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Inhibition of influenza virus replication via small molecules that induce the formation of higher-order nucleoprotein oligomers.

ABSTRACT: Influenza nucleoprotein (NP) plays multiple roles in the virus life cycle, including an essential function in viral replication as an integral component of the ribonucleoprotein complex, associating with viral RNA and polymerase within the viral core. The multifunctional nature of NP makes it an attractive target for antiviral intervention, and inhibitors targeting this protein have recently been reported. In a parallel effort, we discovered a structurally similar series of influenza replication inhibitors and show that they interfere with NP-dependent processes via formation of higher-order NP oligomers. Support for this unique mechanism is provided by site-directed mutagenesis studies, biophysical characterization of the oligomeric ligand:NP complex, and an X-ray cocrystal structure of an NP dimer of trimers (or hexamer) comprising three NP_A:NP_B dimeric subunits. Each NP_A:NP_B dimeric subunit contains two ligands that bridge two composite, protein-spanning binding sites in an antiparallel orientation to form a stable quaternary complex. Optimization of the initial screening hit produced an analog that protects mice from influenza-induced weight loss and mortality by reducing viral titers to undetectable levels throughout the course of treatment.

SUBMITTER: Gerritz SW

PROVIDER: S-EPMC3174639 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Inhibition of influenza virus replication via small molecules that induce the formation of higher-order nucleoprotein oligomers.

Gerritz Samuel W SW Cianci Christopher C Kim Sean S Pearce Bradley C BC Deminie Carol C Discotto Linda L McAuliffe Brian B Minassian Beatrice F BF Shi Shuhao S Zhu Shirong S Zhai Weixu W Pendri Annapurna A Li Guo G Poss Michael A MA Edavettal Suzanne S McDonnell Patricia A PA Lewis Hal A HA Maskos Klaus K Mörtl Mario M Kiefersauer Reiner R Steinbacher Stefan S Baldwin Eric T ET Metzler William W Bryson James J Healy Matthew D MD Philip Thomas T Zoeckler Mary M Schartman Richard R Sinz Michael M Leyva-Grado Victor H VH Hoffmann Hans-Heinrich HH Langley David R DR Meanwell Nicholas A NA Krystal Mark M

Proceedings of the National Academy of Sciences of the United States of America 20110906 37

Influenza nucleoprotein (NP) plays multiple roles in the virus life cycle, including an essential function in viral replication as an integral component of the ribonucleoprotein complex, associating with viral RNA and polymerase within the viral core. The multifunctional nature of NP makes it an attractive target for antiviral intervention, and inhibitors targeting this protein have recently been reported. In a parallel effort, we discovered a structurally similar series of influenza replication ...[more]

PMID: 21896751

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Project description:UnlabelledThe Arenaviridae are enveloped, negative-sense RNA viruses with several family members that cause hemorrhagic fevers. This work provides immunofluorescence evidence that, unlike those of New World arenaviruses, the replication and transcription complexes (RTC) of lymphocytic choriomeningitis virus (LCMV) colocalize with eukaryotic initiation factor 4E (eIF4E) and that eIF4E may participate in the translation of LCMV mRNA. Additionally, we identify two residues in the LCMV nucleoprotein (NP) that are conserved in every mammalian arenavirus and are required for recombinant LCMV recovery. One of these sites, Y125, was confirmed to be phosphorylated by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). NP Y125 is located in the N-terminal region of NP that is disordered when RNA is bound. The other site, NP T206, was predicted to be a phosphorylation site. Immunofluorescence analysis demonstrated that NP T206 is required for the formation of the punctate RTC that are typically observed during LCMV infection. A minigenome reporter assay using NP mutants, as well as Northern blot analysis, demonstrated that although NP T206A does not form punctate RTC, it can transcribe and replicate a minigenome. However, in the presence of matrix protein (Z) and glycoprotein (GP), translation of the minigenome message with NP T206A was inhibited, suggesting that punctate RTC formation is required to regulate viral replication. Together, these results highlight a significant difference between New and Old World arenaviruses and demonstrate the importance of RTC formation and translation priming in RTC for Old World arenaviruses.ImportanceSeveral members of the Arenaviridae cause hemorrhagic fevers and are classified as category A pathogens. Arenavirus replication-transcription complexes (RTC) are nucleated by the viral nucleoprotein. This study demonstrates that the formation of these complexes is required for virus viability and suggests that RTC nucleation is regulated by the phosphorylation of a single nucleoprotein residue. This work adds to the body of knowledge about how these key viral structures are formed and participate in virus replication. Additionally, the fact that Old World arenavirus complexes colocalize with the eukaryotic initiation factor 4E, while New World arenaviruses do not, is only the second notable difference observed between New and Old World arenaviruses, the first being the difference in the glycoprotein receptor.

Dataset Information

Inhibition of influenza virus replication via small molecules that induce the formation of higher-order nucleoprotein oligomers.

Publications

Inhibition of influenza virus replication via small molecules that induce the formation of higher-order nucleoprotein oligomers.

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