NiV, a highly pathogenic zoonotic agent with fatal outcomes in humans, remains a critical public health concern due to the absence of approved therapeutics. Elucidating the molecular architecture of its RNA polymerase machinery is pivotal for developing targeted antiviral strategies.
Two apo-state cryo-EM structures of the NiV L-P complex were resolved, delineating the RdRp and PRNTase domains within the L protein. The P protein tetramer was observed anchored to the RdRp domain through a unique interface. Functional validation confirmed two evolutionarily conserved zinc-binding motifs within the PRNTase domain as indispensable for enzymatic activity. Structural analyses further highlighted the flexibility of the C-terminal regions of the L protein and revealed a specialized positioning of the P protein’s XD linker near the nucleotide entry channel, suggesting a regulatory role in template RNA accessibility.
Targeted mutagenesis disrupting L-P interactions significantly attenuated polymerase activity, emphasizing their mechanistic necessity. Comparative structural studies across Mononegavirales identified conserved hydrophobic interfaces and a critical tyrosine residue central to P-bundle stabilization. These findings establish a structural framework for rational inhibitor design and advance mechanistic understanding of NiV replication, providing critical insights to mitigate henipavirus outbreaks.
Key findings from the study include:
1 Cryo-EM structural resolution of the nipah virus polymerase complex: The study successfully resolved two cryo-EM structures of the Nipah virus L protein in complex with the P protein, including full-length and truncated L-P complexes. The structures revealed well-defined RdRp and PRNTase domains of the L protein, while the C-terminal domains (CD, MTase, and CTD) remained unresolved due to flexibility. The P protein formed a tetrameric bundle, interacting with the L protein via hydrophobic interactions, hydrogen bonds, and salt bridges.
2 Critical role of Zinc-binding sites in PRNTase domain for polymerase activity:​​
Two conserved zinc-binding sites were identified within the PRNTase domain. Alanine substitution assays demonstrated that disrupting these sites completely abolished polymerase activity, confirming their essential role in catalysis. These sites are conserved in most mononegaviruses but absent in pneumoviruses.
3 Unique L-P interaction mechanism and functional implications: The NiV P tetramer engaged the L protein through multiple interfaces: The XD domain of P1 stably bound to the RdRp surface of L, with its linker region covering the NTP entry channel. The XD linkers of P3 and P4 anchored to the L surface via hydrophobic interactions, hydrogen bonds, and cation-π interactions. Mutations disrupting L-P interfaces significantly reduced polymerase activity and L protein stability.
4 Comparative structural analysis of mononegavirus polymerase complexes: The study highlighted divergent binding positions and dynamics of the P protein CTD across viral polymerases. Additionally, conserved tyrosine residues (e.g., Y732 in NiV, Y651 in NDV, Y642 in EBOV) on the L surface were identified as critical anchoring sites for P tetramers.

This study resolved the cryo-EM structures of the Nipah virus polymerase complex, unveiling the binding mode between the RdRp/PRNTase domains of the L protein and the P tetramer. It identified two conserved zinc-binding sites essential for polymerase activity and demonstrated the unique L-P interaction mechanism in paramyxoviruses. These findings significantly advanced our understanding of viral RNA synthesis and provided a structural foundation for developing broad-spectrum antivirals targeting conserved polymerase regions, offering critical insights for combating NiV outbreaks and related viral threats. The work entitled “Cryo-EM structures of Nipah virus polymerase complex reveal highly varied interactions between L and P proteins among paramyxoviruses” was published on Protein & Cell (published on Feb. 18, 2025).
DOI：10.1093/procel/pwaf014