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The representative models fit with electron microscopy densities in Le10 and TrC10-bound RSV polymerase complexes Electron microscopy density segments for representative regions of the Le10 (a-g) and TrC10 (h-n) bound RSV polymerase complexes. The models of RdRp domain (a, h), supporting loop (b, i), supporting helix (c, j), Cap domain (d, k), priming loop (e, l), POD (f, m), and PCTD (g, n), fitting with the density maps at the counter level of 1.5 σ, 2.5 σ or 3.5 σ, were shown with the same color scheme as Fig. 1.
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The respiratory syncytial virus (RSV) polymerase is a multifunctional RNA-dependent RNA polymerase composed of the large (L) protein and the phosphoprotein (P). It transcribes the RNA genome into ten viral mRNAs and replicates full-length viral genomic and antigenomic RNAs¹. The RSV polymerase initiates RNA synthesis by binding to the conserved 3′-...
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Introduction
Whole Genome Sequencing (WGS) of the SARS-CoV-2 virus is crucial in the surveillance of the COVID-19 pandemic. Several primer schemes have been developed to sequence nearly all of the ~30,000 nucleotide SARS-CoV-2 genome, using a multiplex PCR approach to amplify cDNA copies of the viral genomic RNA. Midnight primers and ARTIC V4.1 pri...
Citations
... Notably, ERDRP-0519, a pan-morbillivirus small molecule inhibitor designed for the treatment of MeV, targets the morbillivirus RdRP domain, inhibiting both de novo initiation and elongation of RNA synthesis 47 . Further alignment of the MeV RdRp catalytic core with the ones from the RNA-bound RdRp structures of RSV 48 and EBOV 49 reveals that the conserved motifs A-F and the catalytic residues are well-matched ( Fig. 2a middle). This finding suggests that the RNA synthesis mechanism in the RdRp domain is likely highly conserved among polymerases in nsNSVs. ...
... Cap addition occurs via a nucleophilic attack by GDP 56 , while a GxxT motif B′ in the priming loop has been hypothesized to bind the capping guanosine 57 . Although loop architectures vary across nsNSVs, they remain similar in the RSV L-P complex, both with and without RNA bound (Fig. 2b right) 36,48 , suggesting that they may not be significantly influenced by RNA binding or that any structural rearrangement of the loops during RNA synthesis may be transient. ...
Measles virus (MeV) is a highly contagious pathogen that causes significant morbidity worldwide. Its polymerase machinery, composed of the large protein (L) and phosphoprotein (P), is crucial for viral replication and transcription, making it a promising target for antiviral drug development. Here we present cryo-electron microscopy structures of two distinct MeV polymerase complexes: Lcore-P and Lfull-P-C. The Lcore-P complex characterizes the N-terminal domain, RNA-dependent RNA polymerase (RdRp), and GDP poly-ribonucleotidyltransferase of the L protein, along with the tetrameric P of varying lengths. The Lfull-P-C complex reveals that C protein dimer binds at the cleft between RdRp and the flexible domains of the L protein: the connecting domain, methyltransferase domain, and C-terminal domain. This interaction results in the visualization of these domains and creates an extended RNA channel, remodeling the putative nascent replicated RNA exit and potentially regulating RNA synthesis processivity. Our findings reveal the architecture and molecular details of MeV polymerase complexes, providing new insights into their mechanisms and suggesting potential intervention targets for antiviral therapy.
... In fact, the overall architectures of the MARV and EBOV L-VP35 complexes are analogous to that of other polymerase complexes determined in nsNSVs. Comparison of the L-VP35 core structure of MARV and EBOV with that of vesicular stomatitis virus (VSV) 26,27 , rabies virus (RABV) 28 , parainfluenza virus type 5 (PIV5) 29 , Newcastle disease virus (NDV) 3 , human parainfluenza virus type 3 (hPIV3) 30 , mumps virus (MuV) 31 , human metapneumovirus (HMPV) 32 , respiratory syncytial virus (RSV) [33][34][35] and EBOV 8,9 results in an R.M.S.D. ranging from 1.6-3.0 Å ( Supplementary Fig. 10), indicating that the core structures of the polymerase complexes are highly conserved during the evolution of nsNSVs. ...
... In contrast, the supporting helices in L proteins in the non-initiation/ elongation state (such as HMPV and EBOV) are shifted away or even absent (as observed in RSV), allowing the growing RNA to pass through the exit channel ( Fig. 3c and Supplementary Fig. 15d). Recently, the cryo-EM structures of EBOV and RSV polymerase in complex with RNA promoters in the pre-initiation state have been reported 9,35 . However, the positions of the priming and intrusion loops are nearly identical to those in the previously published elongation state ( Fig. 3d and Supplementary Fig. 15e). ...
... Recently, two studies reported the pre-initiation state of the EBOV and RSV polymerase complexes, in which the replication promoters (either leader or trailer) from the viral genomes were bound to the template entry channels with certain nucleotides in the promoters were specifically recognized by the residues within the RNA entry channels 9,35 . Upon promoter binding, RSV polymerase experiences conformational changes including the stabilization of the supporting helix and the supporting loop as well as subtle inward movement of the PRNTase domain 35 , whereas there is no obvious overall structural remodeling of EBOV polymerase upon the association of the 3'-leader 9 . ...
The Ebola and the Marburg viruses belong to the Filoviridae family, a group of filamentous, single-stranded, negative-sensed RNA viruses. Upon infection, uncontrolled propagation of the Ebola and the Marburg viruses causes severe hemorrhagic fevers with high mortality rates. The replication and transcription of viral genomes are mediated by a polymerase complex consisting of two proteins: L and its cofactor VP35. However, the molecular mechanism of filovirus RNA synthesis remains understudied due to the lack of high-resolution structures of L and VP35 complexes from these viruses. Here, we present the cryo-EM structures of the polymerase complexes for the Marburg virus and the Ebola virus at 2.7 Å and 3.1 Å resolutions respectively. Despite the similar assembly and overall structures between these two viruses, we identify virus-specific L–VP35 interactions. Our data show that intergeneric exchange of VP35 would diminish these interactions and prevent the formation of a functional chimeric polymerase complex between L protein and heterologous VP35. Additionally, we identify a contracted conformation of the Ebola virus polymerase structure, revealing the structural dynamics of the polymerase during RNA synthesis. These insights enhance our understanding of filovirus RNA synthesis mechanisms and may facilitate the development of antiviral drugs targeting filovirus polymerase.
... Recent structural analyses using cryogenic electron microscopy revealed the L protein structures of RABV and VSV in complex with the N-terminal fragment of the P protein (14,15), providing structural insights into the molecular functions of the L proteins. Similar to the L proteins of other viruses in the order Mononegavirales (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26), these L proteins are composed of three functional domains with enzymatic active sites for RNA syntheses and mRNA maturation (RdRp domain [RDRP], capping domain [CAP], and methyltransferase domain [MT]) and two structural domains without enzymatic activity (connector domain [CD] and C-terminal domain [CTD]) (14,15,27,28). Furthermore, these structures illustrated the binding mode of the P protein to the L protein; the P protein fragment wraps around the CTD of the L protein and binds with the RDRP, CD, and CTD (14,15) (Fig. 1A and B). ...
The rabies virus large (L) protein interacts with its cofactor phosphoprotein (P protein) to function as an RNA-dependent RNA polymerase (RdRp). The C-terminal domain (CTD) of the L protein plays a critical role in P protein binding. We previously reported that the highly conserved NPYNE sequence in the hydrophilic region of the CTD (positions 1929–1933 of the L protein [L1929–1933]) is important for both P protein binding and RdRp function. To elucidate the functional role of the CTD in detail, we examined the importance of each of the hydrophilic residues in the NPYNE sequence (underlined). A rabies virus mutant with Ala substitutions in these hydrophilic residues showed low replication capacity. Comprehensive analyses of a revertant of the mutant virus and a series of L protein mutants revealed that Asn at L1929 is crucial for both P protein binding and RdRp function. Analyses of the L protein mutants also showed that Asn at L1932 and Glu at L1933 have roles in RdRp function and P protein binding, respectively. Furthermore, we demonstrated that the NPYNE sequence is essential for stabilizing the L protein through the L-P interaction. In a previously determined L protein structure, all of the hydrophilic residues in the NPYNE sequence form the first α-helix in the CTD. Therefore, our findings indicate that this helix is important for P protein-binding ability, RdRp function, and stabilization of the L protein, thereby contributing to efficient viral replication.
IMPORTANCE
Although RNA-dependent RNA polymerase of rhabdoviruses, which is composed of the large (L) protein and its cofactor phosphoprotein (P protein), has a high potential as a target for therapeutics against the viruses, the relationship between the structure and molecular functions is poorly understood. In this study, we functionally examined the C-terminal domain (CTD) of the rabies virus L protein as a model for the rhabdovirus L protein. We showed that the first α-helix in the CTD is important for the P protein-binding ability, RdRp function, and stability of the L protein. Since in the L-P complex structure, this helix does not form an interface with the P protein, we provide here the first evidence of an indirect contribution of the L protein CTD to the L-P interaction in rhabdoviruses. The findings in this study will be useful for developing therapeutics targeting the L-P interaction.
... Over the past years, structures of L-P complexes from several members of the Paramyxoviridae family as well as from other nsNSVs have been determined in the apo state without nucleic acids bound 8-15 . In addition, structures of EBOV and RSV L-P complexes in complex with leader RNA have recently been obtained 16,17 . These structures have shed light on the overall architecture of nsNSV L-P complexes and provided first insights into how they interact with the template RNA during initiation. ...
... Over the past years, structures of L-P complexes from several members of the Paramyxoviridae family as well as from other nsNSVs have been determined in the apo state without nucleic acids bound [8][9][10][11][12][13][14][15] . In addition, structures of EBOV and RSV L-P complexes in complex with leader RNA have recently been obtained 16,17 . These structures have shed light on the overall architecture of nsNSV L-P complexes and provided first insights into how they interact with the template RNA during initiation. ...
... 588-600) and the supporting loop (res. 579-587), which have been observed to adopt different conformations associated with distinct functional states in other nsNSV polymerase complexes 17,27 . While the supporting loop is partially ordered in the apo NiV L-P complex, the supporting helix appears to be flexible ( Supplementary Fig. 3b). ...
Nipah virus (NiV) is a non-segmented negative-strand RNA virus (nsNSV) with high pandemic potential, as it frequently causes zoonotic outbreaks and can be transmitted from human to human. Its RNA-dependent RNA polymerase (RdRp) complex, consisting of the L and P proteins, carries out viral genome replication and transcription and is therefore an attractive drug target. Here, we report cryo-EM structures of the NiV polymerase complex in the apo and in an early elongation state with RNA and incoming substrate bound. The structure of the apo enzyme reveals the architecture of the NiV L-P complex, which shows a high degree of similarity to other nsNSV polymerase complexes. The structure of the RNA-bound NiV L-P complex shows how the enzyme interacts with template and product RNA during early RNA synthesis and how nucleoside triphosphates are bound in the active site. Comparisons show that RNA binding leads to rearrangements of key elements in the RdRp core and to ordering of the flexible C-terminal domains of NiV L required for RNA capping. Taken together, these results reveal the first structural snapshots of an actively elongating nsNSV L-P complex and provide insights into the mechanisms of genome replication and transcription by NiV and related viruses.
... Although we observed low-resolution features in 3D volumes during the earlier steps of data processing that are consistent with the presence of the L CD, MTase domain, and CTD, these features were lost during additional steps of data processing ( Figure S1C). Therefore, like in the structures of the L proteins of respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) (Pneumoviridae), the three L C-terminal globular domains in NiV L are probably too flexible to be visualized by cryo-EM [18][19][20][21][22][23] (Data S2). For the RdRp and CAP domains that could be resolved, root-meansquare deviation values based on the Ca ranged from 1.7 to 4.7 Å between NiV and other nsNSV L proteins ( Figure S1F), indicating structural conservation, consistent with the generally similar transcription and replication mechanisms of nsNSVs. ...
... In the assembled NiV L-P complex, there are channels for NTP entry, template entry, template exit, and nascent RNA exit ( Figures 2E and 2F). The template entrance channel, identified by analogy to the Ebola virus (EBOV) and RSV L protein-promoter RNA-bound structures, 18,31 involves surfaces of the RdRp and CAP domains ( Figure 2F). The putative template exit channel, identified by analogy with other viral RdRps, is within the CAP domain ( Figure 2F). ...
Nipah virus (NiV) is a bat-borne, zoonotic RNA virus that is highly pathogenic in humans. The NiV polymerase, which mediates viral genome replication and mRNA transcription, is a promising drug target. We determined the cryoelectron microscopy (cryo-EM) structure of the NiV polymerase complex, comprising the large protein (L) and phosphoprotein (P), and performed structural, biophysical, and in-depth functional analyses of the NiV polymerase. The L protein assembles with a long P tetrameric coiled-coil that is capped by a bundle of ⍺-helices that we show are likely dynamic in solution. Docking studies with a known L inhibitor clarify mechanisms of antiviral drug resistance. In addition, we identified L protein features that are required for both transcription and RNA replication and mutations that have a greater impact on RNA replication than on transcription. Our findings have the potential to aid in the rational development of drugs to combat NiV infection.
... It comprises several α helices and an important β-sheet region with the active site, contributing to the formation of the nascent RNA exit and NTP entrance channels, as well as the L-P interactions. The palm subdomain also has an important supporting helix (Fig. 3a), which is positioned close to the priming loop and was proposed to coordinate with it 34,46 . ...
... The overall architectures and orientations of both the priming and intrusion loops vary across the different structures of mononegaviruses (Fig. 3c). However, these loops in the RSV L-P complex, with and without RNA bound, show similar architectures 38,46 , suggesting that structural rearrangement of the loops may be transient and occur only during the process of RNA synthesis. The PRNTase domain also contributes to the formation of the template exit and nascent RNA exit channels. ...
The Nipah virus (NiV), a member of the Paramyxoviridae family, is notorious for its high fatality rate in humans. The RNA polymerase machinery of NiV, comprising the large protein L and the phosphoprotein P, is essential for viral replication. This study presents the 2.9-Å cryo-electron microscopy structure of the NiV L-P complex, shedding light on its assembly and functionality. The structure not only demonstrates the molecular details of the conserved N-terminal domain, RNA-dependent RNA polymerase (RdRp), and GDP polyribonucleotidyltransferase of the L protein, but also the intact central oligomerization domain and the C-terminal X domain of the P protein. The P protein interacts extensively with the L protein, forming an antiparallel β-sheet among the P protomers and with the fingers subdomain of RdRp. The flexible linker domain of one P promoter extends its contact with the fingers subdomain to reach near the nascent RNA exit, highlighting the distinct characteristic of the NiV L-P interface. This distinctive tetrameric organization of the P protein and its interaction with the L protein provide crucial molecular insights into the replication and transcription mechanisms of NiV polymerase, ultimately contributing to the development of effective treatments and preventive measures against this Paramyxoviridae family deadly pathogen.
... Structural modeling based on the recently reported EBOV 17 and RSV 18 initiation complexes shows that no major structural rearrangements are necessary for this, as the template strand can be accommodated in the . CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. ...
Nipah virus (NiV) is a non-segmented negative-strand RNA virus (nsNSV) with high pandemic potential, as it frequently causes zoonotic outbreaks and can be transmitted from human to human. Its RNA-dependent RNA polymerase (RdRp) complex carries out viral genome replication and transcription and is therefore an attractive drug target. However, to date no structural data is available on the NiV RdRp complex. Here, we report cryo-EM structures of NiV RdRp in the apo and in an early elongation state with RNA and incoming substrate bound. The structure of the apo enzyme reveals the architecture of the NiV RdRp complex, which shows a high degree of similarity to other nsNSV RdRps. The structure of the RNA-bound NiV RdRp shows how the enzyme interacts with template and product RNA during early replication and how nucleoside triphosphates are bound in the active site. Comparisons show that RNA binding leads to rearrangements of key elements in the RdRp core and to ordering of the flexible C-terminal domains of NiV L required for RNA capping. Taken together, these results reveal the first structural snapshots of an actively replicating nsNSV RdRp and provide insights into the mechanisms of genome replication and transcription by NiV and related viruses.
... Several apo structures of mononegavirus polymerase complexes have been reported (26,(29)(30)(31)(32)(33)(34)(35)(36), and recently, RSV L-P (37) and EBOV VP35-L (38) bound to RNA have been solved. However, the resolution of individual domains that are subject to, or enable, conformational rearrangements such as the MTase and connector domain remains limited due to their inherent structural pliability, undermining therapeutic exploitation through structure-guided drug design (16,18,33,34). ...
... Based on currently published L protein structures, we propose that the polymerase transitions through five distinct stages: (i) pre-initiation, (ii) initiation, (iii) elongation-1, (iv) elongation-2, and (v) elongation-3. In the pre-initiation state, as demonstrated by the RSV L-P-RNA complex (Fig. 2B), the priming and intrusion loops of the polymerase are extended away from the GDN site which allows space for incoming promoter RNA (Fig. 2C) (37). Within the central cavity, a supporting loop, located within the RdRP domain, stabilizes the first nucleotides of the incoming RNA promoter, resulting in a slight inward shift (~1.8 Å) of the PRNTase domain, generating a more compact and stable catalytic pocket (37). ...
... In the pre-initiation state, as demonstrated by the RSV L-P-RNA complex (Fig. 2B), the priming and intrusion loops of the polymerase are extended away from the GDN site which allows space for incoming promoter RNA (Fig. 2C) (37). Within the central cavity, a supporting loop, located within the RdRP domain, stabilizes the first nucleotides of the incoming RNA promoter, resulting in a slight inward shift (~1.8 Å) of the PRNTase domain, generating a more compact and stable catalytic pocket (37). Subsequently, the polymerase transitions into an initiation state, as seen in the RABV and VSV polymerase structures; the priming loop moves within proximity of the GDN to properly position the initial NTP and the intrusion loop is extended into a cavity within the PRNTase domain ( Fig. 2D) (31,32). ...
Small-molecule antivirals can be used as chemical probes to stabilize transitory conformational stages of viral target proteins, facilitating structural analyses. Here, we evaluate allosteric pneumo- and paramyxovirus polymerase inhibitors that have the potential to serve as chemical probes and aid the structural characterization of short-lived intermediate conformations of the polymerase complex. Of multiple inhibitor classes evaluated, we discuss in-depth distinct scaffolds that were selected based on well-understood structure-activity relationships, insight into resistance profiles, biochemical characterization of the mechanism of action, and photoaffinity-based target mapping. Each class is thought to block structural rearrangements of polymerase domains albeit target sites and docking poses are distinct. This review highlights validated druggable targets in the paramyxo- and pneumovirus polymerase proteins and discusses discrete structural stages of the polymerase complexes required for bioactivity.
... There are three nsNSV polymerase-promoter RNA structures: RSV L-P-le RNA, RSV L-P-tr RNA, and EBOV L-VP35-le RNA (Fig. 5) (43, 168). These structures reveal an extensive network of protein-RNA and RNA-RNA interactions that function to position the promoter appropriately within the polymerase (43,168). Although the core promoter sequences of different virus families are distinctive, it is likely that other nsNSVs have also evolved a network of polymerase-promoter interactions to stabilize the template in the initiation complex. ...
... Consistent with this being a priming loop, a tryptophan at the tip is essential for initiation of RNA synthesis at the 3´ end of the template, but not from an internal site (51). The equivalent loop of the RSV L protein also contains amino acid residues required for RNA synthesis initiation (172), but in RSV polymerase structures, the loop is folded into the capping domain (Fig. 6B) (36,37,168,173,174). Direct evidence that this loop can adopt different conformations comes from comparison of the different EBOV L-VP35 structures. ...
... The polymerase has a template entrance and template exit channel that threads the template through the active site, and a transcript exit channel (Fig. 6C). The template channel that has been identified in the nsNSV polymerase structures runs through the interior of the polymerase (43,45,168). Although the possibility exists that the polymer ase can "open up" to expose the template channel, allowing it to attach directly at an internal site on an RNA template, such a significant structural change would appear to be energetically unfavorable, suggesting that the polymerase is more likely to thread onto the 3´ end of the template like a bead being threaded onto a string. ...
The Mononegavirales, or non-segmented negative-sense RNA viruses (nsNSVs), includes significant human pathogens, such as respiratory syncytial virus, parainfluenza virus, measles virus, Ebola virus, and rabies virus. Although these viruses differ widely in their pathogenic properties, they are united by each having a genome consisting of a single strand of negative-sense RNA. Consistent with their shared genome structure, the nsNSVs have evolved similar ways to transcribe their genome into mRNAs and replicate it to produce new genomes. Importantly, both mRNA transcription and genome replication are performed by a single virus-encoded polymerase. A fundamental and intriguing question is: how does the nsNSV polymerase commit to being either an mRNA transcriptase or a replicase? The polymerase must become committed to one process or the other either before it interacts with the genome template or in its initial interactions with the promoter sequence at the 3´ end of the genomic RNA. This review examines the biochemical, molecular biology, and structural biology data regarding the first steps of transcription and RNA replication that have been gathered over several decades for different families of nsNSVs. These findings are discussed in relation to possible models that could explain how an nsNSV polymerase initiates and commits to either transcription or genome replication.
... As observed in the results, the defining variations are in the interacting domains of the replication-associated proteins. It is known that L, but not P, interacts directly with the RNA template and P interacts with L as a cofactor during replication (52). Established protein prediction also shows that L binds to the C-terminal domain (CTD) of P rather than its NTD (16,38). ...
Respiratory syncytial virus (RSV) is a common cause of respiratory infection that often leads to hospitalization of infected younger children and older adults. RSV is classified into two strains, A and B, each with several subgroups or genotypes. One issue with the definition of these subgroups is the lack of a unified method of identification or genotyping. We propose that genotyping strategies based on the genes coding for replication-associated proteins could provide critical information on the replication capacity of the distinct subgroups, while clearly distinguishing genotypes. Here, we analyzed the virus replication-associated genes N, P, M2, and L from de novo assembled RSV A sequences obtained from 31 newly sequenced samples from hospitalized patients in Philadelphia and 78 additional publicly available sequences from different geographic locations within the United States. In-depth analysis and annotation of variants in the replication-associated proteins identified the polymerase protein L as a robust target for genotyping RSV subgroups. Importantly, our analysis revealed non-synonymous variations in L that were consistently accompanied by conserved changes in its co-factor P or the M2-2 protein, suggesting associations and interactions between specific domains of these proteins. Similar associations were seen among sequences of the related human metapneumovirus. These results highlight L as an alternative to other RSV genotyping targets and demonstrate the value of in-depth analyses and annotations of RSV sequences as it can serve as a foundation for subsequent in vitro and clinical studies on the efficiency of the polymerase and fitness of different virus isolates.
IMPORTANCE
Given the historical heterogeneity of respiratory syncytial virus (RSV) and the disease it causes, there is a need to understand the properties of the circulating RSV strains each season. This information would benefit from an informative and consensus method of genotyping the virus. Here, we carried out a variant analysis that shows a pattern of specific variations among the replication-associated genes of RSV A across different seasons. Interestingly, these variation patterns, which were also seen in human metapneumovirus sequences, point to previously defined interactions of domains within these genes, suggesting co-variation in the replication-associated genes. Our results also suggest a genotyping strategy that can prove to be particularly important in understanding the genotype-phenotype correlation in the era of RSV vaccination, where selective pressure on the virus to evolve is anticipated. More importantly, the categorization of pneumoviruses based on these patterns may be of prognostic value.
