Structural insights into measles virus RNA synthesis regulation and pan-paramyxoviral polymerase inhibition by ERDRP-0519

Significance

Nonsegmented negative-sense RNA viruses (nsNSVs), including measles and Nipah viruses, pose major health threats yet lack effective antivirals. These pathogens rely on a large RNA-dependent RNA polymerase (RdRP) complex to replicate and transcribe their genomes. Using cryoelectron microscopy (cryo-EM) and biochemical assays, we reveal how the measles virus C protein directly binds and stabilizes the polymerase, shifting it into a replication-competent state and enhancing viral RNA encapsidation. We further determine high-resolution structures of measles and Nipah virus polymerases bound to ERDRP-0519, uncovering a previously unknown allosteric pocket that blocks RNA synthesis. These findings provide fundamental insights into polymerase regulation and establish a structural framework for developing broad-spectrum antivirals against clinically important nsNSVs.

Abstract

Nonsegmented negative-sense RNA viruses (nsNSVs) rely on a multifunctional RNA-dependent RNA polymerase (RdRP) complex for transcription and replication. In measles virus (MeV), the nonstructural protein C has long been implicated in regulating RNA synthesis, yet its precise role remains unclear. Here, we show that the MeV C protein directly associates with the RdRP complex. Using cryoelectron microscopy, we determined atomic-resolution structures of the MeV polymerase with and without C, revealing that C binding stabilizes the C-terminal region of L and locks the complex into a replication-competent elongation state. Biochemical data further show that C promotes N protein recruitment, enhancing polymerase processivity through facilitating encapsidation during replication. Additionally, we also resolved high-resolution structures of MeV and Nipah virus (NiV) polymerases bound to ERDRP-0519, an orally available morbillivirus inhibitor. Unexpectedly, the compound occupies an allosteric pocket within the RdRp domain rather than the previously predicted PRNTase domain, overlapping conserved resistance sites. This binding induces conformational changes in palm subdomain, blocking RNA template and nucleotide engagement, thereby halting RNA synthesis. These findings uncover distinct regulatory and inhibitory mechanisms in paramyxovirus polymerases and provide a structural framework for the rational design of broad-spectrum antivirals targeting MeV, NiV, and potentially other clinically relevant nsNSVs.

Nonsegmented, negative-strand RNA viruses (nsNSVs) constitute the order Mononegavirales, which includes 11 viral families. Among them, members of the Paramyxoviridae, Pneumoviridae, Filoviridae, Rhabdoviridae, and Bornaviridae families have been extensively investigated (1, 2). These viruses cause a broad spectrum of diseases in humans and other mammals, ranging from mild infections to severe and often fatal illnesses, thereby posing a significant threat to public health and the livestock industry (1, 2). The genomes of nsNSVs consist of linear, single-stranded, and negative-sense RNA, encapsidated by nucleocapsid proteins (NPs) to form filamentous ribonucleoprotein (RNP) complexes, which serve as templates for both transcription and replication (1–4). The genomes are typically 11 to 19 kb in length and are flanked by a 3′ leader and a 5′ trailer sequence, both functioning as promoters for RNA synthesis (1–4). The viral genes are arranged sequentially and transcribed in tandem, each flanked by cis-acting gene start (GS) and gene end (GE) signals within their untranslated regions (UTRs), which are essential for regulating viral RNA synthesis (1–4). Upon entry into the host cell and release of viral RNPs into the cytoplasm, the nsNSV polymerase initiates transcription at the 3′ promoter. Following synthesis and release of a short noncoding RNA corresponding to the promoter-proximal leader region, the polymerase remains engaged with the template to scan for the GS signal of the first gene. Transcription of each gene produces a capped and methylated monocistronic mRNA, which is elongated until a GE signal is encountered, triggering polyadenylation and release of the transcript. The polymerase then resumes scanning and reinitiates transcription at the next downstream GS signal. This transcriptional strategy, known as the “stop–start” mechanism, leads to a positional gradient of mRNA abundance, with genes proximal to the 3′ leader (e.g., NP) transcribed more frequently, whereas genes near the 5′ trailer (e.g., L), are produced at significantly lower levels due to polymerase attenuation at gene junctions (2–4). In contrast, replication also initiates at the 3′ end of the viral genome, but the polymerase proceeds with high processivity along the entire template without responding to internal cis-acting signals. This results in the synthesis of a full-length, uncapped, and nonpolyadenylated positive-sense antigenome, which serves as a faithful complementary template for generating progeny genomes. Continuous encapsidation of nascent viral RNA by NPs is essential for productive replication, promoting functional RNP assembly and shielding the viral RNAs from host innate immune recognition (2–4).

Viral RNA synthesis in nsNSVs is mediated by the RNA-dependent RNA polymerase (RdRP) complex, which comprises the large (L) protein and a cofactor—phosphoprotein (P) in most nsNSVs or VP35 in filoviruses (1–4). The L protein harbors all enzymatic activities essential for transcription and replication, including ribonucleotidyl transferase activity, polyribonucleotidyl transferase (PRNTase) activity for mRNA capping, and S-adenosylmethionine (SAM)-dependent N⁷-guanosine- and 2′-O-methyltransferase (MTase) activity (4, 5). The cofactor P or VP35 is thought to maintain NP in a monomeric, RNA-free state (N⁰P) and to facilitate its delivery to the polymerase complex, thereby ensuring efficient encapsidation of nascent RNA and supporting polymerase processivity during replication (2, 6). Although the nsNSV RdRP complex carries out both transcription and replication, the molecular mechanism underlying the switch between these two mechanistically distinct processes and their regulation remain poorly understood. Several viral-encoded nonstructural proteins and host factors have been reported to play a role in the regulation of these processes. In viruses including Sendai virus (SeV), measles virus (MeV), Nipah virus (NiV), and human parainfluenza virus type 3 (HPIV3), the L, P, and N genes are sufficient to support reporter gene expression in minigenome assays, suggesting that the transcription of viral mRNAs does not rely on additional viral factors (7–10). The C protein, a nonstructural product translated from an alternative initiation site on the P mRNA, is known to act as a virulence factor by antagonizing host innate immune signaling pathways (11). During infection with MeV, SeV, and HPIV1, the absence of C protein expression strongly induces the production of nonencapsidated copy-back defective interfering RNAs (cbDI-RNAs), which potently activate the innate immune response and can consequently restrict viral propagation (12–20). In addition to its immunomodulatory role, the C protein is also thought to enhance the processivity of the viral RNA polymerase during replication; however, whether it acts directly on the polymerase and the precise mechanism underlying this regulation remain unclear.

As representative members of the morbillivirus and henipavirus genera within the Paramyxoviridae family, MeV and NiV are highly contagious respiratory pathogens that typically cause acute disease and pose a significant threat to human health (21, 22). MeV is transmitted via the respiratory route and initially presents with fever, cough, coryza, and conjunctivitis, followed by the appearance of a characteristic rash. Complications may affect multiple organ systems, with pneumonia and subacute sclerosing panencephalitis being the major contributors to measles-associated morbidity and mortality (23, 24). Although measles is a vaccine-preventable disease, it continues to impose a substantial global disease burden, causing an estimated 100,000 deaths annually. This persistence is attributed to declining vaccination coverage below the 95 percent threshold required for herd immunity, enabling the reestablishment of endemic transmission and driving widespread outbreaks (23–29). NiV, a recently emerged zoonotic paramyxovirus, is transmitted through contact with infected animals or via human-to-human transmission. In humans, NiV infection causes systemic vasculitis that leads to symptoms such as fever, headache, dizziness, and confusion, which can rapidly progress to fatal encephalitis, with case fatality rates ranging from 40 to 90% (22, 30–33). Due to its broad host range, high pathogenicity, and the absence of approved vaccines or antiviral therapies, NiV represents a particularly serious pandemic threat (34), underscoring the urgent need for effective antiviral drug development against such high-risk pathogens.

Due to the absence of a cellular counterpart and the fundamental differences between host and viral RNA synthesis mechanisms, viral RdRP complexes are considered promising targets for the development of antiviral drugs. Previous cell-based high-throughput screening (HTS) and structure–activity relationship (SAR) studies led to the discovery of a series of non-nucleoside inhibitors with a shared core scaffold, including 16677, AS-136a, and ERDRP-0519, all of which exhibit potent antiviral activity against MeV, with subnanomolar EC₅₀ values in cell-based assays, and directly inhibit the RNA synthesis activity of the MeV polymerase (35–39). Importantly, they are effective against a broad spectrum of wild-type MeV genotypes that are currently circulating globally (35, 36, 38). Among them, ERDRP-0519 stands out due to its favorable water solubility and high oral bioavailability, making it a particularly promising candidate for therapeutic development (39). Additionally, ERDRP-0519 has demonstrated strong prophylactic and therapeutic efficacy in animal models, protecting ferrets from otherwise 100% lethal canine distemper virus (CDV) infection and preventing measles disease in squirrel monkeys, thereby confirming its broad activity against morbilliviruses and potential for clinical use (40, 41). A combination of biochemical and computational studies has shown that ERDRP-0519 uniquely inhibits MeV polymerase activity by blocking all phosphodiester bond formation—including de novo initiation and primer-based extension—through simultaneously engaging the PRNTase domain and a flexible intrusion loop within the L protein, thereby locking the polymerase in a preinitiation conformation (42, 43). However, resistance mutations predominantly cluster in the RdRp domains of the L proteins in both MeV and CDV (35, 42), raising unresolved questions about the precise binding mode and inhibitory mechanism of the compound. Thus, structural elucidation is required to clarify how ERDRP-0519 suppresses RNA synthesis and whether its inhibitory scope can extend to other paramyxoviruses or even broader nsNSVs. GHP-88309, another non-nucleoside compound identified through HTS, has been reported to inhibit the polymerase activity of multiple paramyxoviruses, including MeV, CDV, and HPIV3, and to confer resistance mutations that map to a conserved cavity in the L protein distinct from the ERDRP-0519 resistance sites (41, 44), raising the question of whether GHP-88309 exerts its antiviral effect through a different mechanism or modulation of polymerase function.

In this study, we combined biochemical assays and cryoelectron microscopy (cryo-EM) to elucidate the structure and regulation of the MeV RdRP complex. High-resolution structures of the apo polymerase and its complex with the viral C protein revealed that C binding stabilizes the C-terminal functional domains of L and locks the enzyme in a replication-competent elongation state, while biochemical data indicate that C preferentially engages N protein to promote efficient encapsidation and polymerase processivity. We further determined cryo-EM structures of MeV and NiV polymerases bound to the small-molecule inhibitor ERDRP-0519, showing that it occupies a conserved allosteric pocket in the RdRp domain rather than the predicted PRNTase site, inducing conformational changes in the active site that block template and nucleotide binding. These findings uncover distinct regulatory and inhibitory mechanisms of paramyxovirus polymerases and provide a structural framework for the rational design of broad-spectrum antivirals targeting MeV, NiV, and related pathogens.

Results

Cryo-EM Structure Determination of RdRP Complexes of Human Measles Virus.

Using optimized P protein construct, we reconstituted three distinct RdRP complexes of MeV: the apo L–P complex (SI Appendix, Fig. S1) and L–P complexes bound to the allosteric inhibitors ERDRP-0519 and GHP-88309, respectively. To assemble the L–P complex bound to nonstructure protein C (hereafter referred to as the LPC complex), we coexpressed MeV L, P, and C proteins in Sf9 insect cells, using excess amounts of P and C relative to L (SI Appendix, Fig. S1). All complexes, including the apo L–P complex, LPC complex, and L–P complexes with inhibitors ERDRP-0519 and GHP-88309, were purified to homogeneity and subjected to cryo-EM sample preparation (SI Appendix, Fig. S1). Multiple datasets were collected for each complex on an FEI Titan Krios transmission electron microscope equipped with a K3 camera (SI Appendix, Table S1). Cryo-EM structure determination using multiple rounds of 2D, 3D classification and refinement yields a number of density maps, including the apo L–P complex, the LPC complex at 2.7 Å resolution, and the ERDRP-0519-bound complex at 3.1 Å resolution (SI Appendix, Figs. S2–S4). Unexpectedly, we observed no discernible density corresponding to GHP-88309 in the L–P–GHP-88309 complex, despite increasing the compound concentration in a second dataset. The resulting map was nearly indistinguishable from that of the apo L–P complex (SI Appendix, Fig. S4B). Therefore, particles from the apo-form, the GHP-88309 datasets, and the LPC datasets lacking bound C protein were pooled and used to generate a composite apo-form map at 2.6 Å resolution (SI Appendix, Fig. S4). The map Fourier shell correlation (FSC) curves and local resolution estimations are in accordance with the gold-standard resolutions between half maps of split data. These high-resolution reconstructions allowed unambiguous assignment of secondary structures and side-chain orientations. Atomic models were built and iteratively refined in real and reciprocal space, showing excellent agreement with their respective maps (SI Appendix, Fig. S5).

Structural Features of the MeV RdRP Complexes.

In the L–P complex, the cryo-EM density map only resolves the RdRp and PRNTase domains of the L protein, whereas the C-terminal regions, including the connector domain (CD), methyltransferase (MTase) domain, and C-terminal domain (CTD), are not visible (Fig. 1 A and G and SI Appendix, Fig. S6), despite the L protein being intact during sample preparation (SI Appendix, Fig. S1E). This absence likely reflects intrinsic flexibility of the C-terminal region relative to the polymerase core under the current conformational state. Intriguingly, the LPC complex reveals nearly complete density for the L protein, including its full C-terminal region, except for several flexible loops (Fig. 1 B, C, and G). Notably, two C protein molecules, which associate with each other, are observed contacting both the RdRp/PRNTase core and the C-terminal domains of L, occupying a position above the polymerase central cavity (SI Appendix, Figs. S7 and S8). Both the MeV L–P and LPC complexes assembled using nearly identical full-length L–P and LPC constructs have previously been shown to perform de novo RNA synthesis and primer-based extension in vitro, confirming that these MeV RdRp complexes adopt active, catalytically competent conformations (42, 45). The ERDRP-0519-bound RdRP complex map closely resembles the apo L–P complex but features a prominent additional density within a deep cleft of the RdRp domain (Fig. 1 D and E). This density is absent in both the L–P and LPC complexes and is attributed to ERDRP-0519 binding. In all three cryo-EM structures we resolved, each P protomer in the tetramer adopts a different conformation, particularly in the regions following the coiled-coil helices. Three of the four P protomers engage with the RdRp domain L protein (SI Appendix, Fig. S9). Structural superposition of all three models revealed similar configurations in the L protein core and P tetramers (Fig. 1F).

Fig. 1.

Overview of the cryo-EM maps and models of MeV RdRP complexes. (A) Overall architecture of the apo L–P complex showing well-resolved RdRp (light blue) and PRNTase (pink) cores, together with four P protomers (salmon, lime, wheat, and limon). (B and C) Cryo-EM map and model of the MeV L–P complex reveals two bound C protomers (marine and slate) interacting with L domains, with visible CD (yellow), MTase (orange), and CTD (red). (D) Cross-sectional view of the cryo-EM map of the L–P complex bound to ERDRP-0519 (highlighted in yellow). (E) The ERDRP-0519 density in the cryo-EM map superimposed with the fitted atomic model. (F) Superposition of the MeV RdRP complex models: apo L–P (pink), LPE (blue), and LPC (light green). The C-terminal functional domains and C protein dimers are shown in the same colors as in B and C. (G) Schematic domain organization of the MeV L, P, and C proteins. Conserved regions I–VI (CR I–VI) in the L protein are labeled based on sequence alignment across paramyxoviruses. Catalytic residues within the RdRp, PRNTase, and MTase domains are annotated. Gray boxes with solid outlines indicate unresolved regions in the structures, while gray boxes with dashed outlines represent regions that are not included in the constructs.

Conformational Rearrangement of C-Terminal Regions of L upon C Dimer Engagement.

To date, the RNA polymerase structures of nsNSVs that contain fully visible C-terminal regions include those of VSV and RABV in the rhabdovirus family, as well as HPIV3, PIV5, NDV, and MuV in the paramyxovirus family (46–51). Interestingly, unlike other paramyxoviruses, the C-terminal functional domains of MeV do not appear to be firmly associated with the core region under either physiological or slightly high salt conditions, indicating a weak interaction between them (SI Appendix, Fig. S10). However, upon the association of the C dimer, the MTase and CTD become stabilized and adhere to the PRNTase domain through distinct interaction interfaces compared to other viruses (SI Appendix, Fig. S11 A and B), whereas the CD has few contacts with the PRNTase domain.

The state of our MeV LPC complex structure is quite different from all the previously determined structures (Fig. 2A and SI Appendix, Fig. S12). Due to the absence of the priming loop, intrusion loop, and supporting helix, the central cavity is largely vacant (Fig. 2B). In addition, as a result of the association of the C protein dimer, the CD-MTase-CTD module is completely displaced from the above of the central cavity and only attaches to the PRNTase domain of the L protein, fully exposing the product RNA exit channel (Fig. 2 A and B). The domain organization of the C-terminal functional region in the MeV LPC complex is similar to that in the PIV5 polymerase structure (PDB: 6V85), in which the CD-MTase-CTD are arranged in an anticlockwise orientation from the aerial view (Fig. 2A and SI Appendix, Fig. S12G), whereas in the polymerase structures of several other viruses mentioned above, these domains are arranged in a clockwise direction (SI Appendix, Fig. S12 A–F). Through superimposing the core of the PIV5 polymerase (PDB: 6V85) onto that of the LPC structure, it appears that the C protein dimer causes a dramatic rotation and shift of the entire CD-MTase-CTD module, in which the CD flips nearly 180°, the MTase domain rotates about 86° and the CTD about 76° (Fig. 2C). The elongating RNA transcript emerging from the product exit channel seems unable to travel around the CD-CTD-MTase periphery to reach the active site of the MTase domain (Fig. 2A). In addition, the product RNA also cannot directly penetrate through the interior tunnel to access the catalytic sites of the MTase domain (route 1) like in the polymerase structure of MuV (48), or pass through the C dimer-MTase (route 2) due to the interaction between C-2 and CD (Fig. 2A and SI Appendix, Fig. S7 E and F). Thus, the state we presented here is unlikely to be the capping or methylation reaction stage during the transcription process.

Fig. 2.

Conformation of the C-terminal region in MeV LPC complex and functional role of MeV C protein. The RdRp and PRNTase cores are shown as light blue and pink surfaces. Priming and intrusion loops are in green and dark blue, respectively. C-terminal functional domains of MeV L (CD, MTase, CTD) are depicted as yellow, orange, and red cartoons, with P tetramers in gray. Top–down views mark CD, MTase, and CTD by colored circles (yellow, orange, red), with arrows indicating their spatial rearrangements. (A) MeV LPC complex with open RNA exit channel. Two possible RNA exit routes toward the MTase are indicated: route 1 (magenta), route 2 (green). (B) Visualization of the product RNA exit channel in the MeV LPC complex, the CD-MTase-CTD module, and C dimer is hidden for clarity. (C) Conformational comparison of CD, MTase, and CTD between PIV5 (PDB: 6V85) and MeV polymerase. PIV5 domains are shown in green (CD), brown (MTase), and salmon (CTD), with arrows indicating domain rotation angles. For clarity, the superimposed PIV5 core structure is omitted. (D) MST analysis of full-length C protein binding to different forms of N proteins, including the monomeric apo N protein, N protein bound to 8 nt ssRNA, and the N⁰P complex. Data represent means ± SD from n = 3 biologically independent replicates. Raw data are provided as a Source Data file.

In order to investigate the role and potential interaction partners of the C protein in the RNA synthesis process, we performed the MST analysis screen using eGFP-tagged C protein as a target. In addition to the MeV L–P complex, we also found that the C protein is able to interact with the N protein directly. The MST analysis revealed that the C protein binds monomeric apo N and the N–8 nt ssRNA complex with comparable affinities (3.92 ± 0.50 μM and 4.57 ± 1.46 μM, respectively), whereas its binding to the N⁰P complex is markedly reduced (Fig. 2D). Thus, the interaction between N and C uncovers another critical function of C that not only opens up the nascent RNA exit channel but possibly plays a role in recruiting the monomeric N protein to the site where the RNA product emerges from the catalytic cavity, which is poised to encapsidate the nascent RNA. Since only the newly synthesized viral genome and antigenome are encapsidated by N proteins to form the nucleocapsid during the RNA synthesis process, our LPC structure determined here likely reflects an elongation state associated with viral replication. Therefore, the LPC complex may represent a replicase-like form of the polymerase, whereas the L–P complex primarily functions as a transcriptase. These observations suggest that the nonstructural protein C may contribute to regulating the transcription-to-replication switch through its interaction with the N protein.

Structural Basis of ERDRP-0519 Recognition by the MeV L Protein.

A series of non-nucleotide small-molecule inhibitors targeting the morbillivirus polymerase complex have been developed, including 16677, AS-136a, and ERDRP-0159 (SI Appendix, Fig. S13 A–C), all of which revealed nanomolar potency. Surprisingly, contrary to previous photo-crosslinking and in silico docking studies suggesting that the binding pocket of ERDRP-0519 is located in the PRNTase domain of L (42), our 3.1 Å-resolution cryo-EM map and structure of the L–P complex bound to ERDRP-0519 (hereafter referred to as the LPE complex) identifies extra density positioned in the cavity surrounded by the fingers and palm subdomain of the RdRp (primarily involving motifs A–D), in the vicinity of the ⁷⁷²GDNQ⁷⁷⁵ catalytic sites (Figs. 1D and 3 A and B). Notably, no additional density is detected in the PRNTase domain of the LPE complex map even at near-atomic resolution, confirming that ERDRP-0519 binds within the RdRp domain rather than the previously proposed PRNTase site (42). The inhibitor occupies a buried pocket (~500 Ų interface area) deep within the RdRp catalytic cavity. Furthermore, ERDRP-0519 and its first-generation analogues fit the density unambiguously when modeled with appropriate geometry restraints (SI Appendix, Fig. S13 D–F).

Fig. 3.

Molecular mechanism of ERDRP-0519 recognition and inhibition of MeV RNA polymerase. (A) Spatial localization of ERDRP-0519 (yellow sticks) within the MeV L RdRp. (B) Close-up view of the ERDRP-0519 binding pocket in the palm and fingers, with cryo-EM density as magenta mesh. (C) Detailed interactions of ERDRP-0519 with palm (magenta) and fingers (cyan) residues (sticks); polar contacts shown as red dashed lines. (D) Structural superposition of the ERDRP-0519 binding pocket in the apo and LPE complexes, showing clashes, conformational changes in motifs C (yellow) and E (dark blue), and apo conformation (light blue). (E) Main/side chain differences of key residues between apo (slate) and bound (cyan), with Y667 densities in light blue (apo) and salmon (bound). (F) Resistance mutations mapped onto the pocket: MeV (black dots) and CDV (black squares) shown as cyan sticks; surrounding residues in slate. (G and H) Surface representation of the ERDRP-0519 binding pocket, spatial location of the pocket (G), sequence conservation calculated using the ConSurf server (H) (52). (I) Structure-based partial sequence alignment of representative paramyxoviral L proteins spanning motifs A–D; binding residues in bold red with red triangles, fully conserved in cyan, relatively conserved in yellow, conservative substitutions in blue, nonconservative in green, and non-MeV pocket residues in magenta.

The LPE structure provides a detailed view of how this compound is specifically recognized by the MeV L protein (Fig. 3C). Notably, the N-ethylmorpholinyl group at the position 2 of the piperidine ring extends into the solvent exposed central cavity, without making direct contact with surrounding residues (Fig. 3C). This hydrophilic group was introduced primarily to enhance aqueous solubility and oral bioavailability, with minimal impact on antiviral potency compared to its lead compound, AS-136a (39). The piperidine ring is partially buried and stabilized through weak Van der Waals’ interactions with the alkyl chains of surrounding polar residues E740, Q744, and D773 from the catalytic ⁷⁷²GDNQ⁷⁷⁵ motif (Fig. 3C). A hydrophobic residue, L664, supports the ring from below, while a hydrogen bond between G741 and the sulfonyl linker further anchors the molecule in place (Fig. 3C). Aromatic residue W747 forms a perpendicular π–π stacking interaction with the central phenyl ring, which is further clamped by L664 via hydrophobic contact from the other side (Fig. 3C). The acylamino linker is additionally stabilized by a hydrogen bond from W671 (Fig. 3C). The most deeply buried portion of the inhibitor, the 1-methyl-3-trifluoromethyl-pyrazole moiety, is inserted into a highly hydrophobic pocket formed by residues L664, Y667, W671, L755, L777, L807, L811, and L818. Within this pocket, Y667 forms another perpendicular π–π interaction with the pyrazole ring (Fig. 3C). Several polar contacts, involving T662, Y667, T751, H816, and Q775 from the ⁷⁷²GDNQ⁷⁷⁵ motif, interact with nitrogen and fluorine atoms of the pyrazole and its CF₃ substituent, further reinforcing the binding (Fig. 3C). Together, these extensive hydrophobic and polar interactions encase ERDRP-0519 with high specificity, explaining its tight binding to MeV L with a reported affinity ~80 nM (42), which is the prerequisite for the antiviral efficacy of the drug in vivo.

Molecular Mechanism of MeV RNA Synthesis Inhibition by ERDRP-0519.

We then explored the molecular mechanism by which the non-nucleotide inhibitor ERDRP-0519 blocks the RNA synthesis activity of MeV and CDV polymerase complexes. Structural superposition of the fingers–palm subdomain in the LPE complex with that of the apo L–P structure revealed a remarkable conformational change in the β-hairpin loop of motif C, where the catalytic ⁷⁷²GDNQ⁷⁷⁵ motif resides (Fig. 3D). Particularly, the Cα atom of residue G772 moves 2.5 Å away from the inhibitor, and the catalytic residue D773 reorients to the opposite side to avoid the steric clash with the piperidine ring of the inhibitor (Fig. 3D). These shifts propagate to motif E, inducing a rearrangement of its β-hairpin loop and resulting in a 4.7 Å displacement of the Cα atom of K834 at its tip (Fig. 3D). Furthermore, inhibitor binding alters the interaction network between motifs C and E. In the apo state, Q771 in motif C forms a hydrogen bond with the side chain of S833, D773 forms a salt bridge with R544 in motif F, and N774 interacts with the backbone of V831 in motif E (SI Appendix, Fig. S13G). Upon inhibitor engagement, Q771 instead interacts with the backbone of S833, while S833 forms a new hydrogen bond with G772. Additionally, D773 shifts to interact with K665 in motif A, further disrupting the native catalytic environment (SI Appendix, Fig. S13G). Similar local conformational changes were observed when the LPC structure was superimposed with the LPE model (SI Appendix, Fig. S13H), indicating that the inhibitor ERDRP-0519 can also bind to the fingers–palm subdomain of LPC complex and block both transcription and replication.

In addition to the structural rearrangements at the catalytic center, several residues within the ERDRP-0519 binding pocket also experience local conformational adjustments upon inhibitor engagement. Notably, Y667 shifts outward by 1.6 Å to maintain favorable Van der Waals interactions (Fig. 3E). Meanwhile, Q775 and H816 move closer to the compound, adopting new side-chain rotamers to optimize binding contacts (Fig. 3E). These subtle but coordinated changes suggest an induced-fit binding mode rather than a rigid lock-and-key mechanism. Such conformational plasticity in the palm subdomain likely distorts the optimal geometry of the active site, thereby compromising the catalytic competence of the polymerase. Additionally, ERDRP-0519 appears to lock the RdRP complex into an elongation-like conformation, preventing the conformational transitions required for initiation, reinitiation, and termination. This structural rigidity may further impair polymerase turnover during RNA synthesis, offering a mechanistic explanation for the potent antiviral efficacy observed in both cell-based assays and animal studies.

To investigate mechanisms of resistance, we analyzed previously reported escape mutations in the MeV and CDV L proteins that emerged during viral adaptation in the presence of ERDRP-0519 or its precursor compounds (35, 42). These resistance mutations show high convergence between MeV and CDV, clustering within functionally critical regions of the polymerase (Fig. 3F and SI Appendix, Fig. S13I). In CDV, the N398D mutation resides in the fingers subdomain, while in MeV, L1170F, R1233Q, and V1239A are found in the PRNTase domain (SI Appendix, Fig. S13 I and J). Additional mutations—T751I, H816L, and G835R/V in CDV, as well as H589Y and T776A/S shared by both viruses—are located in the palm subdomain (Fig. 3F).

Our LPE complex structure provides insight into the resistance mechanism. Residues T751 and H816 form direct polar contacts with the pyrazole moiety of ERDRP-0519, while S768 stabilizes Q775, which in turn hydrogen-bonds with the CF₃ group of the compound (Fig. 3F). Thus, mutations at these sites are likely to disrupt binding through either steric hindrance or loss of polar interactions. Indeed, the S768A mutation causes over 400-fold decrease in binding affinity and 28-fold increase in EC₅₀ in the cell-based assays (42). Similarly, T776, part of the β-strand in motif C, contributes to local structural integrity (53). Its mutation (e.g., T776A or T776S) can destabilize the β-sheet, leading to an ∼80-fold reduction in compound sensitivity (42). Residue G835, located in motif E and surrounded by Y832, K834, and L842 from the thumb subdomain, is particularly sensitive to side-chain volume changes (Fig. 3F). Substitution with bulkier residues like valine or arginine may disrupt local packing, altering the geometry of motifs E and C and diminishing compound efficacy. H589, which is not resolved in our elongation-state structure, is predicted to be located in the supporting helix and form a hydrogen bond with G840 (SI Appendix, Fig. S13I), a highly conserved residue connecting the motif E of the palm and the thumb subdomain. The H589Y mutation may weaken this interaction and impact structural communication between palm and thumb subdomains. Outside the immediate binding pocket, N398 in the fingers subdomain (mutated to D in CDV) interacts with the main chain of L431 (SI Appendix, Fig. S13J). Another three mutants, L1170F, R1233Q, and V1239A, located in the PRNTase domain, are distant from the inhibitor-binding cavity and mutations in these sites are unlikely to directly affect the architecture of the palm subdomain but might induce allosteric effects, subtly altering polymerase conformation or enhancing RdRP basal activity, thereby reducing inhibitor susceptibility (35).

Viruses within the Paramyxoviridae family typically exhibit a high degree of structural and sequence conservation, particularly in the L protein (SI Appendix, Fig. S14). In order to explore whether the non-nucleotide inhibitor ERDRP-0519 could potentially bind and inhibit the polymerases from other paramyxoviruses beyond morbilliviruses, we performed structure-guided sequence alignments based on conserved motifs and residues directly involved in compound binding within the MeV L protein. Both sequence alignment and binding pocket analysis revealed a remarkable conservation of residues forming the inhibitor-binding cavity and their immediate neighbors (Fig. 3 G–I), suggesting that the antiviral efficacy may extend beyond MeV and CDV to the other genera within the family. However, several site-specific variations were observed. For example, T662 in MeV is replaced by alanine in RDV, potentially disrupting a hydrogen bond with the inhibitor (Fig. 3I). In addition, L811 in MeV is substituted by methionine in HPIV3, SeV, and HPIV1, a conservative change that likely preserves hydrophobic interactions (Fig. 3I). Intriguingly, in rubellavirus, L811 is replaced by asparagine (Fig. 3I), which may reduce hydrophobic contact but introduce a new potential hydrogen bond with the CF₃ group of the compound. Notably, the highly conserved glutamine in the GDNQ motif of nsNSV is substituted by glutamate in NiV and HeV (Fig. 3I), possibly weakening polar interactions with the inhibitor. Additionally, H816 in MeV is replaced by leucine in HeV (Fig. 3I), altering the local electrostatic environment of the pocket. These residue-level differences could modulate the binding affinity of ERDRP-0519 across Paramyxoviridae polymerases and highlight the potential to expand this pan-morbillivirus inhibitor into a broader-spectrum pan-paramyxovirus antiviral agent.

Mechanistic Insights of ERDRP-0519 Engagement with NiV Polymerase.

Given that several amino acid substitutions are present in the putative ERDRP-0519 binding site of NiV and HeV polymerases, we aimed to investigate whether ERDRP-0519 can still engage these viral polymerases and retain its inhibitory effect. To this end, we determined the cryo-EM structure of the NiV polymerase bound to this compound at 2.8 Å resolution (Fig. 4 A and B and SI Appendix, Fig. S15). The resulting map revealed an additional density located within a narrow cavity formed between the fingers and palm subdomains of the L protein (Fig. 4A), consistent with the binding of ERDRP-0519 (Fig. 4 B–D). Overall, the compound adopts a binding mode in the NiV polymerase that is highly similar to that observed in the MeV polymerase, although subtle differences in residue interactions are evident (Fig. 4E).

Fig. 4.

Mechanistic insights into ERDRP-0519 binding to and the inhibition of NiV polymerase. (A) Cross-sectional view of NiV L–P bound to ERDRP-0519; RdRp (blue), PRNTase (pink), P protomers in light orange, wheat, limon, lime, and ERDRP-0519 in yellow. (B) Zoomed-in view with atomic model overlaid. (C) Comparison of ERDRP-0519 localization in MeV (yellow) and NiV (salmon) RdRp; NiV subdomains: NTD (gray), fingers (light slate), palm (light pink), thumb (limon). (D) Close-up view of the ERDRP-0519 binding pocket with ERDRP-0519 density (dark blue mesh). (E) Detailed interactions of ERDRP-0519 with palm (magenta) and fingers (light slate) residues; polar contacts shown as red and blue dashed lines. (F) Superposition of apo (yellow, PDB: 9BDQ) and inhibitor-bound (light pink) NiV RdRp shows clashes (red arrows) and conformational rearrangements in the palm subdomain. (G) Side chain differences of key binding residues between apo (limon sticks) and inhibitor-bound (magenta sticks) structures. (H) Superposition of dsRNA-bound (limon, PDB: 9GJU) and inhibitor-bound (light pink) NiV L–P complexes highlights clashes (red arrows) and palm conformational changes. (I) Steric hindrance between ERDRP-0519 and bound GMPPNP (magenta sticks); catalytic residues shown in cyan, G831 Cα in limon (dsRNA-bound) and light pink (inhibitor-bound), Mg²⁺ as green sphere; inhibitor-bound structure omitted for clarity. (J) Steric hindrance between ERDRP-0519 and template/product RNA (wheat/firebrick); inhibitor-bound structure hidden for clarity.

Specifically, the ethylmorpholinyl group at position 2 of the piperidine ring is fully solvent-exposed, with only the carboxyl group of residue E799 forming a weak salt bridge with the protonated tertiary amine of the morpholine ring at neutral pH (Fig. 4E). The piperidine ring is surrounded by the side chains of E799, Q803, Q830, and D832, primarily through Van der Waals’ interactions (Fig. 4E). As seen in the MeV polymerase, the main chain nitrogen atom of conserved G800 forms a hydrogen bond with the connecting sulfonyl group (Fig. 4E). The central phenyl ring is clamped by residues W806 and L723 from the top and bottom via π–π stacking and hydrophobic interactions, respectively (Fig. 4E). The connecting acylamino bond also forms a hydrogen bond with W730 (Fig. 4E). However, unlike in the MeV structure, the equivalent residue, E834 from the ⁸³¹GDNE⁸³⁴ catalytic motif does not generate a hydrogen bond with the compound, instead, it establishes an unusual charge interaction with the carbon atom of the CF₃ group, likely driven by the strong electronegativity of the fluorine atoms (Fig. 4E). The 1-methyl-3-trifluoromethyl-pyrazole moiety is deeply embedded in a hydrophobic core formed by residues L723, F726, W730, F813, L814, I836, L866, L870, L873, and H875 (Fig. 4E). Additionally, T810 forms a hydrogen bond with N2 atom in the pyrazole ring, while T721 interacts with one of the fluorine atoms in the CF₃ group (Fig. 4E). Together, these polar interactions within the hydrophobic cavity further enhanced the binding affinity of ERDRP-0519 to the NiV polymerase.

Similar to the conformational changes observed in the MeV polymerase upon ERDRP-0519 binding, the NiV polymerase also appears to undergo comparable structural rearrangements in response to the compound (Fig. 4F and SI Appendix, Fig. S16 A and B). The most striking conformational change is the structural remodeling of the motif C within the palm subdomain, particularly the flipping out of the catalytic ⁸³¹GDNE⁸³⁴ motif and a noticeable bulging of the main chain in one of the β-strands (Fig. 4F and SI Appendix, Fig. S16 B and C). Additionally, motif A and motif D exhibit slight outward shifts upon compound engagement. These collective movements result in the complete disappearance of the supporting helix, which is likely repelled during conformational rearrangement. Furthermore, residues such as L723, F726, and W806, which are involved in compound binding, also experience corresponding adjustments to facilitate the interaction (Fig. 4G).

To investigate whether and how ERDRP-0519 binding affects RNA synthesis by the NiV polymerase, we superimposed the ERDRP-0519-bound NiV polymerase structure with the RNA- and incoming nucleotide-bound NiV polymerase structure in the early elongation state (PDB: 9GJU) (54), using the fingers-palm subdomains for structural alignment (Fig. 4H). Notably, in the RNA- and NTP-bound structure (PDB: 9GJU), the catalytic ⁸³¹GDNE⁸³⁴ motif flips toward the fingers subdomain, positioning the carboxyl groups of residues D722 from motif A, D832, the carbonyl group of L732, along with the phosphodiester moiety of the incoming GTP analogue, to chelate a Mg²⁺ ion (54). In contrast, in the ERDRP-bound structure, the ⁸³¹GDNE⁸³⁴ motif instead flips toward motif E (Fig. 4H and SI Appendix, Fig. S16 D–F), and the catalytic residues are misaligned, rendering them unable to participate in such coordination (Fig. 4I and SI Appendix, Fig. S16E). Strikingly, the piperidine ring of ERDRP-0519 directly clashes with the ribose moiety of the GTP analogue (Fig. 4I), whereas the ethylmorpholinyl group, previously considered to play a negligible role in RNA synthesis inhibition, introduces steric hindrance with both the template and product RNA bases (Fig. 4J). These findings reveal that the compound inhibits RNA synthesis not only by disrupting the active site conformation but also by blocking the binding of the incoming nucleotide and interfering with the positioning of the template and product RNA strands, mechanisms that had not been fully appreciated before.

Broad-Spectrum Targeting of Paramyxoviral Polymerases by ERDRP-0519.

To gain deeper insights into the interaction mechanisms of ERDRP-0519 at its binding site, we conducted a series of molecular dynamics (MD) simulations using the MeV L/ERDRP-0519 and NiV L/ERDRP-0519 cryo-EM structures. Three independent 200-ns simulations were performed for each system. In the MeV simulations, ERDRP-0519 forms stable hydrogen bonds with residues T662, Y667, T751, H816, W671, and Q775, and π–π stacking interactions with Y667 and W747 (Fig. 5A). In the NiV simulations, ERDRP-0519 exhibited a comparable interaction network, indicating a conserved mode of binding across both viruses (Fig. 5B). Notably, the ligand maintained a binding pose highly consistent with the initial cryo-EM model throughout the trajectories, validating the observed interaction geometry.

Fig. 5.

Dynamics of ERDRP-0519 in representative paramyxoviral polymerase complexes. MD simulations and docking analyses of ERDRP-0519 bound to the L–P complex structures of MeV (A, this study), NiV (B, this study), PIV5 (C, docking model), HPIV3 (D, docking model), MuV (E, docking model), and NDV (F, docking model). Three independent 200-ns simulations were conducted for each system. Left panels: RMSD plots of ERDRP-0519 over time across the three replicates, illustrating the stability of the compound within the binding pocket. Right panels: Representative structural snapshots extracted at 20-ns intervals throughout the simulations. Key conserved residues (e.g., G741, W747) mediating ERDRP-0519 binding are shown as green sticks. ERDRP-0519 is depicted in salmon, highlighting its consistent binding pose across different Paramyxoviridae RdRp domains.

Beyond its established efficacy against MeV polymerase complex, whether ERDRP-0519 exhibits broad-spectrum inhibitory activity against other Paramyxoviridae members such as PIV5, HPIV3, MuV, and NDV remains incompletely understood. To explore the potential for cross-viral inhibition, we performed molecular docking and MD simulations using the L proteins of these viruses. Docking results revealed that ERDRP-0519 adopts highly similar binding poses across these polymerases (SI Appendix, Fig. S17 A–D), and MD simulations demonstrated that the compound remained stably bound within the RdRp domain in each case. In the PIV5 system, residues G741, T662, Y667, S751, H816, and Q775 formed hydrogen bonds with ERDRP-0519, while W747 engaged in a perpendicular π–π interaction with its central phenyl ring (Fig. 5C). A nearly identical interaction pattern was observed in HPIV3, involving the same set of residues (Fig. 5D). In the MuV system, ERDRP-0519 formed hydrogen bonds with G747, T668, Y673, H822, W677, and Q781, and π–π stacking interactions with W753 and H822 (Fig. 5E). Similarly, in the NDV polymerase, the inhibitor engaged G719, T640, Y645, H794, W671, and Q753 through hydrogen bonds and formed a π–π stacking interaction with W725 (Fig. 5F). Notably, interactions involving G741 (or its equivalent), W671, and W747 are conserved across these systems, including MeV (G741, W671, W747), PIV5 (G741, W671, W747), HPIV3 (G741, W671, W747), MuV (G747, W677, W753), and NiV (G719, W649, W725). These results suggest that ERDRP-0519 engages a structurally conserved allosteric pocket within the RdRp domain, supported by a recurrent network of conserved residues. While further experimental validation is needed, this structural conservation may underlie the potential of ERDRP-0519 as a broad-spectrum inhibitor targeting multiple Paramyxoviridae polymerases. However, structural comparisons further revealed that ERDRP-0519 does not accommodate polymerases from other virus families such as pneumoviruses and rhabdoviruses, consistent with its specificity for paramyxovirus L proteins (SI Appendix, Fig. S18). In addition, molecular docking of GHP-88309 was performed at an alternative binding site partially overlapping the ERDRP-0519 pocket, as detailed in SI Appendix, Fig. S18 H and I.

Discussion

nsNSVs rely on both the L and P proteins to assemble functional RdRP complexes for mRNA transcription and genome replication, with viral nonstructural proteins and host factors often contributing to the regulation of these two distinct processes (2, 4). In this study, we determined two atomic-resolution cryo-EM structures of human measles virus RdRP complexes, each representing distinct functional states in the viral life cycle. In addition, we solved the cryo-EM structures of MeV and NiV RdRP bound to the non-nucleotide allosteric inhibitor ERDRP-0519 at near-atomic resolution, revealing the molecular mechanism by which this leading antiviral candidate inhibits RNA synthesis. Our structural and molecular dynamics analyses identified a conserved, druggable pocket within the RdRp domain of the L protein and demonstrated that ERDRP-0519 effectively targets both MeV and NiV polymerases. These findings underscore its potential to inhibit a broad range of paramyxoviruses and provide a blueprint for the rational design of broad-spectrum antiviral therapeutics against nsNSVs.

The mechanisms by which nsNSV polymerases coordinate mRNA transcription and genome replication remain incompletely understood and are under debate. Based on studies across multiple virus families, including paramyxoviruses, filoviruses, RSV, and VSV, two primary models have been proposed (55). The first model derived from VSV posits that transcription and replication are carried out by distinct polymerase complexes: a “transcriptase” and a “replicase.” Both complexes are based on the L–P assembly, but the transcriptase additionally depends on several host factors (56). Similar regulatory roles have been observed in filoviruses and pneumoviruses, where VP30 (in EBOV and MARV) and M2-1 (in RSV and HMPV) are required for transcription initiation and elongation, respectively (57, 58). While these studies suggest that viral or host cofactors are essential for transcription, it remains unclear whether additional factors specifically regulate replication. The second model, supported by work in RSV, EBOV, and paramyxoviruses, proposes a unified polymerase that switches between transcription and replication. Transcription is initiated through a “stop-start” mechanism at the 3′ end of the genome and proceeds through sequential gene expression without encapsidation of the nascent RNA. In contrast, replication requires abundant N protein, which packages the nascent RNA cotranscriptionally, thereby protecting cis-acting signals and preventing transcription termination or mRNA modifications (2, 4, 55).

In paramyxoviruses such as MeV, NiV, and SeV, the L, P, and N alone are sufficient to drive reporter gene expression in minigenome systems, indicating that the L–P complex functions as the transcriptase. However, in RSV minigenome assays, elevated levels of N protein enhance replication but do not shift the transcription–replication balance, suggesting that N availability is necessary but insufficient to drive this switch (59). Notably, in SeV and MeV, the nonstructural C protein has been implicated in enhancing polymerase processivity during replication and suppressing the generation of immune-activating cbDI-RNAs (12, 13, 15–18). Our cryo-EM structures of MeV L–P (transcriptase) and LPC (replicase) complexes reconcile these models by revealing that C protein binding induces a replication-competent conformation of the polymerase. These structures reveal a previously unrecognized function of the C protein: In addition to antagonizing host innate immunity, C promotes a replication-competent conformation of the polymerase and facilitates N protein recruitment to the RNA exit channel. Collectively, these findings establish the C protein as a multifunctional molecular switch that regulates the transcription–replication transition, enhances polymerase processivity, and safeguards RNA synthesis fidelity. Our study unifies existing models and provides a structural framework for understanding how paramyxoviral polymerases are regulated by viral cofactors.

In paramyxoviruses, C genes are encoded only by members of the morbillivirus (e.g., MeV, CDV, RPV), henipavirus (e.g., NiV, HeV), and respirovirus (e.g., SeV, HPIV1, HPIV3) genera, but are notably absent in rubulavirus (e.g., PIV5, MuV), orthorubulavirus (e.g., PIV2), and avulavirus (e.g., NDV). This phylogenetic distribution raises an intriguing biological question: How do C-lacking viruses coordinate the transcription–replication switch and execute efficient RNA synthesis without this regulatory protein? Upon analyzing the domain architecture of P proteins across these genera, we observed a notable pattern: P proteins from C-encoding viruses tend to be substantially longer, primarily due to the presence of an extended intrinsically disordered region (IDR) between the NP-binding peptide (NPBP) and the oligomerization domain (SI Appendix, Fig. S19A). In contrast, C-lacking viruses encode shorter P proteins with minimal disordered regions (SI Appendix, Fig. S19B). This structural difference likely reflects functional adaptation. In viruses with long, disordered P proteins, the extended N-terminal region may reduce the efficiency of N protein delivery to the nascent RNA, as the P-bound N must diffuse through a larger conformational space before reaching the product RNA emerging from the polymerase. This spatial and dynamic limitation could slow the transfer of N to the growing RNA chain. In contrast, shorter or more structured linkers are likely to promote a more efficient and timely handoff. Our MST result supports this model. We found that the C protein preferentially binds monomeric or ssRNA-bound N protein rather than the N⁰P complex and is likely unable to efficiently extract the monomeric N from the P chaperone and deliver it to the elongating polymerase, thereby compensating for the functional limitations imposed by the long P-protein linker. Together, these observations suggest a functional division of labor in C-encoding viruses: the P protein orchestrates RdRP complex assembly and RNP template engagement, whereas the C protein supports N protein recruitment and enhances polymerase processivity. In C-lacking viruses, however, the P protein may perform all these roles, acting as a multifunctional coordinator of RNA synthesis. Together, these findings propose a mechanistic basis for how viruses with or without the C gene may adapt their replication strategies through structural divergence of their P proteins, highlighting the evolutionary flexibility of the paramyxoviral polymerase machinery.

To date, clinically approved antivirals that directly target essential viral enzymes remain limited, especially for highly pathogenic and lethal RNA viruses. The development of potent small-molecule inhibitors against such pathogens is therefore both urgent and technically challenging. Although previous studies have identified candidate inhibitors for morbilliviruses, including the ERDRP-0519 series and GHP-88309, the pharmacological basis of their binding specificity and inhibitory mechanisms has not been fully elucidated (42, 44). By contrast, our cryo-EM structure of the MeV L–P complex bound to ERDRP-0519 reveals a previously unrecognized allosteric pocket at the interface of the RdRp palm and fingers subdomains. The inhibitor inserts deeply into this cleft like a nail, with its head positioned near the catalytic center of the polymerase. Binding induces substantial conformational rearrangements in the active site, distorting its geometry and locking the enzyme in an inactive state. Importantly, this binding pocket is highly conserved across the Paramyxoviridae family. Despite sequence variations at the inhibitor binding site in NiV, ERDRP-0519 still binds the same pocket and induces similar inhibitory conformational changes. It is worth noting that the early analogue of ERDRP-0519, 16677, which fitted well into the ERDRP-0519 density in both the MeV and NiV LPE complex (SI Appendix, Figs. S13F and S18J), was reported to inhibit MeV but not NiV in minigenome assays (35), raising the question of whether ERDRP-0519 might similarly lack activity outside morbillivirus. In addition, 16677 also exhibited weak and highly variable activity even within morbillivirus, with an EC₅₀ for CDV more than 100-fold higher than for MeV, despite these two viruses sharing ~61% sequence identity for the L protein (35). Subsequent optimization produced ERDRP-0519, which demonstrates markedly improved cross-morbillivirus potency, including only a ~two-fold difference between MeV and CDV (40). Furthermore, we show that the N-ethylmorpholinyl group on the piperidine ring of ERDRP-0519, originally designed to enhance solubility and oral bioavailability, extends into the central cavity of the polymerase and introduces steric clashes with the template, product RNA, and incoming NTP in the elongation state, suggesting an additional mechanism of inhibition in ways not achievable by 16677 and also providing valuable insights for further optimization. Importantly, a recent independent study reports that ERDRP-0519 binds to NiV polymerase with affinity comparable to MeV and inhibits polymerase activity under in vitro conditions (60). Together with the similar conformational rearrangements observed in both studies, these findings help clarify the earlier discrepancy, while highlighting the need for quantitative and cell-based assays to determine the physiological relevance and true antiviral potency against NiV. Finally, our molecular dynamics simulations further support the broad-spectrum potential of ERDRP-0519 within the Paramyxoviridae, revealing favorable and stable binding across multiple genera. However, its binding to polymerases of other nsNSVs, such as rhabdoviruses, filoviruses, and pneumoviruses, appears suboptimal, consistent with previous finding that ERDRP-0519 fails to block the replication of RSV, a distant member of the paramyxovirus family (40). Nonetheless, our structural findings define a conserved and druggable vulnerability in viral RdRPs, offering a valuable blueprint for the future design of next-generation antivirals.

Together, this study provides mechanistic insights into viral RNA synthesis regulation and presents a structural and pharmacological foundation for the development of broad-spectrum inhibitors targeting MeV, NiV, and potentially a wider range of nsNSVs. Moreover, ERDRP-0519 has demonstrated potent protective efficacy against virus-induced lethality in animal models without apparent toxicity (40, 41), further supporting its potential for clinical development and veterinary applications.

Materials and Methods

The MeV L–P, LPC, and the NiV L–P complexes were coexpressed in Sf9 insect cells following the Bac-to-Bac instructions, while MeV N_ΔN-arm and P_NPBP proteins were expressed in Escherichia coli BL21 (DE3) cells. Polymerase inhibitors were added to the apo MeV and NiV L–P complexes prior to cryo-EM grid preparation. Cryo-EM data were collected on a 300 kV Titan Krios transmission electron microscope and processed using cryoSPARC. MST assays were employed to quantify the binding of the MeV C protein to the L–P complex and various forms of N proteins/complexes. Molecular docking and simulation studies were performed to validate the binding of ERDRP-0519 to MeV and NiV polymerases and to predict the binding for the related paramyxoviruses. Detailed experimental procedures are provided in SI Appendix, Methods.

Supplementary Material

Appendix 01 (PDF)

Dataset S01 (XLSX)

Data, Materials, and Software Availability

The cryo-EM maps of the MeV LPC complex, the MeV apo L–P complex in 300 mM salt buffer, the MeV apo L–P complex in 150 mM salt buffer, the MeV L–P complex bound to the allosteric inhibitor ERDRP-0519, and the NiV L–P complex bound to ERDRP-0519 have been deposited in the Electron Microscopy Data Bank (EMDB) with the accession codes EMD-65364 (61), EMD-65365 (62), EMD-65366 (63), EMD-65367 (64), and EMD-65368 (65), respectively. The corresponding atomic coordinates have been deposited in the Protein Data Bank (PDB) under the accession codes 9VUI (66), 9VUJ (67), 9VUK (68), 9VUL (69), and 9VUM (70), respectively. Source data are provided with this paper. All other data are included in the manuscript and/or SI Appendix.