ABSTRACT
[This corrects the article DOI: 10.1371/journal.pone.0063083.].
ABSTRACT
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.
Subject(s)
Influenza A Virus, H1N1 Subtype/physiology , Mutation, Missense , RNA Cap Analogs/metabolism , RNA Cap-Binding Proteins/genetics , RNA Cap-Binding Proteins/metabolism , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Evolution, Molecular , Humans , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/isolation & purification , Influenza, Human/virology , Models, Molecular , Molecular Conformation , Molecular Docking Simulation , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Binding , RNA Cap-Binding Proteins/chemistry , RNA-Dependent RNA Polymerase/chemistry , Viral Proteins/chemistryABSTRACT
Avian influenza viruses of subtype H5N1 circulating in animals continue to pose threats to human health. The binding preference of the viral surface protein hemagglutinin (HA) to sialosaccharides of receptors is an important area for understanding mutations in the receptor binding site that could be the cause for avian-to-human transmission. In the present work, we studied the effect of two receptor binding site mutations, S221P singly and in combination with another mutation K216E in the HA protein of influenza A H5N1 viruses. Docking of sialic acid ligands corresponding to both avian and human receptors and molecular dynamics simulations of the complexes for wild and mutant strains of H5N1 viruses were carried out. The H5N1 strain possessing the S221P mutation indicated decreased binding to α2,3-linked sialic acids (avian receptor, SAα2,3Gal) when compared to the binding of the wild-type strain that did not possess the HA-221 mutation. The binding to α2,6-linked sialic acids (human receptor, SAα2,6Gal) was found to be comparable, indicating that the mutant strain shows limited dual receptor specificity. On the other hand, the S221P mutation in synergism with the K216E mutation in the binding site, resulted in increased binding affinity for SAα2,6Gal when compared to SAα2,3Gal, indicative of enhanced binding to human receptors. The in-depth study of the molecular interactions in the docked complexes could explain how co-occurring mutations in the HA viral protein can aid in providing fitness advantage to the virus, in the context of host receptor specificity in emerging variants of H5N1 influenza viruses.
Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Influenza A Virus, H5N1 Subtype , Molecular Dynamics Simulation , Mutation , Protein Conformation , Animals , Codon , Humans , Hydrogen Bonding , Ligands , Models, Molecular , Protein Binding , Receptors, Virus/chemistry , Receptors, Virus/metabolism , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Antiviral Agents/pharmacology , Drug Resistance, Viral/genetics , Genetic Fitness , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Influenza A Virus, H1N1 Subtype/drug effects , Influenza A Virus, H1N1 Subtype/genetics , Oseltamivir/pharmacology , Amino Acid Sequence , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antibody Affinity/genetics , Antibody Affinity/immunology , Binding Sites, Antibody/genetics , Binding Sites, Antibody/immunology , Crystallography, X-Ray , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Humans , Molecular Docking Simulation , Molecular Sequence Data , Mutation/genetics , Neuraminidase/genetics , Sequence Alignment , Sequence Analysis, ProteinABSTRACT
Extremophiles are the microorganisms which can survive under extreme conditions of temperature, pressure, pH, salinity etc. They have gained much attention for their potential role in biotechnological and industrial applications. The large amount of experimental data in the literature is so diverse, that it becomes difficult and time consuming for the researcher to implement it in various areas of research. Therefore, a systematic arrangement of data and redirection in a similar fashion through web interface can assist researchers in analyzing the data as per their requirement. ExtremeDB is a freely available web based relational database which integrates general characteristics, genome-proteome information, industrial applications and recent scientific investigations of the seven major groups of 865 extremophillic microorganisms. The search options are user friendly and analyses tools such as Compare and Extreme BLAST have been incorporated for comparative analysis of two or more extremophiles and determining the sequence similarity of a given protein/nucleotide in relation to other extremophiles respectively. The effort put forth herein in the form of database, would open up new avenues on the potential utility of extremophiles in applied research. ExtremeDB is freely accessible via http://extrem.igib.res.in.