Influenza RNA-dependent RNA Polymerase Research Articles

High-throughput identification of compounds targeting influenza RNA-dependent RNA polymerase activity

As influenza viruses have developed resistance towards current drugs, new inhibitors that prevent viral replication through different inhibitory mechanisms are useful. In this study, we developed a screening procedure to search for new antiinfluenza inhibitors from 1,200,000 compounds and identified previously reported as well as new antiinfluenza compounds. Several antiinfluenza compounds were inhibitory to the influenza RNA-dependent RNA polymerase (RdRP), including nucleozin and its analogs. The most potent nucleozin analog, 3061 (FA-2), inhibited the replication of the influenza A/WSN/33 (H1N1) virus in MDCK cells at submicromolar concentrations and protected the lethal H1N1 infection of mice. Influenza variants resistant to 3061 (FA-2) were isolated and shown to have the mutation on nucleoprotein (NP) that is distinct from the recently reported resistant mutation of Y289H [Kao R, et al. (2010) Nat Biotechnol 28:600]. Recombinant influenza carrying the Y52H NP is also resistant to 3061 (FA-2), and NP aggregation induced by 3061 (FA-2) was identified as the most likely cause for inhibition. In addition, we identified another antiinfluenza RdRP inhibitor 367 which targets PB1 protein but not NP. A mutant resistant to 367 has H456P mutation at the PB1 protein and both the recombinant influenza and the RdRP expressing the PB1 H456P mutation have elevated resistance to 367. Our high-throughput screening (HTS) campaign thus resulted in the identification of antiinfluenza compounds targeting RdRP activity.

Structures of influenza A proteins and insights into antiviral drug targets

The world is currently undergoing a pandemic caused by an H1N1 influenza A virus, the so-called 'swine flu'. The H5N1 ('bird flu') influenza A viruses, now circulating in Asia, Africa and Europe, are extremely virulent in humans, although they have not so far acquired the ability to transfer efficiently from human to human. These health concerns have spurred considerable interest in understanding the molecular biology of influenza A viruses. Recent structural studies of influenza A virus proteins (or fragments) help enhance our understanding of the molecular mechanisms of the viral proteins and the effects of drug resistance to improve drug design. The structures of domains of the influenza RNA-dependent RNA polymerase and the nonstructural NS1A protein provide opportunities for targeting these proteins to inhibit viral replication.

Molecular Basis of the Interaction for an Essential Subunit PA−PB1 in Influenza Virus RNA Polymerase: Insights from Molecular Dynamics Simulation and Free Energy Calculation

The emergence of the extremely aggressive influenza recently has highlighted the urgent need for new effective treatments. The influenza RNA-dependent RNA polymerase (RdRp) heterotrimer including PA, PB1 and PB2 has crucial roles in viral RNA replication and transcription. The highly conserved PB1 binding site on PA can be considered as a novel potential drug target site. The interaction between PB1 binding site and PA is crucial to many functions of the virus. In this study, to understand the detailed interaction profile and to characterize the binding hot spots in the interactions of the PA-PB1 complex, an 8 ns molecular dynamics simulation of the subunit PA-PB1 combined with MM-PBSA (molecular mechanics Poisson-Boltzmann surface area), MM-GBSA (molecular mechanics generalized Born surface area) computations and virtual alanine scanning were performed. The results from the free energy decomposition indicate that the intermolecular van der Waals interaction and the nonpolar solvation term provide the driving force for binding process. Through the pair interaction analysis and virtual alanine scanning, we identified the binding hot spots of PA and the basic binding motif of PB1. This information can provide some insights for the structure-based RNA-dependent RNA polymerase inhibitors design. The identified binding motif can be used as the starting point for the rational design of small molecules or peptide mimics. This study will also lead to new opportunities toward the development of new generation therapeutic agents exhibiting specificity and low resistance to influenza virus.

Crystal structure of the polymerase PAC–PB1N complex from an avian influenza H5N1 virus

The recent emergence of highly pathogenic avian influenza A virus strains with subtype H5N1 pose a global threat to human health. Elucidation of the underlying mechanisms of viral replication is critical for development of anti-influenza virus drugs. The influenza RNA-dependent RNA polymerase (RdRp) heterotrimer has crucial roles in viral RNA replication and transcription. It contains three proteins: PA, PB1 and PB2. PB1 harbours polymerase and endonuclease activities and PB2 is responsible for cap binding; PA is implicated in RNA replication and proteolytic activity, although its function is less clearly defined. Here we report the 2.9 ångström structure of avian H5N1 influenza A virus PA (PA(C), residues 257-716) in complex with the PA-binding region of PB1 (PB1(N), residues 1-25). PA(C) has a fold resembling a dragon's head with PB1(N) clamped into its open 'jaws'. PB1(N) is a known inhibitor that blocks assembly of the polymerase heterotrimer and abolishes viral replication. Our structure provides details for the binding of PB1(N) to PA(C) at the atomic level, demonstrating a potential target for novel anti-influenza therapeutics. We also discuss a potential nucleotide binding site and the roles of some known residues involved in polymerase activity. Furthermore, to explore the role of PA in viral replication and transcription, we propose a model for the influenza RdRp heterotrimer by comparing PA(C) with the lambda3 reovirus polymerase structure, and docking the PA(C) structure into an available low resolution electron microscopy map.