Block2020). Individual HA–SIA interactions are relatively weak, but multivalent engagement by multiple HA trimers and sialylated glycans supports stable yet reversible virion attachment. This interaction remains dynamic because individual HA molecules repeatedly associate with and dissociate from sialylated receptors (de Vries et al., Reference de Vries, Du, Guo and de Haan2020; Xiong et al., Reference Xiong, Coombs, Martin, Liu, Xiao, McCauley, Locher, Walker, Collins, Kawaoka, Skehel and Gamblin2013). Concurrently, NA engages and cleaves accessible SIAs through its catalytic site, with the 2SBS contributing to substrate binding in NAs that retain this site. Local receptor depletion generated by NA activity biases repeated HA-mediated attachment towards receptor-rich regions and thereby promotes directional movement across receptor-coated surfaces. Depending on the experimental context, this motion has been described as rolling, crawling, or gliding (Du et al., Reference Du, Guo, Nijman, Doedt, van der Vries, van der Lee, Li, Boons, Van Kuppeveld, de Vries, Matrosovich and de Haan2019b; Guo et al., Reference Guo, He, Xu, Benegal, Brody and Vahey2023, Reference Guo, Rabouw, Slomp, Dai, van der Vegt, van Lent, McBride, Paulson, de Groot, van Kuppeveld, De Vries and de Haan2018; Liu et al., Reference Liu, de Vries and de Haan2025; Momont et al., Reference Momont, Dang, Zatta, Hauser, Wang, Di Iulio, Minola, Czudnochowski, De marco, Branch, Donermeyer, Vyas, Chen, Ferri, Guarino, Powell, Spreafico, Yim, Balce, Bartha, Meury, Croll, Belnap, Schmid, Schaiff, Miller, Cameroni, Telenti, Virgin, Rosen, Purcell, Lanzavecchia, Snell, Corti and Pizzuto2023; Vahey and Fletcher, Reference Vahey and Fletcher2019; Wallace et al., Reference Wallace, de Vries, van Kuppeveld and de Haan2023; Yoshida et al., Reference Yoshida, Uekusa, Suzuki, Bauer, Sakai and Yamauchi2025). After traversing mucus, virions continue to sample sialylated receptors on the epithelial surface, which can facilitate access to sites permissive for endocytosis (Fig. 1) (Guo et al., Reference Guo, Rabouw, Slomp, Dai, van der Vegt, van Lent, McBride, Paulson, de Groot, van Kuppeveld, De Vries and de Haan2018; Vahey and Fletcher, Reference Vahey and Fletcher2019). Following replication and assembly, HA on progeny virions can bind SIA on the infected-cell surface and on neighbouring virions, whereas NA-mediated receptor cleavage promotes virion detachment and limits aggregation (Fig. 1). HA-mediated receptor binding and NA-mediated receptor cleavage are therefore functionally coupled across multiple stages of the viral life cycle, providing the mechanistic basis for HA/NA functional balance.
Schematic representation of IAV movement and infection in the respiratory tract. (A) After entering the respiratory tract, IAV encounters a mucus barrier containing O-glycan-rich mucins. Sialylated glycans on mucins can bind HA and trap virions before they reach the epithelial surface. Through repeated HA-mediated receptor binding and NA-mediated receptor cleavage, virions generate local differences in receptor density that bias their movement across mucus and ciliated surfaces towards epithelial cells. (B) At the epithelial surface, continued coordination between HA and NA supports virion movement along the plasma membrane and access to SIA-rich sites permissive for entry. Following replication and assembly, NA-mediated cleavage of SIA promotes progeny virion release and limits aggregation, enabling subsequent rounds of infection. HA, hemagglutinin; IAV, influenza A virus; NA, neuraminidase; SIA, sialic acid.
Figure 1 Long description
The interaction of influenza A virus with the mucus layer, cilia and epithelial cells. Virions bind to sialylated glycans on mucins, facilitated by hemagglutinin (HA) and are trapped before reaching the epithelial surface. Neuraminidase (NA) cleaves receptors, aiding movement across mucus and ciliated surfaces. The virus at the epithelial surface, where HA and NA coordinate to support movement along the plasma membrane. HA binds to sialic acid-rich sites, while NA cleaves sialic acid to promote progeny virion release and limit aggregation. The diagram includes labels for sialic acid, galactose, N-acetylglucosamine, glucose and mannose, illustrating the virus′s path from binding to endocytosis and release.
Biological significance of HA/NA functional balance
Effects on viral attachment
HA/NA functional balance directly influences the efficiency of IAV attachment and movement on host surfaces. When HA receptor-binding avidity is excessively high or NA activity is insufficient, virions may become trapped by decoy receptors, impairing their movement through mucus and reducing access to sites permissive for internalization at the epithelial surface (Guo et al., Reference Guo, Rabouw, Slomp, Dai, van der Vegt, van Lent, McBride, Paulson, de Groot, van Kuppeveld, De Vries and de Haan2018; Wallace et al., Reference Wallace, Liu, van Kuppeveld, de Vries and de Haan2021). Conversely, excessive NA activity can prematurely remove receptors during repeated HA–receptor interactions and thereby prevent stable attachment (Guo et al., Reference Guo, Rabouw, Slomp, Dai, van der Vegt, van Lent, McBride, Paulson, de Groot, van Kuppeveld, De Vries and de Haan2018; Liu et al., Reference Liu, de Vries and de Haan2025; Vahey and Fletcher, Reference Vahey and Fletcher2019; Wallace et al., Reference Wallace, de Vries, van Kuppeveld and de Haan2023). An appropriate HA/NA functional balance therefore supports attachment while avoiding excessive retention, facilitating the transition from receptor engagement to cell entry (de Vries et al., Reference de Vries, Du, Guo and de Haan2020; Liu et al., Reference Liu, de Vries and de Haan2025; Vahey and Fletcher, Reference Vahey and Fletcher2019; Wallace et al., Reference Wallace, de Vries, van Kuppeveld and de Haan2023, Reference Wallace, Liu, van Kuppeveld, de Vries and de Haan2021).
Regulation of viral release and transmission
HA/NA functional balance also influences the efficiency of progeny virion release. When NA activity is insufficient relative to HA receptor-binding avidity, newly formed virions may remain attached to the cell surface or aggregate with one another, reducing their capacity to spread. Conversely, excessive NA activity may remove receptors needed for subsequent attachment. HA and NA must therefore remain functionally matched to coordinate efficient virion release with continued infection of susceptible cells.
The emergence of the 2009 pandemic H1N1 virus illustrates the adaptive optimization of HA/NA balance (Lakdawala et al., Reference Lakdawala, Lamirande, Suguitan, Wang, Santos, Vogel, Matsuoka, Lindsley, Jin and Subbarao2011; Yen et al., Reference Yen, Liang, Wu, Forrest, Ferguson, Choy, Jones, Wong, Cheung, Hsu, Li, Yuen, Chan, Poon, Chan, Nicholls, Krauss, Wong, Guan, Webster, Webby and Peiris2011). Comparative analyses indicated that substitutions in the HA receptor-binding region during early adaptation of the pandemic lineage reduced receptor-binding avidity and improved functional compatibility with NA (Xu et al., Reference Xu, Zhu, McBride, Nycholat, Yu, Paulson and Wilson2012). These findings show that the optimal relationship between HA and NA is context-dependent and may differ among strains. Evidence from ferret transmission models further supports this principle: replacing the NA gene of the swine-origin H1N1 virus A/swine/Hong Kong/915/2004 with that of the 2009 pandemic virus markedly enhanced respiratory-droplet transmissibility (Yen et al., Reference Yen, Liang, Wu, Forrest, Ferguson, Choy, Jones, Wong, Cheung, Hsu, Li, Yuen, Chan, Poon, Chan, Nicholls, Krauss, Wong, Guan, Webster, Webby and Peiris2011). This result suggests that the original HA/NA pairing was less well adapted for transmission in ferrets and underscores the contribution of functional compatibility between the two glycoproteins to host adaptation. More broadly, host-specific HA/NA coevolutionary trajectories may help explain why many reassortant viruses fail to achieve sustained transmission after crossing species barriers.
Effects on viral pathogenicity
HA/NA functional balance can also influence viral pathogenicity, although its effects are strongly dependent on genetic background and host context. Tumpey et al. showed that recombinant viruses bearing both the 1918 pandemic HA and NA in an A/WSN/33 background were highly virulent in mice, whereas viruses bearing only one of these glycoproteins were attenuated (Tumpey et al., Reference Tumpey, García-sastre, Mikulasova, Taubenberger, Swayne, Palese and Basler2002, Reference Tumpey, García-sastre, Taubenberger, Palese, Swayne and Basler2004). These findings indicate that functional compatibility between the 1918 HA and NA contributed to the observed phenotype in this experimental background. Diederich et al. further showed that a reassortant H5N3 virus containing highly pathogenic H5N1 HA and low-pathogenicity H7N3 NA displayed reduced virulence and transmissibility in chickens (Diederich et al., Reference Diederich, Berhane, Embury-hyatt, Hisanaga, Handel, Cottam-birt, Ranadheera, Kobasa and Pasick2015). After serial passage in vivo, an adaptive NA substitution (R293K) emerged, increasing NA activity together with viral transmissibility and pathogenicity. This phenotypic change is consistent with the interpretation that the initial HA/NA pairing was functionally suboptimal and that adaptive modulation of NA activity restored a more favourable balance.
Overall, the interplay between HA-mediated receptor binding and NA-mediated receptor cleavage makes HA/NA balance an important determinant of viral attachment, motility, entry, release, and transmission. A change in either glycoprotein may confer an advantage at one stage of infection – for example, enhanced attachment or more efficient release – while imposing a fitness cost at another stage. Such trade-offs constrain the extent to which HA and NA can evolve independently and favour compensatory changes that preserve functional compatibility across the viral life cycle.
Reshaping HA/NA functional balance under adaptive selective pressures
HA/NA coevolution during cross-species transmission
During host adaptation and cross-species transmission, HA/NA functional balance must be recalibrated to the receptor landscape of the new host. HA receptor specificity and avidity, together with NA catalytic activity and substrate specificity, must remain compatible with the distribution, density, and presentation of sialylated glycans in the relevant tissues. These features differ markedly among species and along the respiratory and intestinal tracts. Waterfowl generally express abundant α2,3-SIA in intestinal and respiratory epithelia; chickens express both α2,3- and α2,6-SIA, with distributions that vary by tissue and cell type; humans and ferrets predominantly express α2,6-SIA in the upper respiratory tract, whereas α2,3-SIA is more prominent in distal airways; and pigs express both receptor types throughout the respiratory tract, again with region- and cell-dependent differences. These broad receptor-distribution patterns and their limitations have been reviewed by de Graaf and Fouchier (Reference de Graaf and Fouchier2014), Long et al. (Reference Long, Mistry, Haslam and Barclay2019), and Wallace et al. (Reference Wallace, Liu, van Kuppeveld, de Vries and de Haan2021). Primary tissue-level studies further indicate that receptor topology, glycan length, sulfation, fucosylation, and mucin presentation influence viral binding and cannot be adequately represented by linkage type alone (Lakdawala et al., Reference Lakdawala, Jayaraman, Halpin, Lamirande, Shih, Stockwell, Lin, Simenauer, Hanson, Vogel, Paskel, Minai, Moore, Orandle, Das, Wentworth, Sasisekharan and Subbarao2015; Walther et al., Reference Walther, Karamanska, Chan, Chan, Jia, Air, Hopton, Wong, Dell, Malik Peiris, Haslam and Nicholls2013).
Efficient infection and transmission in a new host require HA to engage appropriate receptors while NA retains sufficient activity to support movement through mucus and release from infected cells (de Vries et al., Reference de Vries, Du, Guo and de Haan2020; Liu et al., Reference Liu, de Vries and de Haan2025; Vahey and Fletcher, Reference Vahey and Fletcher2019; Wallace et al., Reference Wallace, de Vries, van Kuppeveld and de Haan2023). During transmission from birds to mammals or from waterfowl to terrestrial poultry, the altered receptor landscape can disrupt a pre-existing HA/NA balance, favouring mutations that recalibrate the relationship. Avian-adapted HAs commonly prefer α2,3-sialylated glycans, and their corresponding NAs often retain a functional 2SBS. By transiently engaging α2,3-sialylated glycans, the 2SBS can enhance NA-mediated cleavage of multivalent substrates and facilitate virion movement and release in avian hosts (Du et al., Reference Du, Dai, Li, Boons, Peeters, van Kuppeveld, de Vries and de Haan2018). In mammalian respiratory environments enriched in α2,6-sialylated glycans, however, the selective value of an α2,3-SIA-preferring 2SBS may diminish. Experimental and evolutionary evidence indicates that attenuation of 2SBS function can precede adaptive changes in the HA RBS. Reduced 2SBS activity changes the substrate-binding and cleavage properties of NA, thereby altering selection on an HA that retains a strong preference for α2,3-SIA. This altered selective landscape may favour HA variants with reduced dependence on α2,3-SIA or increased recognition of α2,6-sialylated glycans (Dai et al., Reference Dai, McBride, Dortmans, Peng, Bakkers, de Groot, Van Kuppeveld, Paulson, de Vries and De Haan2017; Du et al., Reference Du, Dai, Li, Boons, Peeters, van Kuppeveld, de Vries and de Haan2018, Reference Du, Wolfert, Peeters, van Kuppeveld, Boons, De Vries and de Haan2020). NA can subsequently re-establish functional coordination with HA through changes in catalytic activity, substrate engagement, or stalk length (Hoffmann et al., Reference Hoffmann, Munier, Larcher, Soubieux, Ledevin, Esnault, Tourdes, Croville, Guérin, Quéré, Volmer, Naffakh and Marc2012; Lai et al., Reference Lai, Karunarathna, Wong, Peiris and Nicholls2019; Liu et al., Reference Liu, Wang, Tan, Wu and Brooke2022; Long et al., Reference Long, Mistry, Haslam and Barclay2019; Xu et al., Reference Xu, Zhu, McBride, Nycholat, Yu, Paulson and Wilson2012).
From a broader evolutionary perspective, the 18 established HA subtypes, the recently proposed H19 subtype, and the 11 reported NA subtypes permit many theoretical combinations, yet the subtype constellations that circulate persistently in nature are highly constrained. Human IAVs are dominated by H1N1 and H3N2, with H1N2 viruses are detected less frequently, whereas diverse but nonrandom constellations circulate in avian populations. As reviewed by Medina and Garcia-Sastre (Medina and Garcia-sastre, Reference Medina and Garcia-sastre2011), the restricted distribution of naturally circulating subtype constellations reflects, among other factors, host-dependent selection for functional compatibility between HA and NA. Coevolutionary analysis further supports long-term coordination between HA and NA in established lineages (Ward et al., Reference Ward, Lycett, Avila, Bollback and Leigh brown2013). Reassortment occurs more frequently in avian populations and can facilitate host jumps, but only a subset of HA/NA combinations establishes sustained transmission (Jang and Bae, Reference Jang and Bae2018; Markin et al., Reference Markin, Macken, Baker and Anderson2025; Qin et al., Reference Qin, Zhu, Ma, Yin, Chen, Peng and Liu2018; Ward et al., Reference Ward, Lycett, Avila, Bollback and Leigh brown2013). Intrinsic differences among HA subtypes in RBS architecture, glycosylation, and virion-surface organization may impose distinct requirements on NA catalytic activity, 2SBS function, stalk length, and abundance. Functionally incompatible HA/NA pairings can disrupt the kinetic coordination of receptor engagement and cleavage, reduce viral fitness, and consequently be removed by selection.
HA/NA rebalancing during immune escape and antiviral resistance
Immune pressure can reshape HA/NA functional balance while selecting for antigenic escape. Antibodies targeting the HA head or NA can drive antigenic change, but escape substitutions may incur functional costs by altering receptor-binding avidity, enzymatic activity, protein stability, or surface expression. Coordinated evolution of HA and NA can partly compensate for these costs and preserve viral fitness. For example, HA escape substitutions that alter receptor-binding avidity may disrupt an established HA/NA balance and thereby modify selection on NA activity or antigenicity (Hensley et al., Reference Hensley, Das, Gibbs, Bailey, Schmidt, Bennink and Yewdell2011; Myers et al., Reference Myers, Wetzel, Linderman, Li, Sullivan and Hensley2013). Conversely, antigenic or functional changes in NA arising through immune selection or natural variation can reshape the accessible mutational landscape of HA and favour compensatory substitutions in the HA head (Liu et al., Reference Liu, Reiser, Tan, Lv, Rivera-Cardona, Heimburger, Wu and Brooke2024, Reference Liu, Wang, Tan, Wu and Brooke2022; Wang et al., Reference Wang, Wan, Wang, Wu, Fu, Gao, Shao, Qian, Ye and Qin2021). Immune escape may therefore involve coordinated evolution of both surface glycoproteins rather than antigenic drift of either protein in isolation. As reviewed by Gubareva and Mohan (Reference Gubareva and Mohan2022) and Xu et al. (Reference Xu, Luo, Huang, Li, Ye, Yan, Zhou, He, Liu and Zhu2024), NAIs occupy the NA catalytic site, thereby reducing SIA cleavage and progeny virion release. NAI use can select resistance-associated substitutions, but resistant variants may also spread in the absence of direct drug exposure when permissive or compensatory substitutions preserve viral fitness. The global emergence of oseltamivir-resistant seasonal H1N1 viruses carrying NA H275Y provides a prominent example. Between 2007 and 2009, the prevalence of H275Y seasonal H1N1 viruses increased from less than 1% to more than 90% in many regions, including populations with limited oseltamivir use (Duan et al., Reference Duan, Govorkova, Bahl, Zaraket, Baranovich, Seiler, Prevost, Webster and Webby2014). Although H275Y can impair NA expression, stability, or activity, secondary substitutions – including V234M and R222Q – can mitigate these costs and support efficient replication and transmission. Other permissive substitutions, including K329E, D344N, and D354G, have also been associated with the emergence or fitness of H275Y variants (Duan et al., Reference Duan, Govorkova, Bahl, Zaraket, Baranovich, Seiler, Prevost, Webster and Webby2014). More recent work has identified additional strain-dependent combinations that can improve the fitness of H275Y-bearing viruses (Duan et al., Reference Duan, Govorkova, Bahl, Zaraket, Baranovich, Seiler, Prevost, Webster and Webby2014; Farrukee et al., Reference Farrukee, Gunalan, Maurer-stroh, Reading and Hurt2022). The broader spectrum of NA substitutions associated with NAI resistance has been reviewed by Xu et al. (Reference Xu, Luo, Huang, Li, Ye, Yan, Zhou, He, Liu and Zhu2024). These observations illustrate how intragenic compensation within NA and broader HA/NA rebalancing can facilitate the persistence and spread of antiviral-resistant variants.
Compensation can also involve HA. For example, glycosylation at HA Asn163 can reduce receptor-binding avidity by partially shielding the RBS, thereby decreasing the level of NA activity required for virion release and altering susceptibility to NA inhibition (Mishin et al., Reference Mishin, Novikov, Hayden and Gubareva2005). NAI resistance may therefore be supported not only by substitutions within NA but also by changes in HA that restore functional compatibility between receptor binding and receptor cleavage.
Challenges and future directions
During circulation in humans and animals, IAV is continually exposed to selective pressures imposed by host adaptation, immune responses, and antiviral use. These pressures can alter HA receptor-binding properties, NA receptor-cleaving activity, and other viral traits, thereby perturbing a pre-existing HA/NA balance and affecting viral fitness, transmission, or pathogenicity. Because HA and NA are functionally interdependent, a change in one glycoprotein may alter the constraints acting on the other and favour adaptive or compensatory changes that restore compatibility. HA/NA functional balance is therefore not a fixed endpoint of IAV evolution but a dynamic relationship that can undergo repeated cycles of ‘balance–imbalance–rebalance’ (Fig. 2). This framework links selective pressures and functional variation to viral phenotypes and may help organize the study of IAV adaptive evolution. Important questions nevertheless remain. First, the mechanisms by which HA/NA balance is maintained during continuing antigenic drift are not fully resolved. Second, the effects of receptor distribution, glycan topology, mucus composition, and tissue microenvironments on HA/NA compatibility remain difficult to quantify. Third, the extent to which this balance constrains evolutionary trajectories and limits the accessibility of adaptive mutations has not been systematically defined. Finally, although compensatory mutations can restore viral fitness, their context-dependent costs and potential trade-offs remain insufficiently understood.
Dynamic coevolution and functional balance between IAV HA and NA. Viral attachment, motility, and release depend on the coordination of HA-mediated receptor binding and NA-mediated receptor cleavage. Genetic variation, reassortment, host receptor landscapes, immune pressure, and antiviral selection can disrupt a pre-existing balance, whereas compensatory changes in HA or NA may restore functional compatibility and establish a new adaptive state. H1–H16 are illustrated, whereas the divergent H17 and H18 subtypes and the recently proposed H19 subtype are excluded because they use noncanonical entry pathways rather than canonical SIA receptors. HA, hemagglutinin; IAV, influenza A virus; NA, neuraminidase; SIA, sialic acid.
Figure 2 Long description
The coevolution of HA receptor-binding and NA receptor-cleaving processes. At the center is a virus, surrounded by arrows indicating functional balance. The top left section shows alpha2,3-SA with H1-H16, N1-N9 and factors like mutations, reassortment, SA type, immune pressure, glycosylation and pH stability. The top right section shows alpha2,3-SA with H1-H13, N1-N9, focusing on virus release and spread. The bottom right section shows alpha2,6-SA/alpha2,3-SA with H1N1, N2, H2N3, H3N2, highlighting mutations, reassortment, SA type, stalk length, glycosylation and drug resistance. The bottom left section shows alpha2,3-SA with H1N1, H2N2, H3N2, H5N1, H7N7, H7N9, emphasizing virus adsorption and entry. Arrows connect these sections, illustrating the dynamic relationship between HA and NA functions.
Elucidating the mechanisms that maintain and reshape HA/NA functional balance could deepen our understanding of IAV evolution and have practical implications for surveillance and countermeasure development. Incorporating measurements of HA receptor-binding properties, NA activity, and HA/NA functional compatibility into viral surveillance could provide phenotypic context for variants suspected of altered host adaptation or transmission. Integrating sequence, structural, and experimental data may similarly improve the risk assessment of emerging strains, although robust predictive models will require standardized assays and validation across viral lineages and host systems. In vaccine research, defining the functional constraints linking HA and NA may help assess the consequences of antigenic variation and support the rational selection of antigen combinations. In antiviral development, resolving the molecular basis of HA/NA coordination and compensation may reveal vulnerabilities that can be exploited by combination therapies or by strategies that disrupt functional coupling. Future studies should integrate structural biology, virology, animal models, and large-scale evolutionary analyses to define the quantitative rules governing HA/NA co-adaptation and the constraints imposed on viral evolutionary trajectories, thereby improving our ability to assess the adaptive potential and cross-host transmission risk of emerging influenza viruses.
Author contributions
Y.T.Z.: Conceptualization; Visualization; Writing – original draft; Writing – review and editing. P.G.C.: Conceptualization; Visualization; Writing – review and editing. Y.X.S. and Y.L.C.: Conceptualization; Funding acquisition; Visualization; Writing – original draft; Writing – review and editing.
Funding statement
This study was supported by the National Natural Science Foundation of China (Grant Nos. 32473061 and 32302862), the ‘Biology +’ High-Level Discipline Innovation Team of the School of Life Sciences, Jilin University (Grant No. BSLS202503), and the National Natural Science Foundation of China Young Student Basic Research Project (Doctoral Students; Grant No. 325B2081).
Competing interest
The authors declare that they have no competing interests.
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