Molecular mechanism of the enhanced viral fitness contributed by secondary mutations in the hemagglutinin protein of oseltamivir resistant H1N1 influenza viruses: Modeling studies of antibody and receptor binding

GENE-40116; No. of pages: 9; 4C: Gene xxx (2014) xxx–xxx

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Gene journal homepage: www.elsevier.com/locate/gene

Molecular mechanism of the enhanced viral fitness contributed by secondary mutations in the hemagglutinin protein of oseltamivir resistant H1N1 influenza viruses: Modeling studies of antibody and receptor binding Abhisek Kumar Behera, Sushmita Basu, Sarah S. Cherian ⁎ Bioinformatics Group, National Institute of Virology, 20-A, Dr. Ambedkar Road, Post Box No. 11, Pune 411001, Maharashtra, India

a r t i c l e

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Article history: Received 11 June 2014 Received in revised form 26 November 2014 Accepted 1 December 2014 Available online xxxx Keywords: H1N1 influenza A Hemagglutinin Oseltamivir resistance Neuraminidase H274Y mutation Antibody and receptor docking Molecular dynamics simulations

a b s t r a c t The envelope protein hemagglutinin (HA) of influenza viruses is primarily associated with host antibody and receptor interactions. The HA protein is known to maintain a functional balance with neuraminidase (NA), the other major envelope protein. Prior to 2007–2008, human seasonal H1N1 viruses possessing the NA H274Y mutation, which confers oseltamivir resistance, generally had low growth capability. Subsequently, secondary mutations that compensate for the deleterious effect of the NA H274Y mutation have been identified. The molecular mechanism of how the defect could be counteracted by these secondary mutations is not fully understood. We studied here the effect of three such mutations (T86K, K144E and R192K) in the HA protein, which are located at either the HA receptor binding site or in the H1N1 antigenic sites. Molecular docking and dynamics studies showed that, of the three mutations, the R192K mutation could have mediated neutralizing antibody escape and decreased receptor binding affinity, either or both of which may have contributed to increased viral fitness. The study suggests the molecular basis of enhanced viral fitness induced by secondary mutations in the evolution of oseltamivir-resistant influenza strains. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Influenza viruses cause severe respiratory infections and are responsible for causing annual epidemics and occasional pandemics worldwide

Abbreviations: HA, hemagglutinin; NA, neuraminidase; H, histidine; Y, tyrosine; T, threonine; K, lysine; E, glutamic acid; R, arginine; Q, glutamine; V, valine; M, methionine; N, asparagine; I, isoleucine; WHO, World Health Organization; RBS, receptor binding site; PDB, Protein Data Bank; Sia-α2,6-Gal, sialic acid α2,6-galactose; PR, Puerto Rico; SI, Solomon Island; NC, New Caledonia; To, Totorri; IEDB, Immune Epitope Database; DS, Discovery Studio; RMSD, root-mean-square deviation; H-bonds, hydrogen bonds; ExPASy, Expert Protein Analysis System; APBS, adaptive Poisson–Boltzmann solver; NBCR, National Biomedical Computational Resource; SAVES, structure analysis and verification server; CDRs, complementarity determining regions; MD, molecular dynamics; GOLD, genetic optimization for ligand docking; PEARLS, program of energetic and receptor ligand system; YASARA, yet another scientific artificial reality application; YAMBER3, yet another model building and energy refinement force field 3; TIP3P, transferable intermolecular potential 3p; PME, particle mesh Ewald; NVT, number of particles (N), system volume (V) and temperature (T) constant; NPT, number of particles (N), system pressure (P) and temperature (T) constant; SA, sialic acid; RMSF, root-mean-square fluctuation; H chain, heavy chain; L chain, light chain ⁎ Corresponding author at: Bioinformatics and Data Management Group, National Institute of Virology, 20-A, Dr. Ambedkar Road, Post Box No. 11, Pune 411001, Maharashtra, India. E-mail addresses: [email protected] (A.K. Behera), [email protected] (S. Basu), [email protected], [email protected] (S.S. Cherian).

(Cox and Subbarao, 2000; Guan et al., 2010). The virus belongs to the Orthomyxoviridae family and contains an eight-segmented negativesense RNA genome. Each segment codes for one or two protein/s essential for viral replication and propagation. In the course of infecting a host cell, the two surface glycoproteins of the virus, hemagglutinin (HA) and neuraminidase (NA), play very significant roles. The viral infection starts with attachment of HA to the terminal sialic acid receptor, which facilitates entry of viral RNA into the host cell (Takemoto et al., 1996; Gambaryan et al., 1997). Thus HA plays an important role in receptor binding and membrane fusion. HA is also the principal antigen on the viral surface, and is thus the primary target for neutralizing antibodies (Stevens et al., 2006). After initial infection and entry of viral RNA into the host cell, NA cleaves the sialic acid of the receptor bound HA by its sialase activity, thereby, aiding viral replication and dissemination within the host cells (Wagner et al., 2002; Gamblin and Skehel, 2010). The primary antiviral drug available for therapy, oseltamivir (Moscona, 2005) inhibits the sialase activity of NA, as a result of which the virus remains attached to the host cell, forms aggregates and finally fails to propagate within the host (Kim et al., 1997). Prior to the year 2007, a mutation of histidine to tyrosine at position 274 of NA (H274Y, N2 numbering) was found to cause oseltamivir resistance (Russell et al., 2006; Collins et al., 2009) in a few seasonal human H1N1 influenza strains (Gubareva et al., 2001). However, it was noted that the H274Y mutation caused impaired growth of the viral strains leading to compromised

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Please cite this article as: Behera, A.K., et al., Molecular mechanism of the enhanced viral fitness contributed by secondary mutations in the hemagglutinin protein of oseltamivir r..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.003

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A.K. Behera et al. / Gene xxx (2014) xxx–xxx

viral fitness (Abed et al., 2004; Herlocher et al., 2004) and hence, was of little clinical significance (Ives et al., 2002). Subsequently, from the period 2007–2008 till the emergence of the 2009 H1N1 pandemic, it was observed that seasonal H1N1 strains predominantly showed the H274Y mutation with significantly improved viral fitness (Rameix-Welti et al., 2008) causing oseltamivir resistance worldwide (Moscona, 2009). As per the World Health Organization (WHO) reports (WHO, 2009) 15% of influenza viruses acquired oseltamivir resistance in the late 2007 and early 2008. The proportion of resistant viruses increased dramatically, during 2008– 09, with 95% of influenza viruses showing oseltamivir resistance. It was shown that the H274Y mutation accounts for decreased amount of NA reaching the cell surface which could be counteracted by secondary mutations that restore viral fitness (Collins et al., 2009; Bloom et al., 2010, 2011; Abed et al., 2011). The evolution of oseltamivir resistance was therefore enabled by “permissive” mutations which allowed the virus to tolerate subsequent occurrences of H274Y. In this regard, mutations R222Q, V234M, D344N (Bloom et al., 2010; Collins et al., 2009; Abed et al., 2011), R257K and T289M (Bloom et al., 2011) in NA have been reported to compensate for the deleterious effect of the H274Y mutation. For efficient viral replication, there should be a functional balance between HA and NA (Ohuchi et al., 1997; Kobasa et al., 1999; Matrosovich et al., 1999; Wagner et al., 2002). Phylogenetic studies of HA and NA showed similar clustering patterns, indicating coevolution of these two proteins. Reverse genetics studies revealed that out of a few mutations that have occurred consistently in HA along with the H274Y mutation in the corresponding NA in strains of the post 2007 period, T86K, K144E and R192K (T82K, K141E and R189K, H1 numbering), have been found to be essential for improved viral fitness (Ginting et al., 2012). Among these three mutations, T86K and K144E have been observed to be present from 2006 onwards, whereas, the R192K mutation was a feature of the antigenically drifted, oseltamivir-sensitive H1N1 viruses of 2007. The molecular mechanism of the enhanced viral fitness due to these secondary mutations has not been fully elucidated. Positions 144 and 192 are located in the H1N1 HA antigenic sites while 86 is in the vicinity of an antigenic site. Position 192 is also a part of the receptor binding site (RBS) of HA. In this study, we made an attempt to determine the antigenic effects of the T86K, K144E and R192K mutations on the HA protein using computational modeling and protein–protein docking with the solved structure of an available antigenic site specific antibody. We also attempted to study the effect of the specific mutation at position 192 on the receptor binding affinity of the HA protein by performing protein–ligand docking and molecular dynamics (MD) simulations. The result of our study would be useful in understanding the molecular evolution of oseltamivir-resistant strains and combating them by designing newer vaccines and antivirals.

2. Materials and methods 2.1. Viral strains, antibody and host receptor For this study, pre 2007 influenza A/H1N1 virus strains: A/Solomon Island/3/2006 (SI/06) and A/New Caledonia/20/99 (NC/99); and a post 2007 influenza A/H1N1 virus: A/Tottori/52/2008 (To/08) were considered. Amino acid sequences for the above strains were obtained from the NCBI Reference Sequence database (RefSeq) (Pruitt et al., 2005). The X-ray crystallographic structure of SI/06 in complex with a human antibody CH65 was obtained from the Protein Data Bank (PDB) (Berman et al., 2003) (PDB ID: 3SM5) (Whittle et al., 2011) and the individual structures of SI/06 and CH65 were extracted from the complex. HA and NA were numbered according to the H3 and N2 numbering schemes, respectively. The structure of the human receptor, sialic acid α 2,6-galactose (Sia-α2,6-Gal) was extracted from the X-ray crystallographic structure of influenza A/H1N1 A/Puerto Rico/8/34 (PR/34), the prototype H1N1 strain, in complex with Sia-α2,6-Gal from PDB (PDB ID: 1RVZ) (Gamblin et al., 2004).

2.2. Sequence-based analyses The immune epitope database and analysis resource (IEDB) server (www.iedb.org) was used considering the parameters of antigenic propensity, hydrophilicity, and surface accessibility to locate the continuous epitopes within a polypeptide chain. Antigenicity prediction is based on the Kolaskar and Tongaonkar method which is a semiempirical method derived from the physicochemical properties of amino acid residues and their frequencies of occurrence in experimentally known segmental epitopes (Kolaskar and Tongaonkar, 1990). The method can detect antigenic peptides with about 75% accuracy at a threshold setting of 1.00. The Parker hydrophilicity prediction method also used to locate linear epitopes (Parker et al., 1986), is based on a hydrophilicity scale, derived from the contribution in high-performance liquid chromatography (HPLC) of each amino acid side chain to the retention time of model synthetic peptides. Further, the Emini surface accessibility (Emini et al., 1985) method used to predict the surface feature of the desired protein sequences is based on indices of surface probability. According to this method, the surface probability (S) at sequence position ‘n’ can be defined as Sn, for a random hexapeptide sequence, with probabilities greater than 1.0 indicating an increased probability of being found on the surface. 2.3. Protein three dimensional (3D) structure visualization and analysis All the 3D structures of the proteins were visualized using Discovery Studio v3.5 (DS) (www.accelrys.com/products). It was also used to induce in silico point mutations in the HA molecule, superimpose structures, calculate root-mean-square deviation (RMSD) and analyze the intra- and inter-molecular hydrogen bonds (H-bonds). For protein–ligand interactions, the intermolecular H-bonds and hydrophobic interactions were monitored using Ligplot + v1.4.4 interface applying the LIGPLOT program (Wallace et al., 1995). Electrostatic surface charge distribution was studied using the Adaptive Poisson–Boltzmann Solver (APBS) calculation (Baker et al., 2001) in the web service of National Biomedical Computational Resource (NBCR) (www.nbcr.ucsd.edu) and was visualized in Chimera to obtain high resolution images (Pettersen et al., 2004; http:// www.cgl.ucsf.edu/chimera). 2.4. Homology modeling The structure of the HA of To/08 strain was obtained by homology modeling using SWISS-MODEL accessed from the ExPASy web server (Guex and Peitsch, 1997; Schwede et al., 2003; Arnold et al., 2006), using the structure of HA of SI/06 (PDB ID: 3SM5) as the template. The structure of the HA of NC/99 was also modeled using the above tool with HA of influenza A/H1N1 virus A/Thailand/Cu41/2006 (PDB ID: 4EDB) as the template. The modeled structures were validated in the Structure Analysis and Verification Server (SAVES) using PROCHECK, WHAT_CHECK, ERRAT, VERIFY3D and PROVE programs (http:// nihserver.mbi.ucla.edu/SAVES). 2.5. Antigen–antibody docking The antibody CH65 was docked with HA of SI/06 and To/08 using the online server ZDOCK v3.0.2 (Pierce et al., 2011). ZDOCK does rigid body docking which is performed using a Fast Fourier Transform algorithm and gives the output by evaluating each binding mode with an energy scoring function (Z-score). To define the binding site for docking as well as for filtering the predicted poses in the output, the entire RBS (residue positions 127–145, 188–200 and 220–230) of HA, which also includes the antigenic site to which CH65 binds, along with the complementarity determining regions (CDRs) of CH65 were selected and the rest of the residues of both the proteins were blocked (Chen and Weng, 2002). The top 10 predicted complexes were considered and the best predicted docked complex was chosen for further analysis.

Please cite this article as: Behera, A.K., et al., Molecular mechanism of the enhanced viral fitness contributed by secondary mutations in the hemagglutinin protein of oseltamivir r..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.003

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2.6. HA protein — Sia-α2,6-Gal ligand docking and MD simulations Small molecule docking of the HA of SI/06 and To/08 was carried out with Sia-α2,6-Gal using GOLD suite v5.2 (Jones et al., 1997). GOLD performs flexible docking and uses a genetic algorithm to dock protein and ligand molecules. Water molecules were removed from the protein and both the molecules were hydrogenated. Constrained docking was performed by selecting a central residue in the RBS (Tyr95) as the reference with atoms in a 10 Å radius. The docked complexes were ranked using Goldscore (Verdonk et al., 2003). The binding energy of the best result was studied using the Program of Energetic and Receptor Ligand System (PEARLS BIDD) server (Wang et al., 2004; Filikov et al., 2000). The total interaction energy between the ligand and the receptor, in this case, includes Van der Waal's energy, electrostatic energy, H-bond energy, solvation energy and conformational entropy terms. The best docked complex of the HA of SI/06 and To/08 with Sia-α2,6Gal was considered for MD simulations using YASARA package version 13.6.16 (YASARA Biosciences). A periodic simulation cell with boundaries extending 20 Å from the surface of the complex was employed with explicit TIP3P water model. A physiological strength (0.15 M) of counter ions (Cl− and Na+) was added to achieve a neutral simulation cell. To remove bumps and correct the covalent geometry, the structure was energy-minimized using the YAMBER3 (Yet Another Model Building and Energy Refinement force field 3) force field (Krieger et al., 2004) with a 7.86 Å cutoff and the Particle Mesh Ewald (PME) method to treat long range electrostatic interactions. Before initiation of simulations, a short steepest descent minimization was followed for removal of conformational stress, and the procedure continued by simulated annealing (integration time steps were set to 2 and 1.25 fs for intra- and inter-molecular forces, respectively, atom velocities scaled down by 0.9 every 10th step) until convergence was reached i.e. the energy improved by less than 0.05 kJ/mol per atom during 200 steps. Minimization was followed by an equilibration procedure using the NVT ensemble to 300 K. The resulting minimized and equilibrated models were subsequently used for MD simulations. The simulations were run at constant pressure (NPT ensemble) and the temperature was controlled by rescaling atom velocities using a Berendsen thermostat (Berendsen et al., 1984). The production simulations were carried out for 10 ns and snapshots of the trajectories were captured at each 1 ps time interval. Analyses of the MD trajectories for the SI/06 and To/08 complexes in terms of RMSD and root-mean-square fluctuation (RMSF) calculations were performed using YASARA and the Grace plotting tool (http://plasma-gate.weizmann.ac.il/Grace/). Further, the Hbond occupancy of amino acid residues during the MD simulation was calculated using Molecular Dynamics Trajectory Analyzer (MDTRA) software (http://bison.niboch.nsc.ru/mdtra.html).

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On the other hand, the RBS of the HA molecule comprises of the 130loop, 190-helix and 220-loop and residues of these structural elements form crucial contacts with the sialic acid of the host receptors. The binding of the terminal sialic acid to the penultimate sugar, galactose, mainly determines the species specificity of the virus. The HA of human viruses recognizes sialic acid bound with a α2-6 glycosidic bond to the galactose (Rogers and D'Souza, 1989) in a cis conformation (Jongkon et al., 2009). These receptors are mainly found in the epithelial surface of the upper respiratory tract, the primary site of infection (Xu et al., 2012). As the three seasonal H1N1 strains (NC/99, SI/06 and To/08) considered in the study had been obtained from human cases, the receptor binding study was carried out with the human receptor, Sia-α2,6-Gal. 3.1. Sequence analyses The secondary mutations in HA of the H1N1 strains, T86K, K144E and R192K, are shown in Fig. 1 with the known antigenic sites marked using red boxes (Brownlee and Fodor, 2001). As can be seen from Fig. 1, SI/06 possesses two mutations, T86K and K144E; To/08 possesses all the three mutations, T86K, K144E and R192K while NC/99 possesses none of these mutations and typically represents an oseltamivir susceptible strain. Stretches of peptide containing residues at positions 86, 144 and 192 were predicted as epitopes and as such no changes were observed in the average antigenic propensity due to the mutations (Table S1). The surface accessibility prediction also showed no such change in surface accessibility due to mutations T86K and R192K, but the stretch of residues around position 144E showed reduced surface accessibility (Fig. 1). 3.2. Three dimensional model construction of HA structures and validation The HA sequence of To/08 shares an identity of 97.2% with the sequence of the template (PDB ID: 3SM5). The built homology model showed an RMSD of 0.07 Å with the template. In case of the constructed HA protein of NC/99, which shared a sequence identity of 97.21% with the template (PDB ID: 4EDB), the backbone RMSD was found to be 0.06 Å. Both the models were validated in SAVES. The models had N95% of the residues in the allowed region, ~ 5% of the residues in the generously allowed region and no residue in the disallowed region of the Ramachandran plot. The models showed similar tertiary folds and secondary structural elements as the templates. However, the electrostatic surface potential calculation revealed that the distribution of surface charge over the proteins showed a drastic change in and around residue position 144 due to the non-synonymous mutation K to E (Fig. 2). No significant change in surface charge was observed around positions 86 and 192. A slight change in surface contour was observed around the R192K mutation.

3. Results 3.3. Docking of antibody CH65 with HA structures Mutations T86K, K144E, and R192K of the HA protein were investigated to understand their possible effects on aspects of antigen–antibody interaction and/or sialic acid receptor binding property. The globular head region of the HA molecule contains the crucial residues for antibody-binding, which generally cluster into five antigenic groups: Sa, Sb, Ca1, Ca2 and Cb (Jackson et al., 1982; Caton et al., 1982). The residue position 144 of HA is a part of the antigenic site Ca2 and 192 is a part of the antigenic site Sb which also falls in the RBS. Though position 86 is not strictly a part of either of the antigenic sites or in the RBS, it is present in the vicinity of the Cb site. Therefore, amino acid physiochemical properties of hydrophilicity, antigenic propensity etc. were predicted for the three H1N1 influenza strains, in order to check for possible changes in these characteristics due to the specific mutations. Further, considering the availability of the structure of an Sb-specific antibody, we carried out antigen–antibody docking studies to understand the possible effect of the R192K mutation on antibody binding.

The crystal structure of the HA of SI/06 complexed with CH65 along with the intermolecular contacts made is shown in Fig. 3A. Residue R192 in the HA of SI/06 is observed to make a salt-bridge with the light (L) chain residue, D95 of CH65. R192 was also found to make a salt-bridge contact with E198 of the HA chain. The other contacts between the antigen and antibody included seven and five H-bonds with the heavy chain and light chain, respectively. On the other hand, among the top ten docked complexes of the HA of To/08 and CH65 obtained from ZDOCK, none of the complexes showed binding of the antibody in the Sb antigenic site. The best ranked docked complex indicated contacts with residues other than the Sb site (Fig. 3B). To substantiate the possible role of K192 in the binding escape of the antibody, we carried out a similar docking with an in silico induced R192K mutation in the SI/06 HA (mutSI/06) and CH65. The docking result showed that the mutated (R192K) HA of SI/06 also failed to bind to CH65 in the Sb site (Fig S1).

Please cite this article as: Behera, A.K., et al., Molecular mechanism of the enhanced viral fitness contributed by secondary mutations in the hemagglutinin protein of oseltamivir r..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.003

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Fig. 1. Sequence alignment of the HA protein of three representative H1N1 influenza viruses (A/New Caledonia/20/99; NC/99, A/Solomon Island/3/2006; SI/06 and A/Tottori/52/2008; To/08) showing the actual antigenic sites (red blocks) and the predicted surface accessibility from IEDB by Emini method (brown underlines); residues 86, 144 and 192 are highlighted in yellow. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.4. Docking of human receptor ligand with HA structures and MD simulations The docking of Sia-α2,6-Gal with the HA of SI/06 showed five H-bonds and seven hydrophobic contacts, whereas, that with the HA of To/08 showed three H-bonds and ten hydrophobic contacts (Fig. 4A, C). The contacts between these two complexes were found to be almost similar and the ligand also maintained the cis conformation in both the complexes. Importantly, the 190 helix of SI/06 formed an H-bond with residue D190 while that of To/08 formed a hydrophobic contact (Table S2). Good

Goldscores (52.15 and 52.28 for SI/06 and To/08, respectively) and interaction energy values (− 7.94 kcal/mol for SI/06 and −9.90 kcal/mol for To/08) were observed. Further, to understand the stability of the protein–ligand complexes, MD simulations were performed for 10 ns. During the simulation, the Cα-RMSD, the Cα-RMSF and the H-bond occupancy as a function of simulation time were calculated. Both SI/06 and To/ 08 attained stability at around 6 ns with average fluctuations of ~ 1 Å (Fig. 5A). The RMSF of the residue R192 in SI/06 was found to be 1.3 Å, and that of K192 in To/08 was found to be 1.7 Å (Fig. 5B). The residues at positions 86 and 144 showed RMSF of ~1.9 Å and ~1.4 Å respectively

Fig. 2. Electrostatic charge on the surface of the HA structures of influenza strains (A) A/New Caledonia/20/99 (B) A/Solomon Island/3/2006 and (C) A/Tottori/52/2008. Blue, white and red colors indicate basic, neutral, and acidic charge distributions, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: Behera, A.K., et al., Molecular mechanism of the enhanced viral fitness contributed by secondary mutations in the hemagglutinin protein of oseltamivir r..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.003

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in both SI/06 and To/08. Among the 130-loop, 190-helix and 220-loop, the 190 helix (residue positions 188–190) showed slightly increased flexibility in To/08 (on average 0.33 Å more fluctuation than that in SI/06). The H-bond occupancy results showed that, R192 of SI/06 and K192 of To/08 formed intramolecular H-bonds with I188 having 100% and 99% H-bond occupancy respectively (Table 1). Moreover, R192 of SI/ 06 formed another intramolecular H-bond with E198 possessing 84% H-bond occupancy, which was not reflected in K192 of To/08 over the period of the MD simulation run. The average bond energy and bond length of the R192-E198 H-bond in SI/06 was observed to be −2.080 kcal/mol and 3.088 Å, respectively. The other sites of interest (residue positions 86 and 144) showed no significant difference in H-bond occupancy. The protein–ligand interaction energy was further calculated for the conformation after 5 ns and 10 ns for both the SI/06 and To/08 complexes (Table S3). For SI/06, the 5 ns structure possessed a ligand interaction energy of − 9.15 kcal/mol and the 10 ns structure, − 9.81 kcal/mol. For To/08, the 5 ns structure showed ligand interaction energy of − 9.46 kcal/mol and that for the 10 ns structure was, −8.42 kcal/mol. Notably, the interactions at 10 ns showed that the H-bond contacts increased to six in SI/06, the additional bond being formed with D190 (Fig. 4B, D; Table S2). 4. Discussions HA and NA, the surface glycoproteins of influenza viruses have significant roles in spreading the viral infection. Particularly, NA has been the major drug target to control the influenza infection over the last decade. Oseltamivir, one of the most effective anti-NA drugs to treat influenza has been the primary drug of choice until oseltamivir-resistant H1N1 seasonal strains (carrying NA-H274Y) predominantly came into circulation (WHO, 2010). Secondary mutations, occurring in NA and HA in the post 2007 strains, were found to compensate for deleterious effects of low growth capability known to arise as a consequence of the H274Y mutation. In this study we attempted to throw some light on the molecular mechanism behind the improved viral fitness due to three such secondary mutations, T86K, K144E and R192K, reported in the HA protein. Seasonal H1N1 viruses cluster into four antigenic clades, three of which are represented by former vaccine strains: the NC/99-like clade 1; the SI/06-like clade 2A; the A/Brisbane/59/2007-like clade 2B and the non-vaccine strain A/St. Petersburg/10/2007-like clade 2C (Hay et al., 2008). Among the compensatory mutations in HA of the seasonal H1N1 viruses, the R192K was one of the key mutations of clade 2B viruses that diverged from clades 2A and 2C (Zaraket et al., 2010). A few other mutations in HA (A193T), NA (H274Y, D357G), and other viral proteins in effect classified clade 2B into oseltamivir-sensitive (clade 2B.1) and oseltamivir-resistant (clade 2B.2) viruses (Yang et al., 2011). To determine the antigenic effects of the R192K mutation, we studied the X-ray crystallographic structure of SI/06 HA bound to the Sb site specific antibody CH65 (PDB ID: 3SM5) and compared it to the docking interaction of To/08 HA (possessing the R192K mutation) with CH65. Analysis of the crystal structure showed that the contacts made by T155 and R192 of SI/06 HA are important as these two contacts made the shortest distance H-bond with CH65; T155 (HA) — Y109 (H chain, CH65): 2.79 Å and R192 (HA) — D95 (L chain, CH65): 2.52 Å. The interaction of R192 with the D95 of the L chain of CH65 (Fig. 3A) forming a salt bridge is significant in the context of antibody binding. R192 was also found to make an intramolecular contact with E198 of the HA molecule which might be further contributing to the stability of the complex. Docking studies of To/08 and CH65 indicated that the antibody failed to bind to the Sb site of the HA of To/08 (Fig. 3B). The alignment of the HA sequences of SI/06 and To/08 showed that the sequences differ in only two positions, T132V and R192K, of which only residue position 192 falls in the specific antigenic site. In order to focus on the role of R192K mutation in the antibody escape, CH65 was also docked with the HA of mutSI/06 (wherein the mutation R192K was induced in HA

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of SI/06). This complex (Fig S1) also failed to show binding in the Sb site. Further, in the mutSI/06-CH65 complex, it was seen that the 2.52 Å separation between D95 of the CH65 light chain and R192 of the HA increased to ~ 4.1 Å after the HA-R192K substitution (Fig. 3C), which exceeds the normal distance for a salt bridge (b4 Å) (Kumar and Nussinov, 2002). It was also noted that the R192K mutation resulted in a loss of an intramolecular contact between R192 and E198 (Fig. 3B, C). The change in antigen surface morphology in the absence of the guanidinium group of arginine, is a vital feature that may explain both the loss of the bond between the antibody and antigen (D95-R192) as well as the loss of the intramolecular contact. These factors may together have contributed to an escape from antibody recognition by the HA molecule. On the other hand, in case of the K144E mutation, the electrostatic surface charge distribution on the HA showed that the surface charge distribution around residue position 144 markedly changed from basic (positive charge) to acidic (negative charge) (Fig. 2). Antigen–antibody interactions majorly involves non-covalent interactions which are very much susceptible to electrostatic charge, hence, the change in surface charge around the 144 position can be expected to influence antibody binding. Further, from the sequence-based analysis of surface accessibility, it was found that the stretch of the peptide around position 144 had reduced surface accessibility due to the K144E mutation, which can also be expected to have some effect on the antibody binding to this position. No other significant changes were observed in the sequence-based analysis. The K144E mutation is located in the Ca2 antigenic site, however due to non-availability of the crystal structure of a Ca2 specific antibody the effect of the mutation on the antibody binding could not be studied. Similarly, in the absence of a Cb specific antibody, the effect of the T86K mutation could not be understood with respect to antibody binding. The effect of the R192K mutation, which is also located in the RBS, on the receptor binding property was studied by performing small molecule docking. The contacts in both SI/06 HA and To/08 HA were found to be similar to that observed in the crystal structure of the complex formed by HA of PR/34 with Sia-α2,6-Gal. The RMSD plot corresponding to the MD simulations of these complexes (Fig. 5) showed that the structures attained stability at around 6 ns, after which the fluctuations were within 1 Å. From the calculated protein–ligand interaction energy, it was found that the SI/06 complex showed a gradual change in the interaction energy from −7.94 kcal/mol to −9.81 kcal/mol and the To/08 complex from −9.90 kcal/mol to −8.42 kcal/mol over the 10 ns simulation. Thus it can be seen that the interaction energy value decreased by almost 2 kcal/mol in the SI/06 complex while by almost −1.5 kcal/mol in the To/08 complex, implying that the binding affinity is energetically more favorable in SI/06 than To/08. Careful examination of the interaction energy terms (Table S3), shows that a major contribution of the interaction energy change in case of SI/06 is from the H-bond energy, which can be correlated to the additional H-bond formed with the 190 helix residue, D190. From another view, the intramolecular H-bond pattern of the complexes had shown that R192 of SI/06 formed a vital contact with E198 through a salt-bridge while this important contact was completely lost in To/08 having the R192K mutation. Thus our simulation study had demonstrated that the R192K mutation abrogates the intramolecular contact between R192 and E198, due to which the K192 residue may have gained relative positional flexibility. Further, the RMSF values for residues 86 and 144 had been observed to be almost same in SI/06 and To/08, while the 192 position and significantly the 190 helix also had shown slightly increased flexibility in To/08 which may also be on account of the lost contact between K192 and E198. Thus, the reduced binding affinity of To/08 HA with Sia-α2,6-Gal may be interpreted as a consequence of the absence of H-bonds with the 190 helix and the increased flexibility of the 190 helix. Considering the functional balance between HA and NA, it was expected that the secondary mutations found in post 2007 strains in HA may cause lesser or weaker binding to the receptor, balancing the reduced number of NA on the viral surface due to H274Y mutation available to cleave the sialic group of the receptor bound HA.

Please cite this article as: Behera, A.K., et al., Molecular mechanism of the enhanced viral fitness contributed by secondary mutations in the hemagglutinin protein of oseltamivir r..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.003

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A.K. Behera et al. / Gene xxx (2014) xxx–xxx

Please cite this article as: Behera, A.K., et al., Molecular mechanism of the enhanced viral fitness contributed by secondary mutations in the hemagglutinin protein of oseltamivir r..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.003

A.K. Behera et al. / Gene xxx (2014) xxx–xxx

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Fig. 4. 2D interaction diagram of Sia-α2,6-Gal with HA of: A. A/Solomon Island/3/2006 at 0 ns; B. A/Solomon Island/3/2006 at 10 ns; C. A/Tottori/52/2008 at 0 ns; and D. A/Tottori/52/2008 at 10 ns using LigPlot + v1.4.4.

There are some experimental data in literature that substantiate our results. Growth kinetics of reverse genetics recombinant viruses based on To/08 in MDCK cells (Ginting et al., 2012), showed low early titre (about 2 log difference at 24 h) but similar 48- and 72-hour titers as in SI/06 oseltamivir sensitive viruses. Further, though the recombinant with the To/08 HA and the SI/06-like NA demonstrated high early titers,

its growth dropped by 72 h. These observations are indicative that the To/08-like HA may be possessing weaker receptor binding properties and also co-operates better with a To/08-like oseltamivir resistant NA. Other reports (Collins et al., 2009; Meijer et al., 2009; Rameix-Welti et al., 2008; Zaraket et al., 2010; Wagner et al., 2002) also suggest that the optimal interplay between the “receptor-binding activity” of

Fig. 3. Antigen–antibody complexes showing the hydrogen bond (H-bond) interactions and significant distances. A: Crystal structure complex of CH65 bound to HA of wild-type A/Solomon Island/3/2006; B: Docking of CH65 with HA of A/Tottori/52/2008 using ZDOCK. A flip of the antibody, distinct from the Sb antigenic site, is indicated with a dashed curve; C: SI/06-CH65 crystal structure complex with R192K point mutation. All HA chains are indicated in purple ribbon, H chain of CH65 in green ribbon and L chain in brown ribbon. The H-bonds are indicated as red dotted lines. Figure insets show close-up views and the interacting residues. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: Behera, A.K., et al., Molecular mechanism of the enhanced viral fitness contributed by secondary mutations in the hemagglutinin protein of oseltamivir r..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.003

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A.K. Behera et al. / Gene xxx (2014) xxx–xxx

Fig. 5. Overall stability of the A/Solomon Island/3/2006 (SI/06) and A/Tottori/52/2008 (To/08) HA in complex with Sia-α2,6-Gal during the 10 ns MD simulation; A. Cα RMSD plot; and B. RMSF of each residue. The black and red lines correspond to SI/06 and To/08 respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Brisbane/59-like HA and the “receptor-destroying activity” of oseltamivirresistant NA is important for efficient viral replication. Further, in a guinea pig transmission model (Bouvier et al., 2012), both an oseltamivir-resistant Brisbane/59-like clinical virus isolate of 2008 as well as an oseltamivir-resistant recombinant virus transmitted significantly better than a similar, contemporaneous oseltamivir-sensitive isolate and a similar oseltamivir sensitive recombinant. Though their study suggested that the improved transmissibility may be attributed to the NA gene possessing mutations H274Y and/or D357G (D354G, N2 numbering), it may be noted that both the viruses considered by them, like To/08, already encoded the HA-R192K change. Thus their result also demonstrated that a To/08-like HA works better with an oseltamivir-resistant NA than an oseltamivir-sensitive one. Based on our HA-antibody docking results, it may be speculated that host antibody failing to bind to the To/ 08-like HA may have a role to play in the resistant Brisbane/59-like viruses out-transmitting the sensitive ones. There are other reports that have also proposed and demonstrated that the influenza A virus evolves by adjusting receptor binding avidity via amino acid substitutions throughout the HA globular domain, many of which simultaneously alter antigenicity (Hensley et al., 2009; Das et al., 2011; Myers et al., 2013).

specifically with modified antibody interactions. Either or both of these factors may have contributed to increased viral fitness. The results are essentially consistent with available experimental data. The deeper understanding of the molecular mechanism gained from this study may also provide a basis for designing antivirals against oseltamivirresistant strains and predicting the evolution of oseltamivir resistance in other influenza strains. Conflict of interest None. Acknowledgments AKB is thankful for the Senior Research Fellowship from the National Institute of Virology, Pune. The authors are thankful to Dr A.C. Mishra, Former Director and Dr D.T. Mourya, Director, National Institute of Virology for the facilities provided, encouragement and support. The authors also acknowledge the anonymous reviewers for their valuable inputs that have helped in considerable improvement of this manuscript.

5. Conclusions

Appendix A. Supplementary data

The results from our in silico studies provide further insight into the mechanisms contributing to the extensive spread and exponential increase of oseltamivir-resistant Brisbane/59-like seasonal H1N1 influenza viruses prior to the emergence of the 2009 H1N1 pandemic. Our studies indicated that the R192K mutation in the HA of H1N1 influenza A strains induced an Sb site-specific antibody escape and altered the receptor-binding avidity of the HA of oseltamivir-resistant viruses, while the two other mutations, K144E and T86K appear to be associated

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.12.003.

Table 1 H-bond occupancy of amino acid residues at positions 86, 144 and 192 during the 10 ns MD simulation for A/Solomon Island/3/2006 and A/Tottori/52/2008 complexes of respective HA proteins with Sia-α2,6-Gal. Strain

Intramolecular H-bond contacts

H-bond occupancy (%)

SI/06

K86…G54 E144…H141 R192…I188 R192…E198 K86…G54 E144…H141 K192…I188

2 99 100 84 1 99 99

To/08

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Please cite this article as: Behera, A.K., et al., Molecular mechanism of the enhanced viral fitness contributed by secondary mutations in the hemagglutinin protein of oseltamivir r..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.003