Analysis of the natural mutation rate of residue 198
An analysis of 4,093 avian H9N2 AIV HA protein sequences at position 198, published in GenBank from 2013 to 2022, revealed three prevalent mutations: 198T, 198A, and 198V. The most frequent was 198T, representing 72.4% of the sequences, followed by 198A (20.7%), while 198V was the least common (6.9%) (Figure 1A). Between 2013 and 2015, the frequency of the 198T mutation steadily increased, reaching over 80% from 2015 to 2017. However, from 2017 to 2020, the prevalence of 198T steadily declined, dropping to 70% in 2021, while the 198V mutation showed an inverse trend, rising in frequency and becoming dominant by 2022 (Figure 1B). The 198A mutation frequency remained stable (10%-15%) throughout the study period, with minor fluctuations.
Rescue and identification of recombinant viruses
Following transfection of COS-1 cells with eight plasmids encoding the full H9N2 genome, the supernatants were inoculated into SPF chicken embryos. After 90 hours, HA titers were measured in the allantoic fluid, and recombinant viruses with HA titers ≥2² were considered positive. The HA titers for rWJ/HA198A and rWJ/HA198T viruses were 2⁶ and 2⁸, respectively. Sequencing confirmed that the HA genes contained the desired mutations at positions 591–594, while all other gene segments (PB2, PB1, PA, NP, NA, M, and NS) were identical to the parental rWJ57 strain.
Mutations at HA position 198 as a key modulator of HI titers
To investigate how mutations at residue 198 influence the antigenic properties of the virus, HI assays were performed using sera from chickens infected with rWJ57. The rWJ57 virus carrying the 198V mutation showed a two-fold lower HI titer compared to rWJ/HA198A and rWJ/HA198T (Figure 1C), suggesting that residue 198 plays a critical role in modulating viral antigenicity.
Mutations at HA position 198 affects the binding affinities to non-neutralizing antibodies
To explore the underlying mechanisms, ELISA assays were conducted to measure the binding affinities of neutralizing and non-neutralizing antibodies. The results demonstrated that the rWJ57 virus had significantly lower affinity for non-neutralizing antibodies compared to rWJ/HA198A (P < 0.001), while rWJ/HA198T displayed significantly higher affinity than both rWJ57 and rWJ/HA198A (P < 0.001) (Figures 2A and 2B). However, no significant differences were observed in the binding affinities of the three viruses to neutralizing antibodies (Figure 2A and 2C).
Mutations at HA position 198 influences the receptor binding activity and specificity.
To assess how receptor binding activity affects HI titers, chicken erythrocytes were treated with α2-3,6,8 neuraminidase to modify sialic acid receptors. Stronger receptor binding was reflected by the virus's ability to agglutinate erythrocytes treated with higher concentrations of neuraminidase. Results showed that rWJ57 exhibited receptor binding activity four-fold higher than rWJ/HA198A and 16-fold higher than rWJ/HA198T (Figure 2D). These findings suggest that the mutation at position 198 alters receptor binding, which primarily drives HI titer fluctuations in the H9N2 virus, rather than neutralizing antibodies.
While the RDE assay demonstrates the virus's binding activity for sialic acids (both α2-3 SA and α2-6 SA), it cannot differentiate the virus's affinity for specific sialic acid types. To further explore the effect of mutations at HA position 198 on the affinity for specific sialic acids, we conducted a solid-phase binding assay. The results revealed distinct receptor preferences among H9N2 virus variants with mutations at HA position 198. HA198V exhibited the highest affinity for 6'SLN, while HA198A showed strongest binding to 3'SLN (Figure 2E). HA198T demonstrated intermediate binding to 6'SLN but the lowest affinity for 3'SLN (Figure 2F). These findings indicate that the amino acid at position 198 of the HA protein plays a crucial role in determining receptor binding specificity.
Mutations at HA position 198 alter in HA protein-sialic acid receptor interactions
The α2,6 SA formed five hydrogen bond interactions with the HA protein of the rWJ57 strain (HA198V protein) at amino acid positions 109, 147, 201, 235, and 236, with a binding energy of -6.1 kcal/mol (Figure 3A). It also formed five hydrogen bond interactions with the rWJ/HA198T HA protein (HA198T protein) at positions 109, 147, 148, 233, and 236, with a binding energy of -5.9 kcal/mol (Figure 3B), and four interactions with the rWJ/HA198A HA protein (HA198A protein) at positions 145, 147, 235, and 236, with a binding energy of -6.0 kcal/mol (Figure 3C).
The α2,3 SA formed six hydrogen bond interactions with the HA198V protein at amino acid positions 109, 147, 148, 233, 235, and 236, with a binding energy of -5.6 kcal/mol (Figure 3D). It also formed six interactions with the HA198T protein at positions 109, 147, 198, 233, 235, and 236, with a binding energy of -5.5 kcal/mol (Figure 3E), and three interactions with the HA198A protein at positions 109, 145, and 147, with a binding energy of -5.5 kcal/mol (Figure 3F).
These results indicate that alterations at amino acid position 198 of the HA protein directly modify the microstructure of the receptor-binding pocket, leading to changes in the binding sites, interaction forces, torque, and binding energy with sialic acid receptors, ultimately influencing the interactions between the HA protein and the receptors.
MD simulation
To explore the molecular mechanisms by which mutations at residue 198 influence receptor binding affinity and viral tropism, molecular dynamics (MD) simulations were employed to evaluate the structural and dynamic behavior of the HA-sialic acid receptor complexes. The simulation focused on how the HA198V, HA198T, and HA198A mutations alter the interaction between the HA protein and both α2,6 SA and α2,3 SA. The key metrics analyzed were root mean square deviation (RMSD), root mean square fluctuation (RMSF), hydrogen bonding patterns, and binding stability, which provided insights into conformational changes and receptor affinity.
MD simulation demonstrates stable binding stability between HA198V and α2,6 SA
During the simulation, the HA protein maintained a stable interaction with the α2,6 SA. The RMSD values for the HA198V protein and the α2,6 SA stabilized around 7.0 Å and 9.0 Å, respectively, after initial fluctuations (Figure 4A), suggesting that the complex underwent minor adjustments before reaching a stable conformation. RMSF analysis (Figures 4B and 4C) revealed minimal fluctuations in key binding residues, particularly Tyr109, Thr147, Met235, and Gly236, highlighting their crucial role in maintaining the interaction’s stability.
Thr147 played a pivotal role in the binding process, forming hydrogen bonds with a high formation frequency of 118%, supported by secondary water bridge interactions with a frequency of 30% (Figures 4D and 4E). Other residues, including Tyr109, Asn145, Trp161, and Gly236, also contributed through hydrophobic and ionic interactions, further stabilizing the complex. The α2,6 SA itself formed several intramolecular hydrogen bonds, which contributed to its conformational stability (Figure 4E).
These findings suggest that the HA198V mutation significantly enhances the binding affinity between the HA protein and α2,6 SA. The combination of hydrogen bonds, hydrophobic interactions, and water bridges forms a robust network that stabilizes the HA -α2,6 SA complex.
MD simulation reveals enhanced binding affinity between HA198T and α2,6 SA
In the MD simulation of the HA198T protein complexed with the α2,6 SA, the RMSD of the HA198T protein stabilized at approximately 9.0 Å, while the α2,6 SA's RMSD stabilized at around 16 Å after initial fluctuations (Figure 5A). These RMSD values indicate that both the protein and α2,6 SA underwent structural adjustments before reaching a stable conformation.
Further analysis using RMSF revealed that the HA198T protein exhibited relatively low fluctuations, indicating minimal conformational changes during the binding process (Figure 5B). In contrast, the α2,6 SA displayed higher RMSF values, highlighting its dynamic and flexible nature (Figure 5C). This flexibility likely facilitated binding interactions with the protein, contributing to the formation of a more stable complex.
A total of 30 interactions were identified between the HA198T protein and the α2,6 SA, with Thr147 playing a critical role, showing a hydrogen bond formation frequency of 92% (Figures 5D and 5E). Other residues, such as Asn145 and Gly233, also contributed significantly to the stability of the complex, reinforcing the hydrogen bonding network. Additionally, the α2,6 SA formed several intramolecular hydrogen bonds, further stabilizing its conformation and strengthening the overall α2,6 SA-protein complex (Figure 5E).
Overall, the HA198T mutation enhances the binding affinity between the HA protein and the α2,6 SA. The hydrogen bond network, particularly involving Thr147, is crucial in stabilizing the complex. The flexibility of the α2,6 SA, along with its intramolecular interactions, plays a significant role in the stability and dynamic behavior of the α2,6 SA-protein interaction. These findings provide valuable insights into the molecular mechanisms underlying the increased receptor binding affinity due to the HA198T mutation, with potential implications for future vaccine or antiviral drug development.
MD simulation reveals unstable binding and dissociation between HA198A and α2,6 SA
During the molecular dynamics simulation, the HA198A protein exhibited significant fluctuations. The RMSD of HA198A eventually stabilized at approximately 4.0 Å, whereas the RMSD of the α2,6 SA fluctuated substantially, finally stabilizing near 90 Å (Figure 6A).
While RMSF of the HA198A protein remained relatively small, the RMSF of the α2,6 SA was notably larger (Figures 6B and 6C), indicating greater movement of the α2,6 SA during the simulation. Despite the formation of 29 interactions, the α2,6 SA eventually moved away from the active binding pocket (Figures 6D and 6E).
These results suggest that the initial binding conformation between the α2,6 SA and the HA198A protein was unstable, characterized by weak binding affinity. During the simulation, the α2,6 SA ultimately dissociated from the binding pocket, failing to maintain a stable interaction with the HA protein.
MD simulation reveals moderate binding affinity between HA198V and α2,3 SA after initial instability
During the MD simulation, the RMSD of the HA198V protein stabilized at at approximately 8.0 Å, while the RMSD of the α2,3 SA, after some fluctuations, settled around 10.0 Å (Figure 7A). The RMSF values for the amino acid residues interacting with the α2,3 SA, highlighted by green lines in Figure 7B, were relatively low, indicating minimal conformational changes in these regions during the binding process. In contrast, the RMSF values for the atoms in the α2,3 SA were relatively high, reflecting substantial fluctuations in the α2,3 SA 's structure (Figure 7C). These findings suggest that the initial binding conformation was unstable, though the system eventually achieved a more stable conformation after significant fluctuations.
The HA198V protein and the α2,3 SA formed 41 interactions, with Phe408 playing a pivotal role by forming hydrogen bonds with a 31% frequency, highlighting its importance in the binding process. Other interaction sites contributed via hydrogen bonds and water bridges, although these interactions were comparatively weak (Figures 7D and 7E). Additionally, the α2,3 SA formed several intramolecular hydrogen bonds that helped stabilize its conformation during binding (Figure 7E).
Overall, these results indicate that the HA198V protein and α2,3 SA optimized their binding conformation through the simulation, achieving a more stable structure with moderate binding affinity.
MD simulation confirms weak binding characteristics between HA198T and α2,3 SA
During the MD simulation, the HA198T protein exhibited minimal fluctuations, with its RMSD stabilizing at approximately 6 Å. In contrast, the RMSD of the α2,3 SA displayed significant variability before eventually stabilizing at around 30 Å (Figure 8A). The RMSF values for the HA198T protein were relatively low, indicating limited conformational changes, while the α2,3 SA exhibited much higher RMSF values, suggesting considerable fluctuations in the α2,3 SA's structure throughout the simulation (Figures 8B and 8C). This pattern suggests that the initial conformation of the HA198T protein and α2,3 SA complex was unstable, with the α2,3 SA gradually moving away from the binding pocket during the course of the simulation.
Although a total of 91 interactions were formed between the protein and α2,3 SA, none of these interactions had frequencies exceeding 30% (Figure 8D), and only those above this threshold are shown in Figures 8D and 8E. The absence of strong, consistent interactions indicates a weak binding affinity between the HA198T protein and the α2,3 SA. Consequently, the α2,3 SA was unable to maintain stable binding within the protein's receptor pocket, which is consistent with its detachment from the binding site.
MD simulation reveals high-affinity stable binding between HA198A and α2,3 SA
During the MD simulation, both the HA198A protein and the α2,3 SA demonstrated high stability. The RMSD of the HA198A protein stabilized at approximately 6.0 Å, and after initial fluctuations, the RMSD of the α2,3 SA also stabilized at around 6.0 Å (Figure 9A). The RMSF values for the interacting amino acid residues of HA198A (indicated in green in Figure 9B) and the α2,3 SA atoms showed minimal fluctuations overall (Figures 9B and 9C), indicating that both the protein and the α2,3 SA had relatively stable initial conformations. After some dynamic movements, a more stable binding conformation was formed.
The HA198A protein and the α2,3 SA formed a total of 33 interactions. Notably, the hydrogen bond and water bridge interaction frequencies of Glu407 reached 159% and 99%, respectively, underscoring the critical role of Glu407 in the binding process (Figures 9D and 9E). Other important residues, including Glu99, Arg275, Ile276, Lys278, and Asp405, also contributed significantly to maintaining the complex's stability (Figures 9D and 9E). Additionally, several intramolecular hydrogen bonds were formed within the α2,3 SA, further stabilizing its binding conformation (Figure 9E).
These results suggest that the α2,3 SA and HA198A protein optimized their initial binding conformations during molecular dynamics simulations, resulting in a more stable complex with high binding affinity.
In summary, MD simulations provided insights into the molecular interactions and conformational changes governing the binding affinity between the HA protein and sialic acids, which are critical for viral receptor specificity. The binding affinity between the 198th residue of the HA protein and the α2,6 SA decreased in the order of HA198V, HA198T, and HA198A. Conversely, for the α2,3 SA, the binding affinity increased in the order of HA198T, HA198V, and HA198A. Furthermore, when interacting with the α2,6 SA, the 198th residue primarily engaged through the formation of water bridges. In contrast, the 198th residue did not directly participate in interactions with the α2,3 SA, suggesting a differential role of this residue in modulating receptor specificity based on SA type.