ABSTRACT
Identification of amino-acid substitutions in the neuraminidase (NA) of low-pathogenic avian influenza (AI) H9N2 viruses is important to study the susceptibility to NA inhibitors (NAI). To identify mutations under NAI selective pressure, the virus was serially passaged with increasing levels of either oseltamivir or zanamivir in ovo, and the growth of the viruses in the presence and absence of NAI's compared. Mutations R292 K in the presence of oseltamivir and E119D in presence of zanamivir were observed within passage one and two respectively. The R292 K mutation reduced oseltamivir susceptibility significantly (2,523-fold) and moderately reduced susceptibility to zanamivir. The E119D mutation significantly reduced susceptibility to zanamivir (415-fold) and remained susceptible to oseltamivir. Genetic stability of the mutations assessed by serial passages of the mutant viruses in eggs without drug pressure resulted in the loss of these mutations, making the virus susceptible to both the drugs. Molecular modeling and dynamics simulations revealed that the R292 K mutation disrupted oseltamivir binding similar to other group 2 NAs, while a different mechanism was noted for zanamivir binding for both R292 K and E119D mutations. The study highlights the need for regular susceptibility screening of circulating AI viruses.
Subject(s)
Antiviral Agents/pharmacology , Influenza A Virus, H9N2 Subtype/drug effects , Influenza A Virus, H9N2 Subtype/genetics , Neuraminidase/antagonists & inhibitors , Oseltamivir/pharmacology , Zanamivir/pharmacology , Amino Acid Substitution , Animals , Chick Embryo , Drug Resistance, Viral , Enzyme Inhibitors/pharmacology , Humans , Molecular Dynamics Simulation , MutationABSTRACT
An F35L mutation in the N-terminal domain of the polymerase acidic protein (PA-Nter), which contains the active site of the endonuclease, has been reported to result in higher polymerase activity in mouse-adapted strains of the 2009 pandemic influenza A H1N1 virus. We modeled wild and mutant complexes of uridine 5'-monophosphate (UMP) as the endonuclease substrate and performed molecular dynamics simulations. The results demonstrated that the F35L mutation could result in a changed orientation of a helix containing active site residues and improve the ligand affinity in the mutant strain. This study suggests a molecular mechanism of enhanced polymerase activity.
Subject(s)
Endonucleases/chemistry , Influenza A Virus, H1N1 Subtype/chemistry , Mutation , RNA-Dependent RNA Polymerase/chemistry , Uridine Monophosphate/chemistry , Viral Nonstructural Proteins/chemistry , Amino Acid Motifs , Amino Acid Substitution , Animals , Catalytic Domain , Crystallography, X-Ray , Endonucleases/genetics , Endonucleases/metabolism , Gene Expression , Humans , Influenza A Virus, H1N1 Subtype/enzymology , Influenza A Virus, H1N1 Subtype/genetics , Leucine , Mice , Molecular Dynamics Simulation , Phenylalanine , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , Substrate Specificity , Uridine Monophosphate/metabolism , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolismABSTRACT
The cap binding domain of the polymerase basic 2 (PB2) subunit of influenza polymerases plays a critical role in mediating the 'cap-snatching' mechanism by binding the 5' cap of host pre-mRNAs during viral mRNA transcription. Monitoring variations in the PB2 protein is thus vital for evaluating the pathogenic potential of the virus. Based on selection pressure analysis of PB2 gene sequences of the pandemic H1N1 (pH1N1) viruses of the period 2009-2014, we identified a site, 344V/M, in the vicinity of the cap binding pocket showing evidence of adaptive evolution and another co-evolving residue, 354I/L, in close vicinity. Modelling of the three-dimensional structure of the pH1N1 PB2 cap binding domain, docking of the pre-mRNA cap analogue m7GTP and molecular dynamics simulation studies of the docked complexes performed for four PB2 variants observed showed that the complex possessing V344M with I354L possessed better ligand binding affinity due to additional hydrogen bond contacts between m7GTP and the key residues His432 and Arg355 that was attributed to a displacement of the 424 loop and a flip of the side chain of Arg355, respectively. The co-evolutionary mutations identified (V344M, I354L) were found to be established in the PB2 gene of the pH1N1 viral population over the period 2010-2014. The study demonstrates the molecular basis for the enhanced m7GTP ligand binding affinity with the 344M-354L synergistic combination in PB2. Furthermore, the insight gained into understanding the molecular mechanism of cap binding in pH1N1 viruses may be useful for designing novel drugs targeting the PB2 cap binding domain.