{"paper_id":"474fed0e-79be-45f2-b0c2-ef73977494ca","body_text":"Structure of the Nipah virus polymerase complex | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Structure of the Nipah virus polymerase complex Jonathan Grimes, Esra Balıkçı1, Franziska Günl, Loic Carrique, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4663080/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Nipah virus poses a recurring threat, causing severe respiratory and neurological disease in Southeast Asia. Since its first identification in Malaysia in 1998 and a subsequent outbreak in Singapore in early 1999, the virus has emerged as a highly virulent zoonotic paramyxovirus. Despite its lethality, there is currently no approved treatment for Nipah virus infection. The viral polymerase complex, composed of the large polymerase protein (L) and the phosphoprotein (P), is responsible for the replication of the viral RNA genome and transcription of viral genes. However, the mechanisms by which the L and P components perform these activities remain unknown. Here, we describe the structures of the Nipah virus L-P polymerase complex at a 2.5 Å resolution and the L protein’s Connecting Domain (CD) at a 1.85 Å resolution, determined by cryo-electron microscopy (cryo-EM) and X-ray crystallography, respectively. The L-P complex structure reveals the organization of the RNA-dependent RNA polymerase (RdRp) and polyribonucleotidyl transferase (PRNTase) domains of the L protein, and how the P protein, which forms a tetramer, interacts with the RdRp domain of the L protein. Furthermore, the CD structure reveals the binding of Mg ions, which likely contribute to the functionality of the PRNTase domain. These findings offer insights into the structural details of the L-P polymerase complex and the molecular interactions between L and P, shedding light on the mechanisms of the replication machinery. This work will underpin efforts to develop antiviral drugs that target the polymerase complex of Nipah virus. Biological sciences/Structural biology/Electron microscopy Biological sciences/Microbiology/Virology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Nipah virus (NiV) infections cause atypical pneumonia and severe febrile encephalitis, with a mortality rate of up to 75% over the past several decades [ 1 , 2 ]. NiV was first identified during an outbreak affecting pigs and pig farmers in Malaysia and Singapore in 1998–1999 [ 3 ]. During this outbreak, 265 cases of encephalitis occurred, resulting in the deaths of 105 people and the culling of nearly one million domestic pigs [ 1 , 4 ]. Later, sporadic outbreaks of NiV occurred in India, including the last one in Kerala in 2023, while Bangladesh has been hit with outbreaks almost every year since 2001 [ 5 ]. Being a recurring threat with high mortality rates and a lack of specific treatments, as well as a zoonosis with the potential of human-to-human transmission, Nipah virus represents a significant public health problem. NiV and the closely related Hendra virus (HeV) are members of the genus Henipavirus within the Paramyxoviridae family. Henipaviruses contain a single-stranded, negative-sense non-segmented RNA (nsNSV) genome that encodes six structural proteins. The RNA genome is encapsidated by nucleoprotein (N), forming a helical nucleocapsid [ 6 ]. The RNA genome is both transcribed into viral genes and replicated by the viral RNA polymerase complex comprising the Large (L) protein and Phospho (P) protein. The L protein, a pivotal component of the polymerase complex, contains three catalytic domains, the RNA-dependent RNA polymerase (RdRp), the polyribonucleotidyl transferase (PRNTase), and methyltransferase (MTase), in addition to two structural domains, the connecting domain (CD) and C-terminal domain (CTD). During transcription, the L protein catalyses the synthesis of a 5’ capped and 3’ polyadenylated mRNA. Viral RNA replication is a two-step process with the initial synthesis of a full-length copy of the genome, known as the antigenomic RNA of positive polarity. This antigenomic RNA, in turn, acts as a template for the subsequent synthesis of the genomic RNA [ 7 ]. These processes rely on the presence of the non-catalytic P protein, which plays a pivotal role as a coordinating hub for the L protein, the nucleocapsid and RNA free N [ 8 ]. P is composed of an ordered N-terminal peptide, separated by an intrinsically disordered region from a central oligomerization domain (OD) and a C-terminal X domain (XD) [ 9 , 10 ]. The N-terminus of the P protein acts as a chaperone by binding to RNA free N protomers (N0), which subsequently encapsidate the newly synthesized antigenomic and genomic viral RNA [ 8 , 11 ]. The C-terminal XD domain has been shown to be important in mediating the interaction between the RNA bound nucleocapsid and the L protein [ 12 ]. Thus, the P protein functions as a central hub in coordinating the assembly of ribonucleoprotein complexes [ 13 ]. Recent years have seen significant advancements in understanding the replication and transcription mechanisms of nsNSVs, with the publication of the structures of L-P complexes from a number of mononegaviruses. These include vesicular stomatitis virus (VSV) [ 14 , 15 ], rabies virus (RABV) [ 16 ], human respiratory syncytial virus (HRSV), [ 17 – 19 ], human metapneumovirus (hMPV) [ 18 ], parainfluenza virus 3 and 5 [ 20 , 21 ], Newcastle disease virus (NDV) [ 22 ], Mumps virus (MuV) [ 23 ] as well as Ebola virus (EBOV) [ 24 , 25 ]. These studies reveal the conserved RdRp and PRNTase domains, the flexibility of the CD, MTase and CTD as well as the conformational changes when bound to their RNA templates. Here, we present the molecular structure of the NiV L-P complex imaged by cryo-electron microscopy (cryo-EM) and the crystal structure of the CD of the L protein. These results reveal the structural arrangement of the RdRp and PRNTase domains of the polymerase complex, and how the tetrameric P protein binds to L. By modelling the CD domain onto an Alphafold 3 model of an RNA-L-P complex our data suggest insights into the early steps of capping and the conformational changes needed for the elongation of the synthesised viral RNA. Results Overall structure of the NiV L-P complex To determine the structure of the NiV polymerase complex, we co-expressed the L and P proteins from the Malaysian strain of NiV in Spodoptera frugiperda 9 (Sf9) cells. Purification of the polymerase complex yielded a stoichiometric preparation suitable for structural and biochemical studies (Supplementary Fig. 1a, b). To confirm that the L-P complex was functional, we performed a template-dependent in vitro polymerase assay using a 12 nucleotide 3′ leader sequence of the NiV genome as a template (Supplementary Fig. 1c). The activity assays revealed that the L-P complex produced RNA products of varying lengths in agreement with earlier studies [ 7 ]. Substituting the catalytic active site aspartic acid with alanine at position 832 (D832A), resulted in no RNA product, indicating a loss of polymerase activity (Supplementary Fig. 1d). Substituting adenosine triphosphate with remdesivir triphosphate did not terminate strand elongation but resulted in the same pattern of RNA products but with slightly reduced mobility (Supplementary Fig. 1e). We determined the structure of the L-P complex at a 2.5 Å resolution using single particle cryo-EM. The final reconstruction (Supplementary Fig. 2) enabled us to build a model for a portion of the L protein bound to an asymmetric tetramer of P (Fig. 1 ). The N-terminal RdRp and PRNTase domains of the L protein were almost entirely resolved with the PRNTase domain bound to two zinc ions. The CD, MTase domain, and the CTD were not visible in our map, despite being present in our construct. The cryo-EM map revealed no density for the N-terminal 475 residues of P, possibly due to the intrinsic disorder of this region. However, we could build models for four separate P monomers of varying lengths, starting from the beginning of the OD domain. The longest visible P monomer spans amino acid residues 477–707; of these, 477–510 correspond to short helical bundles located at the N-terminal end of the OD, 510–575 form the OD, while 660–706 represent the XD region of P (Fig. 1 ). Analysis of the L-P complex by SDS-PAGE after size exclusion chromatography shows that both L and P have maintained their integrity suggesting that the missing domains reflect the highly dynamic nature of the L-P complex (Fig. 1 and Supplemantary Fig. 1 ). Structural details of the polymerase complex The structural analysis revealed the conserved motifs and residues critical for the catalytic function and the regulatory mechanisms of L. The RdRp domain of the L protein folds into the right-handed “fingers-palm-thumb” architecture found in many RNA virus polymerases (Supplementary Fig. 3a) and contains seven specific structural motifs (A-G) (Fig. 2 a, b). Motif A is involved in binding divalent cations via the conserved aspartate residues (Asp722 in NiV), essential for the catalytic activity (Supplementary Fig. 9) [ 26 , 27 ]. Motif B binds template RNA and incoming nucleoside triphosphates (NTPs), and together with motifs A, D and E, it contributes to the structural integrity and the NTPs selection [ 28 , 29 ].The active site residues ( 831 GDNE 834 ) are found at the tip of a β-hairpin formed by motif C, buttressed by structural elements from the fingers subdomain and the N-terminal domain (NTD) of the RdRp that form the tunnel that allows the template RNA to move towards the catalytic site. Motifs F and G, bridging the palm and fingers regions, contribute to the RdRp’s structural stability and flexibility, facilitating effective accommodation of template RNA and NTPs [ 30 ]. The structural elements, including the priming loop (residues 1256–1290), which is thought to support the initiating nucleotide [ 13 , 31 ], and the intrusion loop (residues 1337–1362) of the PRNTase domain, which contains the HR motif that forms a covalent bond with the RNA [ 13 ], as well as the supporting helix (residues 588–600) of the RdRp, which provides structural support and stability to the active site [ 32 ], lacked sufficient density to be fully modelled in our L-P complex structure. Structures of previously solved L-P polymerase complexes from nsNSVs reveal two possible conformational states of the catalytic chamber. In the initiation state, the priming loop and the supporting helix largely occupy the product RNA binding groove [ 14 – 16 ]. After reaching the elongation state, the flexible supporting helix becomes disordered, while the priming loop is retracted and repositioned towards the PRNTase domain to accommodate the nascent double stranded RNA duplex formed between the template and nascent product (Supplementary Fig. 3b) [ 17 , 18 , 24 ]. The absence of sufficient density for these structural elements in the NiV L-P map suggests a degree of flexibility, which is consistent with the absence of bound RNA template and NTP. AlphaFold3 prediction suggests that the missing residues of these structural elements would indeed be stabilized in the presence of a bound template RNA, which is consistent with a composite model based on the EBOV RNA bound L protein structure (Supplementary Fig. 4b, d). Interestingly, the RdRp domain of the NiV harbours a 105 amino acids (605–710) insertion within the palm subdomain, a feature unique to Henipaviruses (Supplementary Fig. 5a). This region is disordered in our maps, indicating its inherent flexibility. Deletion of this sequence resulted in the inhibition of L-P replication in a cell-based minireplicon assay, suggesting its importance for polymerase activity (Supplementary Fig. 5b). The entry and exit channels for template RNA are located adjacent to the fingers and thumb subdomains of the RdRp domain. As the nascent RNA progresses, it passes through a positively charged tunnel towards the product exit channel, where the MTase domain and CTD are located (Fig. 2 c, d). The MTase domain and CTD exhibit dynamic behaviour and can adopt multiple conformations where the CD acts as a hinge between the PRNTase domain and the MTase and CTD portion. Despite the lack of observable density for the C-terminal domains in our cryo-EM map, we determined the structure of the CD by X-ray crystallography at a resolution of 1.85 Å (Fig. 3 a, b). Structural analysis revealed that CD adopts a fold similar to the structures observed within the Paramyxovirus and Rhabdovirus families (Fig. 3 c, d), despite displaying minimal sequence conservation (Supplementary Fig. 9). Interestingly we observed 3 structural magnesium ions (designated as Mg1, Mg2, and Mg3), with Mg1 bound to the N-terminal α2 helix. This part of the CD is known to be dynamic from the structures of other nsNSV CD domains (Fig. 3 d). This flexibility is consistent with its potential role in orchestrating conformational alterations of the C-terminal domains, essential for coordinating the diverse stages of RNA synthesis. Molecular basis for P binding The structure shows that P exhibits a high degree of structural flexibility in which each monomer can adopt different conformations when interacting with different regions of the RdRp domain of L (Fig. 4 a, Supplementary Fig. 8). All four P monomers contain an OD from residues 510 to 575, which form a tetrameric four-helix bundle, as shown previously [ 9 ]. However, the N-terminal and C-terminal regions outside the OD have not been structurally well characterized due to their intrinsic flexibility. Our structure reveals that one of the P monomers (P1) has a complete ordered XD domain that interacts with L through a series of hydrophobic and electrostatic interactions (Fig. 4 b). P1 forms a tentacle-like structure consisting of three consecutive α-helices that surrounds the channel through which NTPs access the active site. Arg669 and Asn702 of P1 form salt bridges with Asp339, and Arg308 of L, respectively (Fig. 4 b), while closer towards the OD domain Arg600 of P1 mediates salt bridges with Glu760 and Glu733 of L (Fig. 4 c). In addition, hydrogen bonds are formed between Pro640, Ala649, Thr670, His671 of P1 and Arg867, Ala879, Asn346, Leu300 of L, respectively (Fig. 4 b, c). Residues 575–578 of P3 form a β-strand which is sandwiched between a parallel β-strand from P1 (597–599) and an anti-parallel strand from L (residues 385–388) to form a three stranded β-sheet. These interactions are mediated by hydrogen bonds and hydrophobic interactions (Fig. 4 d). Residues 570–595 of P2 bind on the opposite side of the RdRp compared to P1. The His570 of P2 mediates a salt bridge with Glu448 of L and is also involved in a hydrogen bond with Tyr389. The residues Asn590 of P2 interact with the main chain of Met459 of L, via a hydrogen bond, while Leu594 and Pro579 of P2 mediate hydrophobic interactions with Leu525 and Tyr732 of L, respectively (Fig. 4 e). A model for RNA binding and processing To gain further insights into the mechanism of RNA synthesis, we constructed a composite model by integrating the cryo-EM structure of RdRp and PRNTase, and the crystal structure of CD, onto an AlphaFold 3 (AF3) model of the full L polymerase (Supplementary Fig. 7) where the MTase and CTD domains are predicted to be ordered (Figs. 5 a, b). This full-length AF3 L model predicts the localization of disordered structural elements—specifically, the intrusion loop, priming loop, and insertion sequence—on the cryo-EM structure of L (Fig. 6 a). The model was further extended by including a 21-nucleotide long 3’-leader sequence as the RNA template and product RNA of varying lengths, ranging from 9 to 15 nucleotides, along with the addition of NTPs and Mg ions. This new composite model predicts with confidence the pathway of the template RNA as it enters and subsequently exits the active site of the RdRp. It also predicts how the product RNA egresses towards the PRNTase and CD domains (Fig. 6 a). The model illustrates how the template RNA is positioned in the active site, where nucleotides are incorporated into the elongating nascent RNA, resulting in the formation of an intermediate RNA duplex (Fig. 6 b). Following this, the two strands are separated, and the template and product are directed towards the template or the product exit channels, respectively (Fig. 6 b). The model also demonstrates the positioning of the conserved GxxT motif ( 1273 GSST 1276 ) of the priming loop, which is thought to bind GTP [ 14 , 33 , 34 ] and the localization of the HR motif ( 1347 HR 1348 ), both of which are crucial for capping of the 5’ end of the nascent RNA (Fig. 6 c) [ 13 ]. Additionally, our composite model predicts the position of the crystallographically determined Mg1 ion bound to the α2 helix of the CD suggesting a role in positioning the GTP required for the capping process (Fig. 6 d). These structural insights underscore the intricate coordination of multiple elements within the polymerase complex necessary for RNA processing. Discussion This study provides structural and functional insights into the NiV polymerase complex. Although the sequences of L differ across the nsNSVs (Supplementary Fig. 9), the architecture and key structural motifs of the L-P complex are conserved. Structural comparison of the RdRp and PRNTase domains across paramyxoviral, pneumoviral, filoviral, and rhabdoviral L proteins yield valuable insights into evolutionary adaptations and species-specific variations in polymerase functionality (Supplementary Fig. 3). The RdRp domain, which plays a central role in RNA synthesis, exhibits high structural conservation among nsNSVs (Supplementary Fig. 3a). However, despite this conservation, the RdRp domain and the NTD can accommodate different insertions, such as the palm insertion observed in NiV, and the NTD insertion in EBOV [ 24 ]. These insertions likely contribute to the functional diversity and regulatory mechanisms observed across the different viral species. In addition, the PRNTase domain, demonstrates significant flexibility in particular functional loops, such as the priming and intrusion loops in performing specific functions during replication and transcription (Supplementary Fig. 3b), contributing to the versatility and adaptability of the polymerase complex in different viral contexts. RNA synthesis by the Nipah virus polymerase complex is a tightly regulated process involving coordinated interactions between the L protein, P protein, and the RNA template encapsidated by N. The 3’ end of the genome is composed of the leader RNA region which is a bipartite promoter that is highly conserved among paramyxoviruses. This sequence is recognized by L to initiate RNA synthesis [ 7 ]. After binding to the leader RNA, the polymerase complex initiates and either proceeds with the replication of full-length antigenomes and genomes or releases the short initial leader RNA product and scans through the genome until it locates one of the ‘gene start’ elements at the beginning of each gene to initiate transcription and produce mRNA. Both replication and transcription use an unprimed initiation mechanism, however, replication initiates terminally as opposed to transcription which initiates internally. Based on our modelling, the active site can accommodate a 9-mer intermediate duplex formed during RNA synthesis, before the two strands—template and product—are separated and directed towards their respective exit channels (Fig. 6 b). Structural studies of L-P complexes from other viruses have revealed the conformation of the CD along with the presence of the C-terminal domains [ 16 , 20 – 23 ]. These studies have demonstrated that the positioning of the CD modulates the organization of the MTase and CTD, thereby allowing the RNA to access the MTase [ 21 , 23 ]. The nascent transcript RNA is initially capped at its first nucleotide, with the cap subsequently methylated by the MTase [ 35 ]. This process involves the covalent attachment of the first nucleotide of the product mRNA to the side chain of the conserved histidine residue in the HR motif, forming an intermediate (L)-(histidyl-Nε2)–pRNA (L–pRNA) [ 13 ]. Following the formation of the L-pRNA complex, RNA is transferred from L-pRNA to GDP and to form the cap structure. The MTase domain subsequently methylates the N7 and O2 of the newly formed cap. Our modelling suggests that a 9 to 10 nts long product is required to be synthesized to reach the conserved HR motif ( 1347 HR 1348 ) for subsequent covalent attachment (Fig. 6 c). Additionally, the model predicts the binding of GTP to the conserved GxxT motif ( 1273 GSST 1276 ) in the priming loop (Fig. 6 c, d). The transfer of the RNA onto the GDP is metal-dependent mainly involving divalent cations [ 36 ]. The crystal structure of the CD reveals the presence of three bound Mg ions. Interestingly, the superimposition of the CD with the AF3 model ideally positions the flexible α2 helix of the CD domain, and the bound Mg1 next to close to the predicted GTP. It is likely that Mg1 likely plays a crucial role in the capping process by aiding the positioning of the GTP during the transfer reaction of PRNTase (Fig. 6 d). This finding suggests that the flexibility of the α2 helix and its interaction with Mg ions is essential for coordinating the conformational changes required for effective RNA synthesis and capping. Additionally, the crystal structure of CD has another bound Mg ion (Mg2) which engages with residues involved in crystal contacts (Supplementary Fig. 6a, b) but – in the context of the full-length polymerase – could have a structural role stabilizing the capping conformation by stabilizing the CD linker1 (Supplementary Fig. 6c, d).This comprehensive model advances our understanding of the spatial arrangement and functional dynamics within the NiV polymerase complex, providing a detailed framework for further investigations into viral replication mechanisms. Based on observations of P tetramers in nsNSVs L-P complexes, both the cartwheeling and sliding models have been proposed to describe the movement of the polymerase complex along the nucleocapsid [ 37 – 41 ]. The cartwheeling model implies that all four XDs must bind sequentially to L during RNA synthesis. Recent studies have challenged this model by showing that P tetramers where one to three XDs are deleted, maintain comparable or even heightened RNA synthesis activity [ 37 ]. Notably, even a single XD capable of binding to N protein is adequate for minigenome transcription [ 37 ]. In addition, our structural studies reveal that around 3500 Å 2 of surface area of L are bound by P, suggesting that the dissociation of P from L is highly unlikely. These observations lend support to the alternative sliding model where the oligomeric P moves along the nucleocapsid without necessitating rotational movement. In the case of the NiV, it is likely that the sliding of the L-P complex along the nucleocapsid is driven primarily by the process of RNA synthesis itself (Fig. 7 ). This movement is facilitated by the continuous association of the polymerase complex with the RNA strand, ensuring that as nucleotides are added to the growing RNA chain. This continuous contact is essential for efficient transcription and replication, stabilizing the polymerase complex and enhancing processivity, ultimately ensuring the fidelity and efficiency of viral RNA synthesis. Nipah virus infections continue to pose a threat to public health. An approved vaccine against the related HeV has been used effectively in horses in Australia to prevent zoonotic transmission to humans [ 42 ], but bats are carriers of NiV and the primary source of human infection, making a similar approach for Nipah virus unlikely. This structural and functional characterization of the NiV L-P complex provides the basis for understanding the molecular details and the function of the polymerase complex and thus accelerates the development of therapeutic anti-viral drugs active against the NiV polymerase complex. Methods Cells and plasmids Human embryonic kidney 293T cells (HEK-293T) were maintained in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS; Sigma-Aldrich) at 37°C and 5% CO2. BSR-T7/5 cells, cells stably expressing the T7 polymerase that are derived from baby hamster kidney cells (BHK-21) [ 43 ] were maintained in DMEM with 10% FBS and 1 mg/ml G418/Geneticin (Roche) every third cell culture passage. The Spodoptera frugiperda Sf9 cells were maintained in Sf-900™ II SFM medium (Thermo Scientific) at 27°C. pCAGGs plasmids expressing NiV L, P and N proteins (Bangladesh, 2004) as well as the pUC57 plasmid expressing T7-dependent NiV bicistronic minigenome (BMG) [ 10 ] were kindly provided by Micheal Lo (CDC). Plasmids expressing truncated versions of L proteins were generated by Gibson assembly (NEB). Protein expression and purification of NiV L-P complex The coding sequences for Malaysian strain NiV L (NCBI: NP_112028.1) with an N terminal twin-Strep tag and P (NP_112022.1) with the C-terminal octa-His tag were codon optimized for insect cell expression and cloned into a pFL plasmid downstream of polyhedrin and p10 promoters, respectively. The preparation of Baculovirus stocks and protein expression were performed following the Bac to Bac manual (Invitrogen). Two litres of Sf9 cells expressing the L-P complex were harvested 72 hours post infection. The cells were resuspended in Buffer A (50 mM HEPES pH 7.4, 500 mM NaCl, 10% vol/vol glycerol, 2 mM tris(2-carboxyethyl) phosphine (TCEP), 5 mM MgCl 2 ) which was further supplemented with 0.05% wt/vol n-octyl beta-d-thioglucopyranoside, one protease inhibitor cocktail tablet (Roche, cOmplete Mini, EDTA-free), 2 mM phenylmethylsulfonyl fluoride (PMSF), Bensonase, and RNase. Cells were lysed using a Dounce homogeniser and clarified with centrifugation. The lysate was incubated with the pre-equilibrated Strep-Tactin® XT Sepharose resin (IBA Lifesciences) for 3 h and resin was washed with the BufferA. The NiV L-P complex was eluted with 50 mM Biotin in Buffer A and further purified with Superose 6 increase 10/300 size exclusion column (GE Healthcare) in Buffer A. The fractions eluting after the void volume (between 10-12ml) was collected and concentrated to 0.5 mg/ml. The L-D832A mutant, in complex with P, was expressed and purified using the same methods as the wild-type L-P complex. Protein expression and purification of CD of NiV L Residues 1480–1742, encoding the CD of L, were cloned into a pET28a vector with an N-terminal hexa-His and Sumo tag. The construct was transformed into BL21(DE3) cells and protein expression was induced with 0.5 mM IPTG at OD 600 = 0.6 and expression was carried out at 18°C for 18 hours. Six litres of bacterial cell pellet were lysed by sonication in a Lysis Buffer containing 50 mM HEPES pH 7.6, 500 mM NaCl, 10% vol/vol glycerol supplemented with protease inhibitors (Roche, cOmplete Mini, EDTA-free), RNase A, and lysozyme (Sigma). Supernatant cleared after ultracentrifugation was filtered and loaded onto HisTrap 5 ml HP columns. Protein was washed and eluted with a gradient increase in imidazole concentration. Elution fractions were combined, desalted into the Final Buffer containing 20 mM HEPES pH 7.6, 500 mM NaCl, 10% vol/vol glycerol and then incubated with Ulp1 overnight at 4°C. Cleaved protein was re-injected into HisTrap 5 ml HP to remove Ulp1 enzyme as well as the cleaved His-Sumo tag. The flowthrough was collected, concentrated, and injected into Superdex 75 16/600 (GE Healthcare) that was pre-equilibrated with the Final Buffer. The protein was concentrated to 10 mg/ml and stored at -80°C. In vitro RNA synthesis assay For the NiV L-P complex in vitro assay, 3 µl reactions were set up containing reaction buffer (20 mM Tris pH 7.5, 10 mM KCl, 2 mM DTT, 0.5% triton, 10% DMSO, 1 U Rnasin (Promega), 5 mM MgCl2), 0.25 µM RNA-template derived from the NiV leader sequence (UGGUUUGUUCCC or UGGUCUGUUCCC), 0.5 µM recombinant L-P complex, 0.5 mM ATP, 0.5 mM CTP, 0.1 µM GTP and 200 µM primer (pACCA). ATP was substituted with remdesivir triphosphate (APExBIO), where indicated. The radioisotope tracer in these reactions was [α 32 P] GTP (Revvity). The reactions were incubated at 30 ̊C for 1 h, stopped with the addition of 3 µl formamide loading buffer and denatured at 95 ̊C for 3 minutes. A 32 P-5’end labelled 20 nucleotide-long DNA served as a marker. RNA products were separated on a 22% polyacrylamide urea gel for 2.5 hours at 35 W and the level of [α 32 P] GMP incorporation was imaged by phosphorimaging on a FLA-5000 scanner (Fuji). Minireplicon assay BSR-T7/5 cells were seeded in 24-well plates at 5 x 10 4 cells/well. 24 hours later, cells were transfected in duplicates with the BMG plasmid (0.4 µg), as well as pCAGGs plasmids expressing for NiV L (0.4 µg), P (0.4 µg), N (0.25 µg) using LT-1 transfection reagent (MirusBio). For negative controls pCAGGs-NiV-L was substituted with a pCAGGs-empty vector. After 48 hours, Gaussia luciferase activity was determined using the Renilla Luciferase Assay System (Promega). For this purpose, cells were lysed in 50 µl Renilla lysis buffer on a microplate shaker at room temperature. After 45 min, 20 µl of the cell lysates were mixed with 50 µl of the Renilla luciferase reagent and RLUs were analysed using a GLOMAX 20/20 luminometer (Promega). Cryo-EM sample preparation, data acquisition and processing 3.5 µl of the NiV L-P complex at 0.5 mg/ml was applied to a freshly glow-discharged UltrAufoil Au 1.2/1.3 300 mesh grid. The sample was blotted for 6 s and plunge frozen in liquid ethane. All grids were prepared using a Vitrobot Mark VI (FEI) under conditions of 100% humidity and 20°C. Cryo-EM data was collected at the Oxford Particle Imaging Centre (OPIC) using a 300 kV G3i Titan Krios microscope (Thermo Fisher Scientific) equipped with a SelectrisX energy filter and Falcon 4i direct electron detector. Automated data collection was setup in EPU 3.4 and a total of 22,859 movies were recorded in EER format, of which 14,319 were collected with a tilt angle of 30°. Data was collected using AFIS with a total dose of ~ 50 e-/Å2, a calibrated pixel size of 0.7303 Å/pix, defocus range of -1.4 to -2.4, and with 10 eV slit. Cryo-EM data collection parameters and refinement statistics are summarized in Table S1. Data processing was performed in CryoSparc v4.4.1 [ 44 ] by following the workflow outlined in Supplementary Fig. 2. Briefly, motion correction and patch CTF estimations were performed for movie frames, and low-quality images were eliminated by manual inspection and excluded from further analysis. A template search was prepared using the PIV5 L-P structure (PDB 6V85) as the template. Following template picking, 2D classes were obtained, and the best-resolved classes were selected for ab-initio model generation. The ab-initio models were refined using Heterogenous refinement. The resulting optimal map was refined using NU-refinement with C1 symmetry. This refined map was then used to train Topaz picking. The picked particles were directly subjected to heterogeneous refinement, and the best resulting map was selected for further refinement using NU-refinement. This entire process, from particle picking to Topaz training, was repeated three times to increase the number of picked particles. The map, containing 490,675 particles, was corrected for reference motion and subjected to 3D classification to identify classes that represented clear densities for the L and P-XD domain. A total of 299,261 particles were selected and refined for the L domain using local NU refinements, resulting in a 2.52 Å resolution map. Meanwhile, 206,718 particles were refined for the P tetramer using local NU refinement, yielding a 2.75 Å resolution map. The local maps were further processed with DeepEMhancer. Cryo EM model building and refinement Initial models were generated for L and P tetramer separately using ModelAngelo [ 45 ]. To improve model geometry, multiple cycles of manual building were performed using COOT [ 46 ] followed by real space refinement against the corresponding local maps in PHENIX [ 47 ]. The model geometry was validated using MolProbity [ 48 ]. Comprehensive statistics for the map and model are presented in the Supplementary Table 1. Structural analysis and figure preparation were conducted using UCSF Chimera [ 49 ] and UCSF ChimeraX [ 50 ]. Crystallization of CD, X-ray data collection and structure solving Freshly purified CD, at a concentration of 10 mg/mL, was mixed with a set of sitting-drop crystallization screens in ratios of 1:1, 2:1, and 1:2, and incubated at 20°C. The crystals were obtained in 25 days from a condition consisting of 35% tert-Butanol and 0.1M tri-Sodium Citrate pH 5.6. The crystals were harvested, cryo-preserved in the crystallization condition containing 20% v/v glycerol and flash frozen in liquid nitrogen. X-ray diffraction data were collected on the I04 beamline at Diamond Light Source (Harwell, U.K.). The data processing was performed using the program DIALS in combination with XIA2 [ 51 ]. The structure was solved via PHASER molecular replacement [ 52 ], employing a search model generated by AlphaFold [ 53 ]. Subsequent manual model building was performed in COOT [ 46 ], followed by refinement in PHENIX [ 47 ]. X-ray data collection and refinement statistics are summarized in Table S2. AlphaFold modelling The RNA-bound NiV L structure was predicted in AlphaFold 3 by providing the leader and product RNA sequences as the input along with the corresponding protein sequences [ 54 ]. For the X-ray crystallography analysis, a reference model for molecular replacement of the CD was prepared using AlphaFold 2 [ 53 ]. Declarations Data availability Structural data generated in this study have been deposited in the Protein Data Bank (PDB) and the Electron Microscopy Data Bank (EMDB) under the following accession codes: PDB 9FTF (crystal structure of the CD), PDB 9FUX (composite model of the NiV L-P complex). The corresponding EMDB entries are EMD- 50781 (composite map), EMD-50808 (consensus map), EMD- 50805 (L focused map), and EMD- 50807 (P focused map). Acknowledgements We thank Michael Lo for providing the plasmids expressing NiV L, P, and N proteins and for the bicistronic minigenome (BMG) plasmid. We thank members of the Grimes and Fodor Laboratories for helpful comments and discussions. We thank Maria Harkiolaki for proofreading the main text. This work was supported by Bill and Melinda Gates Foundation INV-048922 (to J.M.G. and to E.F ). Access to electron microscopes was provided by the OPIC Electron Microscopy Facility (funded by Wellcome JIF (060208/Z/00/Z) and equipment (093305/Z/10/Z) grants). Access to computational resources was supported by the Wellcome Trust Core Award Grant Number 203141/Z/16/Z with additional support from the NIHR Oxford BMRC. Molecular graphics and analyses were performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. Contributions E.B., F.G., E.F. and J.M.G. conceived and designed the study. E.B. generated recombinant baculoviruses, purified protein, performed crystallization and solved structure. E.B., L.C. and J.R.K. performed structural analyses. E.B and J.R.K performed AF3 predictions. F.G. generated plasmids and performed functional assays. E.B., E.F. and J.M.G wrote the paper with input from all authors. Ethics declarations Competing interests The authors declare no competing interests. References Chua, K.B., et al., Nipah virus: a recently emergent deadly paramyxovirus . Science, 2000. 288(5470): p. 1432–5. Eaton, B.T., et al., Hendra and Nipah viruses: different and dangerous . Nat Rev Microbiol, 2006. 4(1): p. 23–35. 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Banerjee, Novel initiation of RNA synthesis in vitro by vesicular stomatitis virus . Nature, 1975. 255(5503): p. 37–40. Ogino, T., S.P. Yadav, and A.K. Banerjee, Histidine-mediated RNA transfer to GDP for unique mRNA capping by vesicular stomatitis virus RNA polymerase . Proc Natl Acad Sci U S A, 2010. 107(8): p. 3463–8. Du Pont, V., Y. Jiang, and R.K. Plemper, Bipartite interface of the measles virus phosphoprotein X domain with the large polymerase protein regulates viral polymerase dynamics . PLoS Pathog, 2019. 15(8): p. e1007995. Kolakofsky, D., et al., Viral DNA polymerase scanning and the gymnastics of Sendai virus RNA synthesis . Virology, 2004. 318(2): p. 463–73. Kolakofsky, D., et al., Sendai Virus and a Unified Model of Mononegavirus RNA Synthesis . Viruses, 2021. 13(12). Kolakofsky, D., Paramyxovirus RNA synthesis, mRNA editing, and genome hexamer phase: A review . Virology, 2016. 498: p. 94–98. Curran, J., A role for the Sendai virus P protein trimer in RNA synthesis . J Virol, 1998. 72(5): p. 4274–80. Middleton, D., et al., Hendra virus vaccine, a one health approach to protecting horse, human, and environmental health . Emerg Infect Dis, 2014. 20(3): p. 372–9. Buchholz, U.J., S. Finke, and K.K. Conzelmann, Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter . J Virol, 1999. 73(1): p. 251–9. Punjani, A., et al., cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination . Nat Methods, 2017. 14(3): p. 290–296. Jamali, K., et al., Automated model building and protein identification in cryo-EM maps . Nature, 2024. 628(8007): p. 450–457. Casanal, A., B. Lohkamp, and P. Emsley, Current developments in Coot for macromolecular model building of Electron Cryo-microscopy and Crystallographic Data . Protein Sci, 2020. 29(4): p. 1069–1078. Afonine, P.V., et al., Real-space refinement in PHENIX for cryo-EM and crystallography . Acta Crystallogr D Struct Biol, 2018. 74(Pt 6): p. 531–544. Davis, I.W., et al., MolProbity: all-atom contacts and structure validation for proteins and nucleic acids . Nucleic Acids Res, 2007. 35(Web Server issue): p. W375-83. Pettersen, E.F., et al., UCSF Chimera–a visualization system for exploratory research and analysis . J Comput Chem, 2004. 25(13): p. 1605–12. Goddard, T.D., et al., UCSF ChimeraX: Meeting modern challenges in visualization and analysis . Protein Sci, 2018. 27(1): p. 14–25. Gildea, R.J., et al., New methods for indexing multi-lattice diffraction data . Acta Crystallogr D Biol Crystallogr, 2014. 70(Pt 10): p. 2652–66. McCoy, A.J., et al., Phaser crystallographic software . J Appl Crystallogr, 2007. 40(Pt 4): p. 658–674. Jumper, J., et al., Highly accurate protein structure prediction with AlphaFold . Nature, 2021. 596(7873): p. 583–589. Abramson, J., et al., Accurate structure prediction of biomolecular interactions with AlphaFold 3 . Nature, 2024. Sievers, F., et al., Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega . Mol Syst Biol, 2011. 7: p. 539. Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-4663080\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":324818884,\"identity\":\"aa4170ec-b09d-4ffe-80c5-3775dfbe57a0\",\"order_by\":0,\"name\":\"Jonathan Grimes\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYBACxgYowQ/hScBlDPBqOQgkJBuYidQCBiAtBgeYoQYQ0sLc3nvw88cddnnG588f+8y7w4KBXyKB8cMPhsPGOB3Wcy5Z4uCZ5GKzG8nMs3nPSDBI9hxgluxhOGyGU8uMHAOJg23MidtuMDMz87ZJMBgcb2CQZmA4bINHi/GPg231iZv7D0O1HGZg/k1AixnQlsOJGxiS4bawgWzB7bCeM2YWZ9uOJ864kWzMOPeMBI9kz8E2yx6DdJzeN2zvMb5R2Vad2N9/8DHD2x11cvwSyYdv/KiwNmzApQVdggcSO3giUh631CgYBaNgFIwCKAAAdStTmuXrWf0AAAAASUVORK5CYII=\",\"orcid\":\"https://orcid.org/0000-0001-9698-0389\",\"institution\":\"University of Oxford\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Jonathan\",\"middleName\":\"\",\"lastName\":\"Grimes\",\"suffix\":\"\"},{\"id\":324818885,\"identity\":\"e62b28d2-d26d-4ec6-9fdb-094dec51f97f\",\"order_by\":1,\"name\":\"Esra Balıkçı1\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Oxford\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Esra\",\"middleName\":\"\",\"lastName\":\"Balıkçı1\",\"suffix\":\"\"},{\"id\":324818886,\"identity\":\"8ba7f519-ac4a-4242-968d-64c3c0a3c863\",\"order_by\":2,\"name\":\"Franziska Günl\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Oxford\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Franziska\",\"middleName\":\"\",\"lastName\":\"Günl\",\"suffix\":\"\"},{\"id\":324818887,\"identity\":\"e196bd1f-267b-4633-8db8-6f2981538a48\",\"order_by\":3,\"name\":\"Loic Carrique\",\"email\":\"\",\"orcid\":\"https://orcid.org/0000-0001-5332-8593\",\"institution\":\"University of Oxford\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Loic\",\"middleName\":\"\",\"lastName\":\"Carrique\",\"suffix\":\"\"},{\"id\":324818888,\"identity\":\"974706f6-b616-446c-a4a4-357c82036ef1\",\"order_by\":4,\"name\":\"Jeremy Keown\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Warwick\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jeremy\",\"middleName\":\"\",\"lastName\":\"Keown\",\"suffix\":\"\"},{\"id\":324818889,\"identity\":\"dba1f731-2ece-4c94-b7c4-579c3636cc50\",\"order_by\":5,\"name\":\"Ervin Fodor\",\"email\":\"\",\"orcid\":\"https://orcid.org/0000-0003-3249-196X\",\"institution\":\"University of Oxford\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Ervin\",\"middleName\":\"\",\"lastName\":\"Fodor\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-06-30 14:10:21\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-4663080/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-4663080/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":60173055,\"identity\":\"c97313af-c1f7-4c89-95b0-15e0af51b364\",\"added_by\":\"auto\",\"created_at\":\"2024-07-12 15:19:28\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":780516,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eStructure of the NiV L-P complex. \\u003cstrong\\u003ea. \\u003c/strong\\u003eSchematic representation of the domain organization of the NiV L and P proteins. The domains of L and the four copies of P resolved in the L-P structure are shown in colour. \\u003cstrong\\u003eb. \\u003c/strong\\u003eCryo-EM density map of the L-P complex. \\u003cstrong\\u003ec. \\u003c/strong\\u003eCartoon representation of the modelled structure; the domains are colour coded as in (a).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4663080/v1/1d0f5a6a4ae3e5e2e49effaa.png\"},{\"id\":60173057,\"identity\":\"f23bc7a7-b330-4634-8f79-6763ea98a4c4\",\"added_by\":\"auto\",\"created_at\":\"2024-07-12 15:19:28\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":714316,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eStructural organization of the NiV L-P complex\\u003cstrong\\u003e. a.\\u003c/strong\\u003e The sub-domains of the RdRp domain of L are shown in different colours. \\u003cstrong\\u003eb.\\u003c/strong\\u003e The catalytic RdRp motifs A-G are highlighted in different colours. The amino acid residues of the \\u003csup\\u003e831\\u003c/sup\\u003eGDNE\\u003csup\\u003e834\\u003c/sup\\u003e sequence are shown as sticks. \\u003cstrong\\u003ec.\\u003c/strong\\u003e Nucleotide entry, template entry, template exit, and product exit channels are indicated by yellow tunnels. \\u003cstrong\\u003ed. \\u003c/strong\\u003eThe electrostatic iso-surface representation shows that the L-P complex is highly negatively charged, while the RNA binding pocket of the polymerase is highly positively charged (blue: positive, red: negative). On the right is the path of the nascent RNA leading towards the CD, MTase and CTD within the positively charged groove.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4663080/v1/33169438060e31d2aedbc348.png\"},{\"id\":60173056,\"identity\":\"9b8d66ca-0af3-4f05-a0eb-397be9fde2a3\",\"added_by\":\"auto\",\"created_at\":\"2024-07-12 15:19:28\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":323533,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eStructure of NiV L CD a. \\u003c/strong\\u003eSchematic representation of domain boundaries of NiV L CD. \\u003cstrong\\u003eb.\\u003c/strong\\u003e The cartoon representation of the NiV CD is shown in rainbow colours from the N- to C-termini, with secondary structures such as helices, β-sheets and 3\\u003csub\\u003e10\\u003c/sub\\u003e helices labelled as α, β and η, respectively. The three Mg ions (Mg1, Mg2 and Mg3) bound to the NiV CD are illustrated as magenta spheres. A close-up view of the Mg1 bound to the α2 helix is shown, highlighting the residues coordinating the Mg1, which are depicted as sticks. Water molecules are shown as black spheres. \\u003cstrong\\u003ec.\\u003c/strong\\u003e Sequence based phylogram of the structurally available CDs generated in Clustal Omega [55]. \\u003cstrong\\u003ed.\\u003c/strong\\u003e Structural comparisons to the CDs of nsNSV L proteins including members of the \\u003cem\\u003eParamyxoviridae\\u003c/em\\u003e (PIV3: PDB 8KDC, PIV5: PIDB 6V85, MUV: PDB 8IZL, NDV: PDB 7YOU) and \\u003cem\\u003eRhabdoviridae\\u003c/em\\u003e (VSIV: PDB 5A22, RABV: PDB 6UEB) families. CDs exhibit structural flexibility of the α2 helix in their N-terminal region, as indicated by the dashed box.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4663080/v1/710046b851d4ed3ea4824ee9.png\"},{\"id\":60173059,\"identity\":\"cfdb3305-8bb8-4ed8-8e00-4ee82550a123\",\"added_by\":\"auto\",\"created_at\":\"2024-07-12 15:19:28\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":758182,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eInteractions of P with the L RdRp domain.\\u003cstrong\\u003e a. \\u003c/strong\\u003eAn overall representation of the L-P interaction sites, which can be divided into four different regions: b, c, d and e. \\u003cstrong\\u003eb. \\u003c/strong\\u003eP1 forms a 3-helix bundle near the NTP entry site and interacts with the RdRp domain through hydrophobic interactions, salt bridges, and hydrogen bonds. \\u003cstrong\\u003ec.\\u003c/strong\\u003e The linker region of P2, located between the OD and XD domains, interacts with multiple residues on the RdRp.\\u003cstrong\\u003e d.\\u003c/strong\\u003e P3 forms a β-sheet, stabilized by interactions with P1 and L at the L binding interface through hydrogen bonds and hydrophobic interactions. \\u003cstrong\\u003ee.\\u003c/strong\\u003e Interactions between P2 and L are mediated by salt bridges, hydrophobic interactions, and hydrogen bonds. The residues involved in hydrogen bond formation and hydrophobic interactions are shown as sticks. Hydrogen bonds and salt bridges are shown as yellow dashed lines.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4663080/v1/7e8eeef76e01966d4d0169fa.png\"},{\"id\":60173052,\"identity\":\"dbd1189e-9349-4a76-a29e-2a1cc80a1785\",\"added_by\":\"auto\",\"created_at\":\"2024-07-12 15:19:28\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":578610,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eA model for the complete NiV L-P complex.\\u003cstrong\\u003e a. \\u003c/strong\\u003eSchematic diagram of L and P with the indication that the structure of CD was determined by x-ray crystallography while AF3 was used to predict the structures of the MTase and CTD. \\u003cstrong\\u003eb.\\u003c/strong\\u003e Composite model of the NiV L-P complex. The RdRp and PRNTase domains are aligned with the full-length AF3 model of the L protein, achieving a root-mean-square deviation (RMSD) of 1.7 Å. Additionally, the CD of the L protein is aligned with the AF3 model, resulting in an RMSD of 0.9425 Å. The insertion sequence, which is disordered in the cryo-EM map, is predicted by AF3 and is highlighted in light ivory colour. Additionally, the residues for the CD linker1 (residues 1464-1480), which connects the PRNTase to the CD, and the CD linker2 (residues 1743-1758), which connects the CD to the MTase, are shown. The missing regions for these linkers are predicted by AF3 and represented in white colour.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4663080/v1/cefb9edae380fd30e3103124.png\"},{\"id\":60173060,\"identity\":\"a38c4ba8-afdf-4e21-9f33-da6c26572554\",\"added_by\":\"auto\",\"created_at\":\"2024-07-12 15:19:28\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":949389,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eModelling of RNA on the composite L-P model. \\u003cstrong\\u003ea.\\u003c/strong\\u003e The RNA template with a growing nascent RNA is modelled on the composite L-P structure. The template and product RNA are represented by surface in 5’-3’ direction. A cartoon representation of the template and the product RNAs is shown on the right. Template RNA used in the AF3 composite models is 5’-UUUGUUGUUAACGCAAAAAAA-3’ and product RNA is 5’-AAAUUGCGUUA-3’. \\u003cstrong\\u003eb.\\u003c/strong\\u003e A close-up view of the AF3 model of the active site pocket where the product RNA is synthesized, highlighting the incorporation of nucleotides complementary to the template RNA. This process results in the formation of an intermediate duplex RNA. The active site residues are shown as sticks, and the Mg ions are in grey. \\u003cstrong\\u003ec.\\u003c/strong\\u003e A close-up view that shows the number of nucleotides that need to be added to the RNA product to reach the HR motif for the covalent attachment of the RNA to the L protein. GTP, the \\u003csup\\u003e1347\\u003c/sup\\u003eHR\\u003csup\\u003e1348\\u003c/sup\\u003e motif and the \\u003csup\\u003e1273\\u003c/sup\\u003eGSST\\u003csup\\u003e1276\\u003c/sup\\u003e motif of the AF3 model are depicted as sticks, while Mg1 ion bound to the CD (yellow) in the crystal structure is represented in magenta. \\u003cstrong\\u003ed.\\u003c/strong\\u003e The crystal structure of CD bound to Mg1 is shown. Water molecules coordinating Mg1 are represented as black spheres. A focused view illustrates the approximate position of the Mg1 in relation to the predicted coordination with GTP, as predicted by AF3.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4663080/v1/e87ac9dabe00d1d156f8472a.png\"},{\"id\":60173053,\"identity\":\"bae7558c-2628-4450-a646-56d13572bf5d\",\"added_by\":\"auto\",\"created_at\":\"2024-07-12 15:19:28\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":444507,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eCartoon for the sliding model for NiV RNA synthesis. \\u003c/strong\\u003eThe XD of P1 interacts with the L while the other XDs tether N. Here, the L-P complex maintains continuous contact with the RNA template while advancing along its length. \\u003cstrong\\u003ea.\\u003c/strong\\u003e During transcription, the synthesized mRNA is capped at the 5’ end, and the cap is subsequently methylated. \\u003cstrong\\u003eb.\\u003c/strong\\u003e During replication, the newly synthesized Ns are chaperoned by the N-terminal domain of the Ps and subsequently incorporated into the growing nascent RNA strand.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4663080/v1/6faf604bcb7ad91a60ff338e.png\"},{\"id\":62343799,\"identity\":\"2d1faffd-6e1c-4077-a954-b3eb173b4f97\",\"added_by\":\"auto\",\"created_at\":\"2024-08-13 06:58:40\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":5308819,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4663080/v1/0a7a4662-a79b-466f-aa1d-91ae86e3ecac.pdf\"},{\"id\":60174142,\"identity\":\"3a591bed-250b-4265-a472-27428f16b4a4\",\"added_by\":\"auto\",\"created_at\":\"2024-07-12 15:27:28\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":8470059,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryInformation.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4663080/v1/f1020403f4d483f70b58e0db.docx\"}],\"financialInterests\":\"There is \\u003cb\\u003eNO\\u003c/b\\u003e Competing Interest.\",\"formattedTitle\":\"Structure of the Nipah virus polymerase complex\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eNipah virus (NiV) infections cause atypical pneumonia and severe febrile encephalitis, with a mortality rate of up to 75% over the past several decades [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. NiV was first identified during an outbreak affecting pigs and pig farmers in Malaysia and Singapore in 1998\\u0026ndash;1999 [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. During this outbreak, 265 cases of encephalitis occurred, resulting in the deaths of 105 people and the culling of nearly one million domestic pigs [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. Later, sporadic outbreaks of NiV occurred in India, including the last one in Kerala in 2023, while Bangladesh has been hit with outbreaks almost every year since 2001 [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. Being a recurring threat with high mortality rates and a lack of specific treatments, as well as a zoonosis with the potential of human-to-human transmission, Nipah virus represents a significant public health problem.\\u003c/p\\u003e \\u003cp\\u003eNiV and the closely related Hendra virus (HeV) are members of the genus Henipavirus within the \\u003cem\\u003eParamyxoviridae\\u003c/em\\u003e family. Henipaviruses contain a single-stranded, negative-sense non-segmented RNA (nsNSV) genome that encodes six structural proteins. The RNA genome is encapsidated by nucleoprotein (N), forming a helical nucleocapsid [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e]. The RNA genome is both transcribed into viral genes and replicated by the viral RNA polymerase complex comprising the Large (L) protein and Phospho (P) protein. The L protein, a pivotal component of the polymerase complex, contains three catalytic domains, the RNA-dependent RNA polymerase (RdRp), the polyribonucleotidyl transferase (PRNTase), and methyltransferase (MTase), in addition to two structural domains, the connecting domain (CD) and C-terminal domain (CTD). During transcription, the L protein catalyses the synthesis of a 5\\u0026rsquo; capped and 3\\u0026rsquo; polyadenylated mRNA. Viral RNA replication is a two-step process with the initial synthesis of a full-length copy of the genome, known as the antigenomic RNA of positive polarity. This antigenomic RNA, in turn, acts as a template for the subsequent synthesis of the genomic RNA [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. These processes rely on the presence of the non-catalytic P protein, which plays a pivotal role as a coordinating hub for the L protein, the nucleocapsid and RNA free N [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. P is composed of an ordered N-terminal peptide, separated by an intrinsically disordered region from a central oligomerization domain (OD) and a C-terminal X domain (XD) [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. The N-terminus of the P protein acts as a chaperone by binding to RNA free N protomers (N0), which subsequently encapsidate the newly synthesized antigenomic and genomic viral RNA [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. The C-terminal XD domain has been shown to be important in mediating the interaction between the RNA bound nucleocapsid and the L protein [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e]. Thus, the P protein functions as a central hub in coordinating the assembly of ribonucleoprotein complexes [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eRecent years have seen significant advancements in understanding the replication and transcription mechanisms of nsNSVs, with the publication of the structures of L-P complexes from a number of mononegaviruses. These include vesicular stomatitis virus (VSV) [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e], rabies virus (RABV) [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e], human respiratory syncytial virus (HRSV), [\\u003cspan additionalcitationids=\\\"CR18\\\" citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e], human metapneumovirus (hMPV) [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e], parainfluenza virus 3 and 5 [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e], Newcastle disease virus (NDV) [\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e], Mumps virus (MuV) [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e] as well as Ebola virus (EBOV) [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. These studies reveal the conserved RdRp and PRNTase domains, the flexibility of the CD, MTase and CTD as well as the conformational changes when bound to their RNA templates. Here, we present the molecular structure of the NiV L-P complex imaged by cryo-electron microscopy (cryo-EM) and the crystal structure of the CD of the L protein. These results reveal the structural arrangement of the RdRp and PRNTase domains of the polymerase complex, and how the tetrameric P protein binds to L. By modelling the CD domain onto an Alphafold 3 model of an RNA-L-P complex our data suggest insights into the early steps of capping and the conformational changes needed for the elongation of the synthesised viral RNA.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eOverall structure of the NiV L-P complex\\u003c/h2\\u003e \\u003cp\\u003eTo determine the structure of the NiV polymerase complex, we co-expressed the L and P proteins from the Malaysian strain of NiV in Spodoptera frugiperda 9 (Sf9) cells. Purification of the polymerase complex yielded a stoichiometric preparation suitable for structural and biochemical studies (Supplementary Fig.\\u0026nbsp;1a, b). To confirm that the L-P complex was functional, we performed a template-dependent in vitro polymerase assay using a 12 nucleotide 3\\u0026prime; leader sequence of the NiV genome as a template (Supplementary Fig.\\u0026nbsp;1c). The activity assays revealed that the L-P complex produced RNA products of varying lengths in agreement with earlier studies [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. Substituting the catalytic active site aspartic acid with alanine at position 832 (D832A), resulted in no RNA product, indicating a loss of polymerase activity (Supplementary Fig.\\u0026nbsp;1d). Substituting adenosine triphosphate with remdesivir triphosphate did not terminate strand elongation but resulted in the same pattern of RNA products but with slightly reduced mobility (Supplementary Fig.\\u0026nbsp;1e).\\u003c/p\\u003e \\u003cp\\u003eWe determined the structure of the L-P complex at a 2.5 \\u0026Aring; resolution using single particle cryo-EM. The final reconstruction (Supplementary Fig.\\u0026nbsp;2) enabled us to build a model for a portion of the L protein bound to an asymmetric tetramer of P (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). The N-terminal RdRp and PRNTase domains of the L protein were almost entirely resolved with the PRNTase domain bound to two zinc ions. The CD, MTase domain, and the CTD were not visible in our map, despite being present in our construct. The cryo-EM map revealed no density for the N-terminal 475 residues of P, possibly due to the intrinsic disorder of this region. However, we could build models for four separate P monomers of varying lengths, starting from the beginning of the OD domain. The longest visible P monomer spans amino acid residues 477\\u0026ndash;707; of these, 477\\u0026ndash;510 correspond to short helical bundles located at the N-terminal end of the OD, 510\\u0026ndash;575 form the OD, while 660\\u0026ndash;706 represent the XD region of P (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eAnalysis of the L-P complex by SDS-PAGE after size exclusion chromatography shows that both L and P have maintained their integrity suggesting that the missing domains reflect the highly dynamic nature of the L-P complex (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e and Supplemantary Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStructural details of the polymerase complex\\u003c/h2\\u003e \\u003cp\\u003eThe structural analysis revealed the conserved motifs and residues critical for the catalytic function and the regulatory mechanisms of L. The RdRp domain of the L protein folds into the right-handed \\u0026ldquo;fingers-palm-thumb\\u0026rdquo; architecture found in many RNA virus polymerases (Supplementary Fig.\\u0026nbsp;3a) and contains seven specific structural motifs (A-G) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea, b). Motif A is involved in binding divalent cations via the conserved aspartate residues (Asp722 in NiV), essential for the catalytic activity (Supplementary Fig.\\u0026nbsp;9) [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. Motif B binds template RNA and incoming nucleoside triphosphates (NTPs), and together with motifs A, D and E, it contributes to the structural integrity and the NTPs selection [\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e].The active site residues (\\u003csup\\u003e831\\u003c/sup\\u003eGDNE\\u003csup\\u003e834\\u003c/sup\\u003e) are found at the tip of a β-hairpin formed by motif C, buttressed by structural elements from the fingers subdomain and the N-terminal domain (NTD) of the RdRp that form the tunnel that allows the template RNA to move towards the catalytic site. Motifs F and G, bridging the palm and fingers regions, contribute to the RdRp\\u0026rsquo;s structural stability and flexibility, facilitating effective accommodation of template RNA and NTPs [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe structural elements, including the priming loop (residues 1256\\u0026ndash;1290), which is thought to support the initiating nucleotide [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e], and the intrusion loop (residues 1337\\u0026ndash;1362) of the PRNTase domain, which contains the HR motif that forms a covalent bond with the RNA [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e], as well as the supporting helix (residues 588\\u0026ndash;600) of the RdRp, which provides structural support and stability to the active site [\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e], lacked sufficient density to be fully modelled in our L-P complex structure. Structures of previously solved L-P polymerase complexes from nsNSVs reveal two possible conformational states of the catalytic chamber. In the initiation state, the priming loop and the supporting helix largely occupy the product RNA binding groove [\\u003cspan additionalcitationids=\\\"CR15\\\" citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e]. After reaching the elongation state, the flexible supporting helix becomes disordered, while the priming loop is retracted and repositioned towards the PRNTase domain to accommodate the nascent double stranded RNA duplex formed between the template and nascent product (Supplementary Fig.\\u0026nbsp;3b) [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e]. The absence of sufficient density for these structural elements in the NiV L-P map suggests a degree of flexibility, which is consistent with the absence of bound RNA template and NTP. AlphaFold3 prediction suggests that the missing residues of these structural elements would indeed be stabilized in the presence of a bound template RNA, which is consistent with a composite model based on the EBOV RNA bound L protein structure (Supplementary Fig.\\u0026nbsp;4b, d). Interestingly, the RdRp domain of the NiV harbours a 105 amino acids (605\\u0026ndash;710) insertion within the palm subdomain, a feature unique to Henipaviruses (Supplementary Fig.\\u0026nbsp;5a). This region is disordered in our maps, indicating its inherent flexibility. Deletion of this sequence resulted in the inhibition of L-P replication in a cell-based minireplicon assay, suggesting its importance for polymerase activity (Supplementary Fig.\\u0026nbsp;5b).\\u003c/p\\u003e \\u003cp\\u003eThe entry and exit channels for template RNA are located adjacent to the fingers and thumb subdomains of the RdRp domain. As the nascent RNA progresses, it passes through a positively charged tunnel towards the product exit channel, where the MTase domain and CTD are located (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ec, d). The MTase domain and CTD exhibit dynamic behaviour and can adopt multiple conformations where the CD acts as a hinge between the PRNTase domain and the MTase and CTD portion. Despite the lack of observable density for the C-terminal domains in our cryo-EM map, we determined the structure of the CD by X-ray crystallography at a resolution of 1.85 \\u0026Aring; (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ea, b). Structural analysis revealed that CD adopts a fold similar to the structures observed within the Paramyxovirus and Rhabdovirus families (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ec, d), despite displaying minimal sequence conservation (Supplementary Fig.\\u0026nbsp;9). Interestingly we observed 3 structural magnesium ions (designated as Mg1, Mg2, and Mg3), with Mg1 bound to the N-terminal α2 helix. This part of the CD is known to be dynamic from the structures of other nsNSV CD domains (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ed). This flexibility is consistent with its potential role in orchestrating conformational alterations of the C-terminal domains, essential for coordinating the diverse stages of RNA synthesis.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMolecular basis for P binding\\u003c/h2\\u003e \\u003cp\\u003eThe structure shows that P exhibits a high degree of structural flexibility in which each monomer can adopt different conformations when interacting with different regions of the RdRp domain of L (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea, Supplementary Fig.\\u0026nbsp;8). All four P monomers contain an OD from residues 510 to 575, which form a tetrameric four-helix bundle, as shown previously [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. However, the N-terminal and C-terminal regions outside the OD have not been structurally well characterized due to their intrinsic flexibility. Our structure reveals that one of the P monomers (P1) has a complete ordered XD domain that interacts with L through a series of hydrophobic and electrostatic interactions (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eb). P1 forms a tentacle-like structure consisting of three consecutive α-helices that surrounds the channel through which NTPs access the active site. Arg669 and Asn702 of P1 form salt bridges with Asp339, and Arg308 of L, respectively (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eb), while closer towards the OD domain Arg600 of P1 mediates salt bridges with Glu760 and Glu733 of L (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ec). In addition, hydrogen bonds are formed between Pro640, Ala649, Thr670, His671 of P1 and Arg867, Ala879, Asn346, Leu300 of L, respectively (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eb, c). Residues 575\\u0026ndash;578 of P3 form a β-strand which is sandwiched between a parallel β-strand from P1 (597\\u0026ndash;599) and an anti-parallel strand from L (residues 385\\u0026ndash;388) to form a three stranded β-sheet. These interactions are mediated by hydrogen bonds and hydrophobic interactions (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ed). Residues 570\\u0026ndash;595 of P2 bind on the opposite side of the RdRp compared to P1. The His570 of P2 mediates a salt bridge with Glu448 of L and is also involved in a hydrogen bond with Tyr389. The residues Asn590 of P2 interact with the main chain of Met459 of L, via a hydrogen bond, while Leu594 and Pro579 of P2 mediate hydrophobic interactions with Leu525 and Tyr732 of L, respectively (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ee).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eA model for RNA binding and processing\\u003c/h2\\u003e \\u003cp\\u003eTo gain further insights into the mechanism of RNA synthesis, we constructed a composite model by integrating the cryo-EM structure of RdRp and PRNTase, and the crystal structure of CD, onto an AlphaFold 3 (AF3) model of the full L polymerase (Supplementary Fig.\\u0026nbsp;7) where the MTase and CTD domains are predicted to be ordered (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ea, b). This full-length AF3 L model predicts the localization of disordered structural elements\\u0026mdash;specifically, the intrusion loop, priming loop, and insertion sequence\\u0026mdash;on the cryo-EM structure of L (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ea). The model was further extended by including a 21-nucleotide long 3\\u0026rsquo;-leader sequence as the RNA template and product RNA of varying lengths, ranging from 9 to 15 nucleotides, along with the addition of NTPs and Mg ions. This new composite model predicts with confidence the pathway of the template RNA as it enters and subsequently exits the active site of the RdRp. It also predicts how the product RNA egresses towards the PRNTase and CD domains (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ea). The model illustrates how the template RNA is positioned in the active site, where nucleotides are incorporated into the elongating nascent RNA, resulting in the formation of an intermediate RNA duplex (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eb). Following this, the two strands are separated, and the template and product are directed towards the template or the product exit channels, respectively (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eb). The model also demonstrates the positioning of the conserved GxxT motif (\\u003csup\\u003e1273\\u003c/sup\\u003eGSST\\u003csup\\u003e1276\\u003c/sup\\u003e) of the priming loop, which is thought to bind GTP [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e] and the localization of the HR motif (\\u003csup\\u003e1347\\u003c/sup\\u003eHR\\u003csup\\u003e1348\\u003c/sup\\u003e), both of which are crucial for capping of the 5\\u0026rsquo; end of the nascent RNA (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ec) [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. Additionally, our composite model predicts the position of the crystallographically determined Mg1 ion bound to the α2 helix of the CD suggesting a role in positioning the GTP required for the capping process (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ed). These structural insights underscore the intricate coordination of multiple elements within the polymerase complex necessary for RNA processing.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eThis study provides structural and functional insights into the NiV polymerase complex. Although the sequences of L differ across the nsNSVs (Supplementary Fig.\\u0026nbsp;9), the architecture and key structural motifs of the L-P complex are conserved. Structural comparison of the RdRp and PRNTase domains across paramyxoviral, pneumoviral, filoviral, and rhabdoviral L proteins yield valuable insights into evolutionary adaptations and species-specific variations in polymerase functionality (Supplementary Fig.\\u0026nbsp;3). The RdRp domain, which plays a central role in RNA synthesis, exhibits high structural conservation among nsNSVs (Supplementary Fig.\\u0026nbsp;3a). However, despite this conservation, the RdRp domain and the NTD can accommodate different insertions, such as the palm insertion observed in NiV, and the NTD insertion in EBOV [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e]. These insertions likely contribute to the functional diversity and regulatory mechanisms observed across the different viral species. In addition, the PRNTase domain, demonstrates significant flexibility in particular functional loops, such as the priming and intrusion loops in performing specific functions during replication and transcription (Supplementary Fig.\\u0026nbsp;3b), contributing to the versatility and adaptability of the polymerase complex in different viral contexts.\\u003c/p\\u003e \\u003cp\\u003eRNA synthesis by the Nipah virus polymerase complex is a tightly regulated process involving coordinated interactions between the L protein, P protein, and the RNA template encapsidated by N. The 3\\u0026rsquo; end of the genome is composed of the leader RNA region which is a bipartite promoter that is highly conserved among paramyxoviruses. This sequence is recognized by L to initiate RNA synthesis [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. After binding to the leader RNA, the polymerase complex initiates and either proceeds with the replication of full-length antigenomes and genomes or releases the short initial leader RNA product and scans through the genome until it locates one of the \\u0026lsquo;gene start\\u0026rsquo; elements at the beginning of each gene to initiate transcription and produce mRNA. Both replication and transcription use an unprimed initiation mechanism, however, replication initiates terminally as opposed to transcription which initiates internally. Based on our modelling, the active site can accommodate a 9-mer intermediate duplex formed during RNA synthesis, before the two strands\\u0026mdash;template and product\\u0026mdash;are separated and directed towards their respective exit channels (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eb).\\u003c/p\\u003e \\u003cp\\u003eStructural studies of L-P complexes from other viruses have revealed the conformation of the CD along with the presence of the C-terminal domains [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e, \\u003cspan additionalcitationids=\\\"CR21 CR22\\\" citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e]. These studies have demonstrated that the positioning of the CD modulates the organization of the MTase and CTD, thereby allowing the RNA to access the MTase [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e]. The nascent transcript RNA is initially capped at its first nucleotide, with the cap subsequently methylated by the MTase [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e]. This process involves the covalent attachment of the first nucleotide of the product mRNA to the side chain of the conserved histidine residue in the HR motif, forming an intermediate (L)-(histidyl-Nε2)\\u0026ndash;pRNA (L\\u0026ndash;pRNA) [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. Following the formation of the L-pRNA complex, RNA is transferred from L-pRNA to GDP and to form the cap structure. The MTase domain subsequently methylates the N7 and O2 of the newly formed cap. Our modelling suggests that a 9 to 10 nts long product is required to be synthesized to reach the conserved HR motif (\\u003csup\\u003e1347\\u003c/sup\\u003eHR\\u003csup\\u003e1348\\u003c/sup\\u003e) for subsequent covalent attachment (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ec). Additionally, the model predicts the binding of GTP to the conserved GxxT motif (\\u003csup\\u003e1273\\u003c/sup\\u003eGSST\\u003csup\\u003e1276\\u003c/sup\\u003e) in the priming loop (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ec, d). The transfer of the RNA onto the GDP is metal-dependent mainly involving divalent cations [\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e]. The crystal structure of the CD reveals the presence of three bound Mg ions. Interestingly, the superimposition of the CD with the AF3 model ideally positions the flexible α2 helix of the CD domain, and the bound Mg1 next to close to the predicted GTP. It is likely that Mg1 likely plays a crucial role in the capping process by aiding the positioning of the GTP during the transfer reaction of PRNTase (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ed). This finding suggests that the flexibility of the α2 helix and its interaction with Mg ions is essential for coordinating the conformational changes required for effective RNA synthesis and capping. Additionally, the crystal structure of CD has another bound Mg ion (Mg2) which engages with residues involved in crystal contacts (Supplementary Fig.\\u0026nbsp;6a, b) but \\u0026ndash; in the context of the full-length polymerase \\u0026ndash; could have a structural role stabilizing the capping conformation by stabilizing the CD linker1 (Supplementary Fig.\\u0026nbsp;6c, d).This comprehensive model advances our understanding of the spatial arrangement and functional dynamics within the NiV polymerase complex, providing a detailed framework for further investigations into viral replication mechanisms.\\u003c/p\\u003e \\u003cp\\u003eBased on observations of P tetramers in nsNSVs L-P complexes, both the cartwheeling and sliding models have been proposed to describe the movement of the polymerase complex along the nucleocapsid [\\u003cspan additionalcitationids=\\\"CR38 CR39 CR40\\\" citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e]. The cartwheeling model implies that all four XDs must bind sequentially to L during RNA synthesis. Recent studies have challenged this model by showing that P tetramers where one to three XDs are deleted, maintain comparable or even heightened RNA synthesis activity [\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e]. Notably, even a single XD capable of binding to N protein is adequate for minigenome transcription [\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e]. In addition, our structural studies reveal that around 3500 \\u0026Aring;\\u003csup\\u003e2\\u003c/sup\\u003e of surface area of L are bound by P, suggesting that the dissociation of P from L is highly unlikely. These observations lend support to the alternative sliding model where the oligomeric P moves along the nucleocapsid without necessitating rotational movement. In the case of the NiV, it is likely that the sliding of the L-P complex along the nucleocapsid is driven primarily by the process of RNA synthesis itself (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e). This movement is facilitated by the continuous association of the polymerase complex with the RNA strand, ensuring that as nucleotides are added to the growing RNA chain. This continuous contact is essential for efficient transcription and replication, stabilizing the polymerase complex and enhancing processivity, ultimately ensuring the fidelity and efficiency of viral RNA synthesis.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eNipah virus infections continue to pose a threat to public health. An approved vaccine against the related HeV has been used effectively in horses in Australia to prevent zoonotic transmission to humans [\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e], but bats are carriers of NiV and the primary source of human infection, making a similar approach for Nipah virus unlikely. This structural and functional characterization of the NiV L-P complex provides the basis for understanding the molecular details and the function of the polymerase complex and thus accelerates the development of therapeutic anti-viral drugs active against the NiV polymerase complex.\\u003c/p\\u003e\"},{\"header\":\"Methods\",\"content\":\"\\u003cp\\u003eCells and plasmids\\u003c/p\\u003e \\u003cp\\u003eHuman embryonic kidney 293T cells (HEK-293T) were maintained in Dulbecco\\u0026rsquo;s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS; Sigma-Aldrich) at 37\\u0026deg;C and 5% CO2. BSR-T7/5 cells, cells stably expressing the T7 polymerase that are derived from baby hamster kidney cells (BHK-21) [\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e] were maintained in DMEM with 10% FBS and 1 mg/ml G418/Geneticin (Roche) every third cell culture passage. The \\u003cem\\u003eSpodoptera frugiperda\\u003c/em\\u003e Sf9 cells were maintained in Sf-900\\u0026trade; II SFM medium (Thermo Scientific) at 27\\u0026deg;C.\\u003c/p\\u003e \\u003cp\\u003epCAGGs plasmids expressing NiV L, P and N proteins (Bangladesh, 2004) as well as the pUC57 plasmid expressing T7-dependent NiV bicistronic minigenome (BMG) [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e] were kindly provided by Micheal Lo (CDC). Plasmids expressing truncated versions of L proteins were generated by Gibson assembly (NEB).\\u003c/p\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eProtein expression and purification of NiV L-P complex\\u003c/h2\\u003e \\u003cp\\u003eThe coding sequences for Malaysian strain NiV L (NCBI: NP_112028.1) with an N terminal twin-Strep tag and P (NP_112022.1) with the C-terminal octa-His tag were codon optimized for insect cell expression and cloned into a pFL plasmid downstream of polyhedrin and p10 promoters, respectively. The preparation of Baculovirus stocks and protein expression were performed following the Bac to Bac manual (Invitrogen). Two litres of Sf9 cells expressing the L-P complex were harvested 72 hours post infection. The cells were resuspended in Buffer A (50 mM HEPES pH 7.4, 500 mM NaCl, 10% vol/vol glycerol, 2 mM tris(2-carboxyethyl) phosphine (TCEP), 5 mM MgCl\\u003csub\\u003e2\\u003c/sub\\u003e) which was further supplemented with 0.05% wt/vol n-octyl beta-d-thioglucopyranoside, one protease inhibitor cocktail tablet (Roche, cOmplete Mini, EDTA-free), 2 mM phenylmethylsulfonyl fluoride (PMSF), Bensonase, and RNase. Cells were lysed using a Dounce homogeniser and clarified with centrifugation. The lysate was incubated with the pre-equilibrated Strep-Tactin\\u0026reg; XT Sepharose resin (IBA Lifesciences) for 3 h and resin was washed with the BufferA. The NiV L-P complex was eluted with 50 mM Biotin in Buffer A and further purified with Superose 6 increase 10/300 size exclusion column (GE Healthcare) in Buffer A. The fractions eluting after the void volume (between 10-12ml) was collected and concentrated to 0.5 mg/ml. The L-D832A mutant, in complex with P, was expressed and purified using the same methods as the wild-type L-P complex.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eProtein expression and purification of CD of NiV L\\u003c/h2\\u003e \\u003cp\\u003eResidues 1480\\u0026ndash;1742, encoding the CD of L, were cloned into a pET28a vector with an N-terminal hexa-His and Sumo tag. The construct was transformed into BL21(DE3) cells and protein expression was induced with 0.5 mM IPTG at OD\\u003csub\\u003e600\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;0.6 and expression was carried out at 18\\u0026deg;C for 18 hours. Six litres of bacterial cell pellet were lysed by sonication in a Lysis Buffer containing 50 mM HEPES pH 7.6, 500 mM NaCl, 10% vol/vol glycerol supplemented with protease inhibitors (Roche, cOmplete Mini, EDTA-free), RNase A, and lysozyme (Sigma). Supernatant cleared after ultracentrifugation was filtered and loaded onto HisTrap 5 ml HP columns. Protein was washed and eluted with a gradient increase in imidazole concentration. Elution fractions were combined, desalted into the Final Buffer containing 20 mM HEPES pH 7.6, 500 mM NaCl, 10% vol/vol glycerol and then incubated with Ulp1 overnight at 4\\u0026deg;C. Cleaved protein was re-injected into HisTrap 5 ml HP to remove Ulp1 enzyme as well as the cleaved His-Sumo tag. The flowthrough was collected, concentrated, and injected into Superdex 75 16/600 (GE Healthcare) that was pre-equilibrated with the Final Buffer. The protein was concentrated to 10 mg/ml and stored at -80\\u0026deg;C.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eIn vitro RNA synthesis assay\\u003c/h2\\u003e \\u003cp\\u003eFor the NiV L-P complex in vitro assay, 3 \\u0026micro;l reactions were set up containing reaction buffer (20 mM Tris pH 7.5, 10 mM KCl, 2 mM DTT, 0.5% triton, 10% DMSO, 1 U Rnasin (Promega), 5 mM MgCl2), 0.25 \\u0026micro;M RNA-template derived from the NiV leader sequence (UGGUUUGUUCCC or UGGUCUGUUCCC), 0.5 \\u0026micro;M recombinant L-P complex, 0.5 mM ATP, 0.5 mM CTP, 0.1 \\u0026micro;M GTP and 200 \\u0026micro;M primer (pACCA). ATP was substituted with remdesivir triphosphate (APExBIO), where indicated. The radioisotope tracer in these reactions was [α\\u003csup\\u003e32\\u003c/sup\\u003eP] GTP (Revvity). The reactions were incubated at 30 ̊C for 1 h, stopped with the addition of 3 \\u0026micro;l formamide loading buffer and denatured at 95 ̊C for 3 minutes. A \\u003csup\\u003e32\\u003c/sup\\u003eP-5\\u0026rsquo;end labelled 20 nucleotide-long DNA served as a marker. RNA products were separated on a 22% polyacrylamide urea gel for 2.5 hours at 35 W and the level of [α\\u003csup\\u003e32\\u003c/sup\\u003eP] GMP incorporation was imaged by phosphorimaging on a FLA-5000 scanner (Fuji).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMinireplicon assay\\u003c/h2\\u003e \\u003cp\\u003eBSR-T7/5 cells were seeded in 24-well plates at 5 x 10\\u003csup\\u003e4\\u003c/sup\\u003e cells/well. 24 hours later, cells were transfected in duplicates with the BMG plasmid (0.4 \\u0026micro;g), as well as pCAGGs plasmids expressing for NiV L (0.4 \\u0026micro;g), P (0.4 \\u0026micro;g), N (0.25 \\u0026micro;g) using LT-1 transfection reagent (MirusBio). For negative controls pCAGGs-NiV-L was substituted with a pCAGGs-empty vector. After 48 hours, Gaussia luciferase activity was determined using the Renilla Luciferase Assay System (Promega). For this purpose, cells were lysed in 50 \\u0026micro;l Renilla lysis buffer on a microplate shaker at room temperature. After 45 min, 20 \\u0026micro;l of the cell lysates were mixed with 50 \\u0026micro;l of the Renilla luciferase reagent and RLUs were analysed using a GLOMAX 20/20 luminometer (Promega).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCryo-EM sample preparation, data acquisition and processing\\u003c/h2\\u003e \\u003cp\\u003e3.5 \\u0026micro;l of the NiV L-P complex at 0.5 mg/ml was applied to a freshly glow-discharged UltrAufoil Au 1.2/1.3 300 mesh grid. The sample was blotted for 6 s and plunge frozen in liquid ethane. All grids were prepared using a Vitrobot Mark VI (FEI) under conditions of 100% humidity and 20\\u0026deg;C. Cryo-EM data was collected at the Oxford Particle Imaging Centre (OPIC) using a 300 kV G3i Titan Krios microscope (Thermo Fisher Scientific) equipped with a SelectrisX energy filter and Falcon 4i direct electron detector. Automated data collection was setup in EPU 3.4 and a total of 22,859 movies were recorded in EER format, of which 14,319 were collected with a tilt angle of 30\\u0026deg;. Data was collected using AFIS with a total dose of ~\\u0026thinsp;50 e-/\\u0026Aring;2, a calibrated pixel size of 0.7303 \\u0026Aring;/pix, defocus range of -1.4 to -2.4, and with 10 eV slit. Cryo-EM data collection parameters and refinement statistics are summarized in Table S1.\\u003c/p\\u003e \\u003cp\\u003eData processing was performed in CryoSparc v4.4.1 [\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e] by following the workflow outlined in Supplementary Fig.\\u0026nbsp;2. Briefly, motion correction and patch CTF estimations were performed for movie frames, and low-quality images were eliminated by manual inspection and excluded from further analysis. A template search was prepared using the PIV5 L-P structure (PDB 6V85) as the template. Following template picking, 2D classes were obtained, and the best-resolved classes were selected for ab-initio model generation. The ab-initio models were refined using Heterogenous refinement. The resulting optimal map was refined using NU-refinement with C1 symmetry. This refined map was then used to train Topaz picking. The picked particles were directly subjected to heterogeneous refinement, and the best resulting map was selected for further refinement using NU-refinement. This entire process, from particle picking to Topaz training, was repeated three times to increase the number of picked particles.\\u003c/p\\u003e \\u003cp\\u003eThe map, containing 490,675 particles, was corrected for reference motion and subjected to 3D classification to identify classes that represented clear densities for the L and P-XD domain. A total of 299,261 particles were selected and refined for the L domain using local NU refinements, resulting in a 2.52 \\u0026Aring; resolution map. Meanwhile, 206,718 particles were refined for the P tetramer using local NU refinement, yielding a 2.75 \\u0026Aring; resolution map. The local maps were further processed with DeepEMhancer.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCryo EM model building and refinement\\u003c/h2\\u003e \\u003cp\\u003eInitial models were generated for L and P tetramer separately using ModelAngelo [\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e]. To improve model geometry, multiple cycles of manual building were performed using COOT [\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e] followed by real space refinement against the corresponding local maps in PHENIX [\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e]. The model geometry was validated using MolProbity [\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e]. Comprehensive statistics for the map and model are presented in the Supplementary Table\\u0026nbsp;1. Structural analysis and figure preparation were conducted using UCSF Chimera [\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e] and UCSF ChimeraX [\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCrystallization of CD, X-ray data collection and structure solving\\u003c/h2\\u003e \\u003cp\\u003eFreshly purified CD, at a concentration of 10 mg/mL, was mixed with a set of sitting-drop crystallization screens in ratios of 1:1, 2:1, and 1:2, and incubated at 20\\u0026deg;C. The crystals were obtained in 25 days from a condition consisting of 35% tert-Butanol and 0.1M tri-Sodium Citrate pH 5.6. The crystals were harvested, cryo-preserved in the crystallization condition containing 20% v/v glycerol and flash frozen in liquid nitrogen.\\u003c/p\\u003e \\u003cp\\u003eX-ray diffraction data were collected on the I04 beamline at Diamond Light Source (Harwell, U.K.). The data processing was performed using the program DIALS in combination with XIA2 [\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e]. The structure was solved via PHASER molecular replacement [\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e], employing a search model generated by AlphaFold [\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e]. Subsequent manual model building was performed in COOT [\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e], followed by refinement in PHENIX [\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e]. X-ray data collection and refinement statistics are summarized in Table S2.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eAlphaFold modelling\\u003c/h2\\u003e \\u003cp\\u003eThe RNA-bound NiV L structure was predicted in AlphaFold 3 by providing the leader and product RNA sequences as the input along with the corresponding protein sequences [\\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e54\\u003c/span\\u003e]. For the X-ray crystallography analysis, a reference model for molecular replacement of the CD was prepared using AlphaFold 2 [\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eData availability\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eStructural data generated in this study have been deposited in the Protein Data Bank (PDB) and the Electron Microscopy Data Bank (EMDB) under the following accession codes: PDB 9FTF (crystal structure of the CD), PDB 9FUX (composite model of the NiV L-P complex). The corresponding EMDB entries are EMD-\\u0026nbsp;50781 (composite map), EMD-50808 (consensus map), EMD-\\u0026nbsp;50805 (L focused map), and EMD-\\u0026nbsp;50807 (P focused map).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe thank Michael Lo for providing the plasmids expressing NiV L, P, and N proteins and for the bicistronic minigenome (BMG) plasmid. We thank members of the Grimes and Fodor Laboratories for helpful comments and discussions. We thank Maria Harkiolaki for proofreading the main text. This work was supported by Bill and Melinda Gates Foundation INV-048922 (to J.M.G. and to E.F ). Access to electron microscopes was provided by the OPIC Electron Microscopy Facility (funded by Wellcome JIF (060208/Z/00/Z) and equipment (093305/Z/10/Z) grants). Access to computational resources was supported by the Wellcome Trust Core Award Grant Number 203141/Z/16/Z with additional support from the NIHR Oxford BMRC. Molecular graphics and analyses were performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eContributions\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eE.B., F.G., E.F. and J.M.G. conceived and designed the study. E.B. generated recombinant baculoviruses, purified protein, performed crystallization and solved structure. E.B., L.C. and J.R.K. performed structural analyses. E.B and J.R.K performed AF3 predictions. F.G. generated plasmids and performed functional assays. E.B., E.F. and J.M.G wrote the paper with input from all authors.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics declarations\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare no competing interests.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eChua, K.B., et al., \\u003cem\\u003eNipah virus: a recently emergent deadly paramyxovirus\\u003c/em\\u003e. Science, 2000. 288(5470): p. 1432\\u0026ndash;5.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eEaton, B.T., et al., \\u003cem\\u003eHendra and Nipah viruses: different and dangerous\\u003c/em\\u003e. 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Mol Syst Biol, 2011. 7: p. 539.\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":false,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":true,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4663080/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4663080/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eNipah virus poses a recurring threat, causing severe respiratory and neurological disease in Southeast Asia. Since its first identification in Malaysia in 1998 and a subsequent outbreak in Singapore in early 1999, the virus has emerged as a highly virulent zoonotic paramyxovirus. Despite its lethality, there is currently no approved treatment for Nipah virus infection. The viral polymerase complex, composed of the large polymerase protein (L) and the phosphoprotein (P), is responsible for the replication of the viral RNA genome and transcription of viral genes. However, the mechanisms by which the L and P components perform these activities remain unknown. Here, we describe the structures of the Nipah virus L-P polymerase complex at a 2.5 \\u0026Aring; resolution and the L protein\\u0026rsquo;s Connecting Domain (CD) at a 1.85 \\u0026Aring; resolution, determined by cryo-electron microscopy (cryo-EM) and X-ray crystallography, respectively. The L-P complex structure reveals the organization of the RNA-dependent RNA polymerase (RdRp) and polyribonucleotidyl transferase (PRNTase) domains of the L protein, and how the P protein, which forms a tetramer, interacts with the RdRp domain of the L protein. Furthermore, the CD structure reveals the binding of Mg ions, which likely contribute to the functionality of the PRNTase domain. These findings offer insights into the structural details of the L-P polymerase complex and the molecular interactions between L and P, shedding light on the mechanisms of the replication machinery. This work will underpin efforts to develop antiviral drugs that target the polymerase complex of Nipah virus.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Structure of the Nipah virus polymerase complex\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-07-12 15:19:22\",\"doi\":\"10.21203/rs.3.rs-4663080/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"ee4fd060-c545-4c04-8beb-470d78941132\",\"owner\":[],\"postedDate\":\"July 12th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[{\"id\":34358423,\"name\":\"Biological sciences/Structural biology/Electron microscopy\"},{\"id\":34358424,\"name\":\"Biological sciences/Microbiology/Virology\"}],\"tags\":[],\"updatedAt\":\"2024-10-07T13:27:44+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2024-07-12 15:19:22\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4663080\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4663080\",\"identity\":\"rs-4663080\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}