Cryo-EM structures of Nipah virus polymerases and high-throughput RdRp assay development enable anti-NiV drug discovery

• Published in: Nature communications Vol. 16; no. 1; pp. 6655 - 14
• Main Authors: Chen, Zhenhang, Quirit Dudley, Jeanne, Deniston, Colin, Buffalo, Cosmo Z., Patra, Debjani, Cao, Dongdong, Hunt, Julia, Rohaim, Ahmed, Sengupta, Debapriya, Wen, Lan, Tsang, Tiffany, Xie, Lili, DiDonato, Michael, Spraggon, Glen, Clifton, Matthew C., Jarrousse, Nadine, Straimer, Judith, Liang, Bo
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 19.07.2025; Nature Publishing Group; Nature Portfolio
• Subjects: 101/28; 101/47; 13/106; 14/34; 14/5; 38/70; 38/71; 631/326/596; 631/45/173; 631/535/1258/1259; 82/16; 82/29; 82/80; 82/83; Antiviral Agents - chemistry; Antiviral Agents - pharmacology; Assaying; Binding; Cryoelectron Microscopy; DNA-directed RNA polymerase; Drug development; Drug discovery; Drug Discovery - methods; Genomes; Henipavirus; Henipavirus Infections - drug therapy; Henipavirus Infections - virology; High-throughput screening; High-Throughput Screening Assays - methods; Humanities and Social Sciences; Humans; L protein; Models, Molecular; Molecular modelling; multidisciplinary; Nipah virus; Nipah Virus - drug effects; Nipah Virus - enzymology; Nipah Virus - genetics; Phosphatase; Polymerization; Priming; Proteins; RNA polymerase; RNA-Dependent RNA Polymerase - antagonists & inhibitors; RNA-Dependent RNA Polymerase - chemistry; RNA-Dependent RNA Polymerase - genetics; RNA-Dependent RNA Polymerase - metabolism; RNA-Dependent RNA Polymerase - ultrastructure; RNA-directed RNA polymerase; Science; Science (multidisciplinary); Strains (organisms); Structural analysis; Transcription; Viral Proteins - chemistry; Viral Proteins - genetics; Viral Proteins - metabolism; Virus Replication - drug effects; Viruses; Bangladesh; Malaysia
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-025-61764-4

Transcription and replication of the Nipah virus (NiV) are driven by the large protein (L) together with its essential co-factor phosphoprotein (P). L encodes all the viral enzymatic functions, including RNA-dependent RNA polymerase (RdRp) activity, while the tetrameric P is multi-modular. Here, we investigate the molecular mechanism of the NiV polymerase and build tools for anti-NiV drug discovery. We analyze and compare multiple cryo-EM structures of both full-length and truncated NiV polymerases from the Malaysia and Bangladesh strains. We identify two conserved loops in the polyribonucleotidyltransferase (PRNTase) domain of L and the binding between RdRp-PRNTase and CD domains. To further assess the mechanism of NiV polymerase activity, we establish a highly sensitive radioactive-labeled RNA synthesis assay and identify a back-priming activity in the NiV polymerase as well as a fluorescence and luminescent-based non-radioactive polymerase assay to enable high-throughput screening for L protein inhibitors. The combination of structural analysis and the development of both high-sensitive and high-throughput biochemical assays will enable the identification of new direct-acting antiviral candidates for treating highly pathogenic henipaviruses. They determine four cryo-EM structures of NiV polymerases from different strains and establish RdRp enzymatic assays for anti-NiV drug discovery. A back-priming RdRp activity and the binding between RdRp core and CD domains in NiV L were identified.