The significance of naturally occurring neuraminidase quasispecies of H5N1 avian influenza virus on resistance to oseltamivir: a point of concern.

Viral adaptability and survival arise due to the presence of quasispecies populations that are able to escape the immune response or produce drug-resistant variants. However, the presence of H5N1 virus with natural mutations acquired without any drug selection pressure poses a great threat. Cloacal samples collected from the 2004-2005 epidemics in Thailand from Asian open-billed storks revealed one major and several minor quasispecies populations with mutations on the oseltamivir (OTV)-binding site of the neuraminidase gene (NA) without prior exposure to a drug. Therefore, this study investigated the binding between the NA-containing novel mutations and OTV drug using molecular dynamic simulations and plaque inhibition assay. The results revealed that the mutant populations, S236F mutant, S236F/C278Y mutant, A250V/V266A/P271H/G285S mutant and C278Y mutant, had a lower binding affinity with OTV as compared with the WT virus due to rearrangement of amino acid residues and increased flexibility in the 150-loop. This result was further emphasized through the IC50 values obtained for the major population and WT virus, 104.74 nM and 18.30 nM, respectively. Taken together, these data suggest that H5N1 viruses isolated from wild birds have already acquired OTV-resistant point mutations without any exposure to a drug.

Role of R292K mutation in influenza H7N9 neuraminidase toward oseltamivir susceptibility: MD and MM/PB(GB)SA study.

The H7N9 avian influenza virus is a novel re-assortment from at least four different strains of virus. Neuraminidase, which is a glycoprotein on the surface membrane, has been the target for drug treatment. However, some H7N9 strains that have been isolated from patient after drug treatment have a R292K mutation in neuraminidase. This substitution was found to facilitate drug resistance using protein- and virus- assays, in particular it gave a high resistance to the most commonly used drug, oseltamivir. The aim of this research is to understand the source of oseltamivir resistance using MD simulations and the MM/PB(GB)SA binding free energy approaches. Both methods can predict the reduced susceptibility of oseltamivir in good agreement to the IC 50 binding energy, although MM/GBSA underestimates this prediction compared to the MM/PBSA calculation. Electrostatic interaction is the main contribution for oseltamivir binding in terms of both interaction and solvation. We found that the source of the drug resistance is a decrease in the binding interaction combined with the reduction of the dehydration penalty. The smaller K292 mutated residue has a larger binding pocket cavity compared to the wild-type resulting in the loss of drug carboxylate-K292 hydrogen bonding and an increased accessibility for water molecules around the K292 mutated residue. In addition, oseltamivir does not bind well to the R292K mutant complex as shown by the high degree of fluctuation in ligand RMSD during the simulation and the change in angular distribution of bulky side chain groups.

Glycan binding and specificity of viral influenza neuraminidases by classical molecular dynamics and replica exchange molecular dynamics simulations.

Two important glycoproteins on the influenza virus membrane, hemagglutinin (HA) and neuraminidase (NA), are relevant to virus replication. As previously reported, HA has a substrate specificity towards SIA-2,3-GAL-1,4-NAG (3SL) and SIA-2,6-GAL-1,4-NAG (6SL) glycans, while NA can cleave both types of linkages. However, the substrate binding into NA and its preference are not well understood. In this work, the glycan binding and specificity of human and avian NAs were evaluated by classical molecular dynamics (MD) simulations, whilst the conformational diversity of 3SL avian and 6SL human glycans in an unbound state was investigated by replica exchange MD simulations. The results indicated that the 3SL avian receptor fits well in the binding cavity of all NAs and does not require a conformational change for such binding compared to the flexible shape of the 6SL human receptor. From the QM/MM-GBSA binding free energy and decomposition free energy data, 6SL showed a much stronger binding towards human NAs (H1N1, H2N2 and H3N2) than to avian NAs (H5N1 and H7N9). This suggests that influenza NAs have a substrate specificity corresponding to their HA, indicating the functional balance between the two important glycoproteins. Both linkages show distinct glycan topologies when complexed with NAs, while the flexibility of torsion angles between GAL and NAG in 6SL results in the various shapes of glycan and different binding patterns. Lower conformational diversities of both glycans when bound to NA compared to the unbound state were found, and were required in order to be accommodated within the NA cavity. Communicated by Ramaswamy H. Sarma.

A 3D-RISM/RISM study of the oseltamivir binding efficiency with the wild-type and resistance-associated mutant forms of the viral influenza B neuraminidase.

The binding affinity of oseltamivir to the influenza B neuraminidase and to its variants with three single substitutions, E119G, R152K, and D198N, is investigated by the MM/3D-RISM method. The binding affinity or the binding free energy of ligand to receptor was found to be determined by a subtle balance of two major contributions that largely cancel out each other: the ligand-receptor interactions and the dehydration free energy. The theoretical results of the binding affinity of the drug to the mutants reproduced the observed trend in the resistivity, measured by IC50 ; the high-level resistance of E119G and R152K, and the low-level resistance of D198N. For E119G and R152K, reduction of the direct drug-target interaction, especially at the mutated residue, is the main source of high-level oseltamivir resistance. This phenomenon, however, is not found in the D198N strain, which is located in the framework of the active-site.