1. Microbiology and Infectious Disease
  2. Structural Biology and Molecular Biophysics
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Self-capping of nucleoprotein filaments protects the Newcastle disease virus genome

  1. Xiyong Song
  2. Hong Shan
  3. Yanping Zhu
  4. Shunlin Hu
  5. Ling Xue
  6. Yong Chen
  7. Wei Ding
  8. Tongxin Niu
  9. Jian Gu
  10. Songying Ouyang  Is a corresponding author
  11. Qing-Tao Shen  Is a corresponding author
  12. Zhi-Jie Liu  Is a corresponding author
  1. Kunming Medical University, China
  2. Institute of Biophysics, Chinese Academy of Sciences, China
  3. University of Chinese Academy of Sciences, China
  4. ShanghaiTech University, China
  5. Yangzhou University, China
  6. Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of Ministry of Education, College of Life Sciences, Fujian Normal University, China
  7. College of Life Sciences, Fujian Normal University, China
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Cite this article as: eLife 2019;8:e45057 doi: 10.7554/eLife.45057

Abstract

Non-segmented negative-strand RNA viruses, such as measles, ebola and Newcastle disease viruses (NDV), encapsidate viral genomic RNAs into helical nucleocapsids, which serve as the template for viral replication and transcription. Here, the clam-shaped nucleocapsid structure, where the NDV viral genome is sequestered, was determined at 4.8 Å resolution by cryo-electron microscopy. The clam-shaped structure is composed of two single-turn spirals packed in a back-to-back mode. This tightly packed structure functions as a seed for the assembly of a nucleocapsid from both directions, facilitating the growth of double-headed filaments with two separate RNA strings inside. Disruption of this structure by mutations in its loop interface yielded a single-headed unfunctional filament.

https://doi.org/10.7554/eLife.45057.001

Introduction

Members of the order Mononegavirales encompass some of the most lethal human and animal pathogens, including ebola, rabies virus, measles, nipah virus and the human respiratory syncytial virus (RSV) (Amarasinghe et al., 2017; Kuhn et al., 2010). Mononegaviruses commonly contain a non-segmented, linear, negative-strand RNA genome, and the replication of this genome is vital for virus survival and pathogenicity (Ruigrok et al., 2011). One remarkable character of negative-strand RNA viruses is that their genomes are enwrapped by the nucleoprotein (N), which results in the formation of helical nucleocapsids (Finch and Gibbs, 1970; Heggeness et al., 1980; Longhi, 2009). During viral RNA synthesis, the assembled nucleocapsid, rather than the naked RNA genome, is opened and unveiled so that it can be recognized by the viral RNA-dependent RNA polymerase (RdRp) and it serves as the template for both replication and transcription (Dochow et al., 2012; Emerson and Wagner, 1972; Emerson and Yu, 1975; Fearns et al., 1997; Perlman and Huang, 1973; Severin et al., 2016). In Paramyxoviridae or Rhabdoviridae viruses, the viral phosphoprotein (P) mediates the ability of RdRp to access nucleoprotein, and the RdRp moves across the nucleocapsid for viral transcription (Blanchard et al., 2004; Bourhis et al., 2006; Kingston et al., 2004). RNA is susceptible to nuclease in vivo, so the virus has evolved a complicated mechanism to protect its viral genome, in which its N plays a major role in enwrapping nascent RNA thereby preventing possible damage (Dortmans et al., 2010; Ruigrok et al., 2011).

A great deal of effort has been expended on understanding the N assembly mechanism that protects the genome of the Mononegavirales. N has two domains, the amino-terminal domain (NTD) and the carboxy-terminal domain (CTD), with a positively charged cleft in between that is suitable for RNA binding. In the presence of RNA, each N can bind 6, 7 or 9 nucleotides and thus can clamp RNA into the cleft, forming a ribonucleoprotein complex (RNP) (Albertini et al., 2006; Gutsche et al., 2015; Tawar et al., 2009). RNP can further assemble into either a helical or ring structure with 10, 11 or 13 protomers per turn (Albertini et al., 2006; Green et al., 2006; Gutsche et al., 2015; Tawar et al., 2009). In RNP oligomers, the NTD and CTD interact successively with adjacent N proteins, forming long helical filaments that efficiently protect the viral genome, and which serve as the template for viral RNA transcription and the replication of new virions (Ge et al., 2010; Zhou et al., 2013).

Detailed structural analyses have shown that measles RNP filaments exhibit more rigid and regular single-headed, herringbone-like characteristics after trypsin treatment, and in this state are seemingly not sufficient to protect RNA genome at the tips of the filaments (Schoehn et al., 2004). The mechanism through which viral RNP protects its tips from digestion by proteases remains to be discovered. Here, the Newcastle disease virus (NDV), a member of the genus Avulavirus, family Paramyxoviridae, which is relatively safe for handling, was selected as the model to look at how NDV RNP protects its viral genome and to provide new insights into the development of nucleocapsid-based antivirus therapies.

Results

Clam-shaped NDV nucleocapsid

Following previous reports (Guryanov et al., 2015; Peng et al., 2016), the NDV N was expressed in an Escherichia coli system and pure protein was obtained after tandem affinity and gel-filtration chromatography. The N was found to be of high purity in SDS-PAGE, with an absorbance of A260/A280 of ∼1.1, suggesting the presence of RNA-bound N (Figure 1—figure supplement 1A). Under negative-stain EM, purified N exhibited round-shaped structures with a small portion of double-headed filaments of different lengths (Figure 1—figure supplement 1B), which are similar to the nucleocapsids of measles that are expressed in Sf21 insect cells (Jensen et al., 2011), of sendai virus in mammalian cells (Buchholz et al., 1993) and of hendra virus in E. coli (Communie et al., 2013). Those two kinds of assemblies were further separated with continuous sucrose-gradient ultracentrifugation. The separated round-shaped sample was quite homogenous with a diameter of ∼200 Å and was used for subsequent structure determination (Figure 1A).

Figure 1 with 4 supplements see all
NDV N assembles into clam-shaped structures with two single-strand spirals packing in a back-to-back manner.

(A) The images show negative-stain EM micrographs of the round-shaped structures (top image, upper fraction) and filaments (bottom image, lower fraction) after sucrose-gradient centrifugation (close-ups of the boxed areas are shown on the right). (B) Various views of the 3D reconstruction of the clam-shaped structure of N from the upper fraction. The C2 symmetry axis enforced during reconstruction is indicated in the center view (middle). The NTD, CTD and RNA are colored in pink, green and gold, respectively. (C) Atomic model of the clam-shaped structure of N shown from the same view as in (B) and using the same color code. The two 5′ ends of the enwrapped RNA and the seam between them are labeled in the middle view.

https://doi.org/10.7554/eLife.45057.002

The cryo-electron microscopy (cryo-EM) images for the round-shaped samples were collected and the single particle analysis was carried out. Two-dimensional (2D) and three-dimensional (3D) classification results showed a clam-shaped rigid body with some flexible extensions (Figure 1—figure supplement 1C–E). Further 3D refinement resolved the clam-shaped structure to 6.4 Å resolution and showed obvious C2 symmetry in the rigid body. The C2 symmetry was then applied to improve the resolution, yielding an overall 4.8 Å resolution of the core structure (Figure 1B, Figure 1—figure supplement 2A–E and Video 1). Each protomer was easily recognized from the reconstruction. Those protomers furthest from the seam were better resolved while those closer to the seam were of lower resolution (Figure 1—figure supplement 2E). However, an atomic resolution structure of NDV N was still missing. Homolog modeling on NDV N, based on the 40% sequence identity of N between NDV and Parainfluenza virus 5 (PIV5) (Alayyoubi et al., 2015), resulted the subunit N model and the model was flexibly docked into the EM density map (Figure 1C, Figure 1—figure supplement 3A,B). The docked model fits the EM density well with only minor modification, and resulted in a reliable initial model of NDV N.

Video 1
3D reconstruction of the clam-shaped structure and the fitting of a pseudoatomic model.
https://doi.org/10.7554/eLife.45057.007

The whole reconstruction revealed a clam-shaped structure with the symmetry axis perpendicular to the spiral axis, where two single-turn spirals pack in a back-to-back manner (Figure 1B,C). In each single-turn spiral, there are around 13 N molecules per turn, and each N uses its N-arm (residues 2–34) and its C-arm (residues 370–398) to interact horizontally with a neighboring N for domain exchange contact (Figure 1C and Figure 1—figure supplement 3C and E), as reported in previous ring structures (Alayyoubi et al., 2015; Albertini et al., 2006; Green et al., 2006; Tawar et al., 2009). More specifically, in a NDV clam-shaped structure, Ni uses the N-arm to interact with the Ni-1 CTD and the C-arm to make extensive contact with the Ni+1 CTD tip, forming an exceedingly stable structure (Figure 1—figure supplement 3C–E). Different from the ring structure of PIV5 N (Alayyoubi et al., 2015), the add-on N shifts upward by ~4.6 Å, which drives NDV N to form a single-turn spiral instead (Video 2).

Video 2
Morphing of the ring structure to form a single-turn spiral.
https://doi.org/10.7554/eLife.45057.008

Endogenous RNA from E. coli can be traced in the EM map. Limited by resolution, poly-Uracil (poly-U) was modeled into the EM map to mimic cellular RNA. In the clam-shaped structure, the RNA follows a relaxed helical pattern and orients outside the N molecule, being more similar to the RSV nucleocapsid than to that of rhabdovirus or vesicular stomatitis virus (Figure 1—figure supplement 3EVideo 3) (Albertini et al., 2006; Green et al., 2006; Tawar et al., 2009). The external RNA is deeply buried in the interdomain cleft between the NTD and the CTD, following the ‘rule of six’ with alternating three-base-in and three-base out conformation (Figure 1—figure supplement 3F) (Calain and Roux, 1993; Kolakofsky et al., 1998). Six nucleotides are covered by one N, and there will be 78 nucleotides per single-turn spiral (Figure 1—figure supplement 3F). On the basis of nucleocapsid structural similarity between NDV and the measles virus, the RNA in NDV is estimated to be left-handed with the 5′ end of RNA, which would be first replicated and enwrapped by N immediately after synthesis, lying inside (as labeled in Figure 1B and C) (Gutsche et al., 2015).

Video 3
RNA enwrapped between the NTD and the CTD.
https://doi.org/10.7554/eLife.45057.009

Of particular note is an obvious seam between the two single-turn spirals, which disconnects two RNA molecules (Figure 1B,C). The separation between the two 5′ ends of the RNAs is ~6 nm and the bending angle of these ends is approximately 120°, which blocks the continuity of the RNA because it is impossible for one RNA to span two back-to-back spirals. Thus, the clam-shaped structure is not an integrated helix at all, but rather is composed of two spirals self-capping each other in a back-to-back mode. To confirm whether the NDV nucleocapsid is packed using this mode in vivo, the negative-stain EM images of highly polymeric RNPs extracted from Newcastle disease virus were obtained. Interestingly, the images showed a filamentous assembly of the genomic RNA with the clam-like structure, similar to that observed in the resolved structure (Figure 1—figure supplement 4).

Double-headed filament derived from clam-shaped nucleocapsid

Importantly, decreasing threshold values to show more EM densities with or without C2 symmetry revealed that each single-turn spiral had the potential to grow further into a longer filament following a helical trajectory (Figure 1—figure supplement 2A,B). The pseudo-model of N could be docked into extra densities following the helical trajectory without any structural conflicts. Iteratively adding N protein in such manner to both single-turn spirals would yield a longer helix with double heads (Video 4).

Video 4
Double-headed spiral derived from clam-shaped structure.
https://doi.org/10.7554/eLife.45057.010

To verify this, the filaments fraction after ultracentrifugation was examined with cryo-EM. Almost every filament had double heads derived from one clam-shaped structure (Figure 2A). Owing to the heterogeneity of the double-headed filaments, their structures could not be directly resolved via the single particle reconstruction approach. Consequently, the filament was split into two parts for structural analysis: the clam-shaped core and the helical part (Figure 2A,B). For the former, 2608 clam-shaped particles truncated from double-headed filaments yielded a 14.0 Å resolution structure (Figure 2—figure supplement 1A). The overall shape of the core fitted very well with the 4.8 Å clam-shaped structure (Figure 2B and Figure 2—figure supplement 1B). The distinctive back-to-back packing mode and the seam between two single-turn spirals were clearly recognizable, suggesting that the clam-shaped core acts as the seed for filament growth (Figure 2D).

Figure 2 with 2 supplements see all
Double-headed filament derived from the clam-shaped structure.

(A) Representative cryo-EM micrograph of the N filament from the lower fraction. One typical double-headed filament was selected, magnified and colored in blue (helical structure) and red (clam-shaped core). (B) 3D reconstructions of the helical structure (top) and the clam-shaped core (bottom). The 4.8 Å clam-shaped structure is docked into the clam-shaped core in the filament. (C) Combination of both helical filaments and the clam-shaped core yields the whole double-headed filament. The position of the clam-shaped core in the composite structure is delineated by the dashed line. (D) Atomic model of the double-headed filament shows the position of the clam-shaped core (dashed line). Corresponding 5′ and 3′ ends from the same RNA are labeled in red and blue. (E) The two helixes in one double-headed filament are of unequal length. The length of each helix is defined as the distance between the helix tip and the center of the clam-shaped core in the cartoon. The length measurements and the RNA direction from 5′ to 3′ are given.

https://doi.org/10.7554/eLife.45057.011

The helical part of the filament was reconstructed at 15.0 Å resolution (Figure 2B and Figure 2—figure supplement 1A). Like the clam-shaped structure, the helix was composed of 13 protomers per turn, with an outer diameter of ∼200 Å, in agreement with the pseudo-atomic model. The helical pitch varied by ~60 Å, which provided flexibility for the helical nucleocapsids to fit into the crowded virus. Thus, the clam-shaped structure was perfectly compatible with the helical filament and could further grow into a helical filament (Figure 2C,D). Following the direction of RNAs in the clam-shaped structure, the 5′ ends of the RNAs of the double-headed filament were depicted similar to those of the clam-shaped structure (Figure 1C and Figure 2C,D).

Interestingly, the lengths of the two helixes in around 90% of the back-to-back spirals were not equal and one helix was obviously longer than the other one in the raw images (Figure 2A and Figure 2—figure supplement 1C). The statistics showed that the shorter helix had an average length of ~14 nm with fewer than two helical turns, while the average length of the longer one was doubled to ~34 nm (Figure 2E), although the factors that determine the length difference are uncertain.

The clam-shaped nucleocapsid affects the function of the viral genome

In the clam-shaped structure or in the derived double-headed filament, the self-capping interface came from loops (residues 114–120) of vertically adjacent N in the clam-shaped core. Distance analysis of the residues in the loop suggested that hydrogen bonds may exist between two pairs of Glysin119 and Arginine117 residues (Figure 3A,B). The Loop114–120 region was involved only in the assembly of the clam-shaped core but not in the helical assembly of the double-headed filament. All of the residues in Loop114–120 were mutated to Alanine to check whether the mutations affected the clam-shaped assembly. The mutated N (NLoop) was purified using the same protocol as that used for NWT and yielded filaments that were an average of 50 nm longer than those of NWT. A zoomed-in view of the NLoop filaments clearly showed a single-headed, herringbone-like filament instead of a double-headed assembly from 2D classification (Figure 3C and Figure 2—figure supplement 2). Direct fast fourier transformation (FFT) analysis of one single-headed NLoop filament showed clean diffraction bands with ~1/60 Å intervals, and the 3D reconstruction of NLoop showed a helical structure that was similar to the single-turn spiral of the double-headed NWT. The evidence suggests that Loop114–120 has no influence on helical assembly but has a crucial role in clam-shaped structure formation (Figure 2—figure supplement 2).

Figure 3 with 1 supplement see all
The clam-shaped nucleocapsid is important for the function of the viral genome.

(A) Loop pairs from the vertically adjacent N form the self-capping interface in the clam-shaped structure. Five loop pairs furthest from the seam are shown. Colors are as in Figure 1. (B) View of one loop pair of the clam-shaped structure. Seven residues (114–120) in the upper loop are labeled and the lower loop is docked into the EM density. (C) Raw micrograph of a single-headed helix from the NLoop and the 2D classification of the filament tip (circled). Zoomed-in view of selected raw filaments (examples in dashed boxes) with two typical 2D classes on the tip shown. (D) NWT was able to form double-headed filaments and functioned well in the minigenome assay. The retention volume of NWT in gel filtration chromatography was ~47 ml (left) and the negative-stain image of this fraction consisted of a clam-shaped structure and filaments was zoomed in (middle). NWT exhibited strong fluorescence signals in a minigenome assay in BSR-T7/5 cells (right). (E) The NLoop formed filaments but was not functional in a minigenome assay. The retention volume of the NLoop was ~47 ml, close to NWT (left). Negative-stain EM showed more filaments than NWT (middle). However, there was no fluorescence signal in the minigenome assay (right).

https://doi.org/10.7554/eLife.45057.014

To further investigate whether the Loop114–120 is functionally relevant in vivo, minigenome analyses of several N mutants (Figure 3D,E and Figure 3—figure supplement 1) were performed. The N-arm and the C-arm had been proven previously to be critical for the assembly of N (Buchholz et al., 1993; Kho et al., 2003) and the truncations of N∆N-arm, N∆N-arm∆C-arm∆C-tail and N∆C-arm∆C-tail disabled or heavily affected the assembly to higher ordered structure that was enabled by NWT, NLoop and N∆C-tail, as shown by size-exclusion chromatography and negative-stain EM images (Figure 3D,E and Figure 3—figure supplement 1). RNA synthesis was fully functional in the presence of wild type N (NWT), but truncation mutants lacking the N-arm (N∆N-arm), the N-arm or the C-arm/C-tail (N∆C-arm∆C-tail and N∆N-arm∆C-arm∆C-tail) were all nonfunctional and lost the ability to express the GFP reporter. Surprisingly, the N∆C-tail were partially functional according to the weak fluorescence signals observed in the minigenome assay. Although the mutation of NLoop could form longer single-headed filaments as mentioned above, it showed a negative result in the fluorescence assay (Figure 3D,E and Figure 3—figure supplement 1 ). RNA replication, transcription or translation was not successful in the minigenome assay of the NLoop, indicating that the clam-shaped structure is critical for the expression of the GFP reporter gene.

The clam-shaped nucleocapsid is resistant to nuclease

The detailed structural analysis showed that the single-headed filament from NLoop exposed the RNP 5′ end to the environment, whereas the double-headed filament from NWT enabled intermolecular self-capping to cover its sensitive 5′ end. (Figure 4A,B). To test the sensitivity of the RNP 5′ end to protease, elastase was incubated with double-headed or single-headed filament samples. The SDS-PAGE gel showed a ~40-kDa band with some smears from elastase-digested NWT (Figure 4C). Peptide mapping of the 40-kDa band via Mass Spectrum showed a residue range of 33 to 361, which suggested that the cleaving site was in the loop in the C-arm (Figure 4D and Figure 4—figure supplement 1A). For the single-headed filament from NLoop with the 5′ end exposed, an obvious difference was that the 40-kDa band was found to be further digested to 30 kDa from the N-arm after elastase treatment, based on the SDS-PAGE and Mass Spectrum results, which strongly indicates that there is another cleavage site in the NTD loop regions.

Figure 4 with 2 supplements see all
Clam-shaped nucleocapsid is resistant to elastase and RNase A.

(A) An atomic model of an NWT double-headed filament from different views shows reciprocal capping between two single-headed spirals. Colors as in Figure 1. One single-headed spiral is highlighted by the black line and labeled ‘capping’. (B) An atomic model of an NLoop single-headed filament from different views with no cap and with the 5′ end of its RNA exposed. The supposed capping spiral, marked by the dashed line, is missing from the single-headed filament. (C) SDS-PAGE gels of NWT and NLoop after elastase digestion. There was a ~40-kDa main band with some smears in the elastase-digested NWT assay (left), while elastase cut the NLoop to form a ~30-kDa band (right). (D) Mass spectrum results identified peptides drawn on the atomic structure of N, indicating one additional cutting site on NLoop (gold scissors). The common regions mapped by Mass Spectrum in NWT and NLoop are colored in blue, and the unique region checked in NWT is shown in red. Five peptides were identified from the 40-kDa band of NWT and marked on the N atomic structure, leaving the CTD loops as the cutting site. Only four peptides were identified from the 30-kDa band of NLoop and marked on N atomic structure. Given the reduced molecular weight and the missing NTD peptide, another cutting site should exist within NTD. (E) Comparison of RNase A digesting NWT and NLoop at different timepoints. Both the clam-shaped structures and the filaments are counted. The numbers of oligomers in NWT and NLoop at 0 s are normalized to 100%. At 180 s, almost 100% disassembly of nucleocapsid was seen in NLoop whereas over 25% of filaments remained in NWT. (F) A cartoon depicts the hypothetical full protection provided to the viral RNA genome by NWT via the self-capping clam-shaped structure. When the clam-shaped structure is broken, nuclease is able to access the RNA 5’ end and can digest the whole RNA strand.

https://doi.org/10.7554/eLife.45057.016

In addition, the influence of nuclease on RNA genome stability was tested. RNase A was added to the solutions containing NWT or NLoop filaments to check the digestion result of the assemblies. The results showed that NLoop was more sensitive to RNase A than that of NWT after 180 s exposure (Figure 4—figure supplement 1B). The statistical results showed that almost all of the NLoop samples were completely disassembled whereas over 25% of the NWT filaments remained intact (Figure 4E). Meanwhile, the NWT rather than the NLoop contained RNA with an absorbance of A260/A280 of ∼0.9, whereas that of NLoop was ∼0.6. The enzyme digestion analysis showed that NWT, rather than NLoop, was resistant to the digestion of nuclease and protease, from which it could be hypothesized that the NLoop exposed its RNP 5′ end without the self-capping protection, and was exposed to protease and became accessible by nuclease. Through self-capping, N can protect the viral genome not only from side attack but also from both ends.

Discussion

N is the key factor for protecting the nascent RNA from degradation during RNA replication. Different from the reported ring-structure and helical spirals (Alayyoubi et al., 2015; Albertini et al., 2006; Green et al., 2006; Tawar et al., 2009; Gutsche et al., 2015), a novel clam-shaped structure of NDV N with two single-turn spirals packing in a back-to-back manner was identified and determined, corresponding to the extracted nucleocapsid assembly of NDV (Figure 1—figure supplement 4). The clam-shaped structure of the NDV nucleocapsid was verified by in vivo transcription and translation experiments with minigenome analysis. The deletion of the N-arm or the C-arm of N disrupted or affected the formation of highly ordered nucleocapsid and resulted the absence of fluorescence signals in the minigenome assay. However, in a similar minigenome assay in which a truncated C-tail, namely N∆C-tail, was used, some weaker fluorescence signals were observed. The previous studies showed that the P protein used its NTD domain (PNTD) to uncoil the nucleocapsid and allowed the RdRp to access the genomic RNA and then tethered the RdRp to the nucleocapsid with its XD domain binding to the C-tail of the N protein (Cox et al., 2014). One possible explanation is that even though the N∆C-tail lacks the C-tail, the P protein can still mediate the formation of the N–RNA–RdRp complex in the minigenome assay due to the interaction of PNTD with the nucleocapsid.

The double-headed spiral uses a self-capping mechanism that involves the clam-shaped core to protect the RNA genome’s integrity. This is the possible reason why the single-headed NLoop was not functional in a minigenome assay. More interestingly, this clam-shaped structure functions as a seed for the assembly of double-headed spirals with two separate RNAs inside. The illumination of the clam-like nucleocapsid expands our understanding of the involvement of N in the assembly of the the helical nucleocapsid, especially introducing the clam-like core as the starting point for N assembly and then elongation on both sides, which maintains genome integrity in vivo. A self-capping mechanism is quite common in filaments that are involved in biological processes. For example, in DNA repair, the Rad51 paralog complex RFS-1/RIP-1 induces remodeling at the tips of Rad51–ssDNA filaments to stimulate Rad51 strand exchange activity (Taylor et al., 2015; Taylor et al., 2016). In microtubule assembly, γ-tubulin pre-assembles into single-turn spirals that serve as the template to nucleate sequential α/β-tubulin assembly (Kollman et al., 2010; Zehr et al., 2014). Previous studies have shown that self-capping is a mechanism that allows proteins or their homologues to fit easily into the spiral assembly and to fine-tune its function efficiently.

The clam-shaped nucleocapsid also provides possible explanations for the pleomorphism and polyploidy of the Mononegavirales. Mononegavirus morphology appears to vary considerably, especially among the Paramyxoviruses and Filoviruses, in the range of about 110–540 nm in diameter for Sendai virus (Loney et al., 2009), 100–250 nm for spherical NDV particles (Battisti et al., 2012), and 50–510 nm for the measles virus (MeV) (Cox and Plemper, 2017). This character of flexible volume of virus could accommodate variation in the copy number of the genome. It is a common observation for mononegaviruses to contain more than one genome; examples include NDV (Dahlberg and Simon, 1969; Goff et al., 2012; Kingsbury and Darlington, 1968), hemagglutinating virus of Japan (HVJ) (Hosaka et al., 1966), Sendai virus (Loney et al., 2009; Lynch and Kolakofsky, 1978), measles virus (Liljeroos et al., 2011; Rager et al., 2002), RSV (Kiss et al., 2014) and ebola virus (Beniac et al., 2012; Booth et al., 2013). The presence of multiple genomes in a virion is essential for their infections, for example, two types of genomic analyses of MeV infections have provided independent evidence of multi-genome MeV transmission (Rager et al., 2002; Shirogane et al., 2012). In addition, the multi-genome in one virion seems to be packaged in continuous mode in ebola virus (Beniac et al., 2012; Booth et al., 2013), and even in a ‘end to end’ mode in HVJ (Hosaka et al., 1966). One interesting aspect on self-capping assemblies is that they provide the possibility of accommodating two copies of viral genomes with different lengths in one virion. So, the double-headed mode provides a possible organizing pattern for the multiple genomes of the polyploidy viruses.

In addition, the C-tail of N may exist inside rather than outside of the nucleocapsids of Mononegavirales. Even though full-length N was purified and used for structural analysis, the C-tail (residues 399–489) is not easily recognized in the EM map because of the long, intrinsically flexible domain reported in other structures (Houben et al., 2007; Longhi et al., 2003). Compared to ring structures, the clam-shaped structure has extra cone-like densities in the center (117 nm3 at the threshold of 0.0054) of the density map, which are apparently from the C-tail (Figure 4—figure supplement 2). The C-tail is located inside the clam-shaped structure, and is only accessible from either end by P or other proteins in order to form the N–RNA–RdRp complex for the replication and transcription of the genomic RNA; this finding differs from those of previous reports that have described outside-orientated C-tails (Jensen et al., 2011; Krumm et al., 2013).

NDV infects many domestic and wild avian species, severely impacting the poultry industries in many countries. The structure of NDV N significantly improves our understanding of how NDV protects itself and infects hosts. It is important to highlight that NDV shares many features with other members of the order Mononegavirales. For example, measles virus nucleocapsid was reported to assemble into herringbone-shaped structures (Gutsche et al., 2015), similar to the NDV NLoop single-headed spiral. Therefore, it is reasonable to predict that nucleocapsid of measles virus, as well as that of other mononegaviruses, might adopt a similar self-capping mechanism in order to keep its genome secure. N is a most conserved viral protein and the vital building block for nucleocapsid assembly, which makes it an ideal target for antivirus drug development (Cox and Plemper, 2016). The positive charged clefts between the CTD and NTD lobes of N, and especially the interaction loop between the vertically adjacent N in the clam-shaped structure, are the possible druggable sites for further structure-based design of small-molecule drugs. The structural study of the NDV provides new insights into the negative-sense RNA virus field and represents the starting point for inspiring new antiviral drug design for mononegavirus diseases.

Materials and methods

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Gene
(Newcastle disease virus)
NDV NSangon Biotech
Company
GenBank ID: HM063424.1Synthetic gene
Strain, strain background
(E. coli)
BL21 (DE3) Star competent cellsThermoFisher ScientificC6010-03Cells for protein expression
Strain, strain background
(Newcastle disease virus)
LaSotaChina Veterinary Culture Collection Center
Cell line
(hamster)
BSR-T7/5PMID: 9847328Gift from Zhigao Bu's lab from Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences
Chemicalcompound, drugelastaseSIGMAE8140-1UN
Chemicalcompound, drugRNase APromegaA7973
Software, algorithmRELION 1.4PMID: 23000701https://www3.mrc-lmb.cam.ac.uk/relion/index.php?title=Main_Page
Software, algorithmRELION 2.0PMID: 27845625https://www3.mrc-lmb.cam.ac.uk/relion/index.php?title=Main_Page
Software, algorithmUCSF Chimerahttp://plato.cgl.ucsf.edu/chimera/RRID:SCR_004097
Software, algorithmCootPMID: 20383002RRID:SCR_014222http://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/
Software, algorithmPHENIXPMID: 22505256RRID:SCR_014224https://www.phenix-online.org/
Software, algorithmImageJhttp://imagej.nih.gov/ij/RRID: SCR_003070
OtherCrystal structure of the paramyxovirus parainfluenza virus 5 nucleoprotein–RNA complexPMID: 25831513PDB: 4XJN

Plasmid construction

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The nucleoprotein (N) gene of the Newcastle disease virus (NDV) (GenBank ID: HM063424.1) was synthesized by the Sangon Biotech Company (China). The gene was cloned into the pMCSG7 vector with a N-terminal 6 × His tag and a C-terminal 8 × His tag (Stols et al., 2002). The transition mutation of amino acids 114–120 to Ala and the truncations caused by deleting the N-arm (residues 1–33), C-tail (residues 399–489), C-arm and C-tail (371-489), as well as combinations of the N-arm, C-arm and C-tail (1–33 and 371–489) of N gene, were also cloned into the pMCSG7 vector and designed as NLoop, N∆N-arm, N∆C-tail, N∆C-arm∆C-tail and N∆N-arm∆C-arm∆C-tail, respectively. All of the recombinant plasmids with target genes were sequenced to verify their sequences.

Protein expression and purification

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NDV N and its derived mutants were expressed in Escherichia coli BL21(DE3) cells and purified using tandem affinity and gel filtration columns. In detail, the cells were grown in LB media at 37°C until the OD600 nm reached 0.6. The target protein was induced at 16°C for an extra 20 hr with the final concentration of IPTG (isopropyl-B-D-1-thiogalactopyranoside) at 0.1 mM. The cells were harvested by centrifugation at 4680 g for 20 min to obtain the sediments. The pellets were resuspended in PBS buffer (137 mM NaCl, 2.7 mM KCl, 50 mM Na2HPO4, and 10 mM KH2PO4 (pH 7.4)) and disrupted with microfluidizer. Cell debris was removed by centrifugation at 38,900 g for 50 min. The clarified supernatant was loaded onto a 5 ml nickel-nitrilotriacetic acid (Ni-NTA) resin gravity column (Qiagen), which was preequilibrated with PBS buffer. The column was washed with 50 ml PBS buffer containing 20 mM imidazole followed by a 100 mM imidazole wash. Finally, the protein was eluted using PBS buffer containing 500 mM imidazole. The proteins with His-tags were concentrated and loaded onto a Superdex G200 size-exclusion chromatography column (120 ml, GE Healthcare Life Sciences, USA) preequilibrated with TRIS buffer at pH 8.0 (20 mM Tris-HCl, 150 mM NaCl and 2 mM DTT). The target proteins with endogenous RNA were collected for the following experiments.

The samples obtained above were loaded onto the top of a continuous 10% to 30% (w/v) sucrose gradient in the same TRIS buffer and centrifuged for 6 hr at 16 × 104 g and 4°C with an SW40 rotor (Beckman). The samples were collected by puncturing the tube and dialyzing in the TRIS buffer.

Negative stain EM

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Grids of N or its mutants for negative-stain EM were prepared as described previously (Ohi et al., 2004). Specifically, 4 μl of samples (0.15 mg/ml) were applied to glow-discharged EM grids covered by a thin layer of continuous carbon film and stained with 2% (w/v) uranyl acetate. Negatively stained grids were imaged on a Tecnai Spirit 120 microscope (Thermo Fisher Scientific, USA) operating at 120 kV. Images were recorded at a magnification of ×43,000 and a defocus set to −2 μm, using a 4K × 4K scintillator-based charge-coupled device camera (UltraScan 4000, Gatan, USA).

Cryo-EM data collection

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To prevent sample aggregation, the N-RNA sample was diluted to 0.65 mg/ml containing 0.018 mg/ml Qβ virus-like particles. A 4 μl sample was applied to a glow-discharged holey carbon grid (Quantifoil, R1.2/1.3, Ted Pella) with a thin layer of continuous carbon film. The grids were blotted using a Vitrobot Mark IV (Thermo Fisher Scientific, USA) with 5 s blotting time, force level of 2 at 100% humidity and 4°C and then immediately plunged into liquid ethane cooled by liquid nitrogen.

The micrographs of the clam-shaped structure samples were recorded on a 300 kV Titan Krios G2 electron microscope equipped with Cs corrector (Thermo Fisher Scientific, USA) and a K2 Summit direct electron detector (Gatan, USA), which was used in counting mode with a pixel size of 1.35 Å. Each movie was exposed for 7.6 s and dose-fractioned into 38 frames with 0.2 s for each frame, generating a total dose of ~41 e-/A2 on the samples. Defocus values during data collection varied from −1.5 μm to −3 μm. All the images were collected under the SerialEM automated data collection software package (Mastronarde, 2005). The micrographs of the filament samples were collected on a 200 kV Talos F200C electron microscope (Thermo Fisher Scientific, USA) equipped with a DE20 Summit direct electron detector (DE, USA) in counting mode with a pixel size of 1.582 Å. Each movie was exposed for 40 s and contained 32 frames, generating a total dose of ~41e-/A2 on the samples. Defocus values for the date collection varied from −1.5 μm to −3 μm. All the images were collected by utilizing the SerialEM automated data collection software package (Mastronarde, 2005).

Cryo-EM data processing and 3D reconstruction

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A total of 3200 micrographs were used for the clam-shaped structure determination. Before further image processing, the images were aligned and summed with MotionCorr software (Li et al., 2013) and the CTF parameters of each image were determined by CTFFIND3 (Mindell and Grigorieff, 2003). The single-particle analysis and reconstruction was mainly executed in Relion1.4 (Scheres, 2012) and Relion 2.0 (Kimanius et al., 2016). First of all, the particles were picked automatically by Gautomatch and bad particles were excluded by manual selection and reference-free two-dimensional (2D) classification, with 167,588 particles selected for further processing. The initial model was produced by EMAN2 using typical 2D classes with different orientations (Tang et al., 2007). The initial model was lowpass-filtered to 60 Å to limit reference bias during three-dimensional (3D) classification and later refinement. No symmetry was applied in the 3D classification process, and one of the three classes with a better structure feature was selected for further 3D auto-refinement. A 3D map with an overall resolution of 6.4 Å was obtained without symmetry by 3D refinement of the cleaned-up 75,290 particles. Then, a soft mask was applied to avoid the influence of the spreading map on the alignment. Meanwhile, the C2 symmetry was also applied and the final resolution was improved to 4.8 Å with the gold-standard Fourier Shell correlation (FSC) 0.143 criteria. The map was filtered and sharpened during a Relion post-processing session and the local resolution was estimated with Resmap (Kucukelbir et al., 2014).

Double-headed filaments were divided into two parts for structure determination: helical filaments and clam-shaped junctions. Both helix and joint parts of the filament were picked manually, 2D classified and 3D reconstructed with Relion2.0 (Kimanius et al., 2016). For helical reconstruction, 4909 good segments were selected with 75% overlap. The 3D refinement using a cylinder as the initial map yielded a 15 Å-resolution helical map with the helical twist of −27.30° and a helical rise of 4.78 Å. For single-particle reconstruction of the clam-shaped junction, 2608 particles were manually picked, and the same cylinder in helical reconstruction was used as the initial model. 3D refinement without symmetry yielded a structure, which was used as reference for the next refinement with C2 symmetry. All of the reference structures were pre-filtered to 60 Å to avoid reference bias during the 3D reconstruction. The C2 refinement yielded a map at the resolution of 14 Å. Both the helical map and the C2 symmetric map were filtered and b-factor sharpened during a Relion post-processing session.

The direct FFT analysis of a single-headed filament was performed with the EMAN2 software package (Tang et al., 2007). In total, 6333 segments of the single-headed filament samples were manually picked and the helical reconstruction was performed in Relion 2.0 (Kimanius et al., 2016).

Model building and validation

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The homology model of N and RNA were generated by Modeller (Eswar et al., 2008) using the crystal structure of parainfluenza virus 5 (PDB accession number 4XJN) as the template. Then the pseudo-atomic model of N was flexibly docked into the protomer furthest from the seam in the EM density map with Rosetta software (Das and Baker, 2008). The extra density excluding N was assigned as RNA enwrapped between NTD and CTD and docked using poly-Uracils due to the uncertain sequence of RNA in Coot (Emsley et al., 2010). The model refinement on an N with six Uracils was carried out using secondary structure restraints to maintain proper stereochemistry in Phenix.refine(v1.12) (Afonine et al., 2012). The model was further optimized manually for better local density fitting using Coot (Emsley et al., 2010). To prevent overfitting, TLS refinement and weight optimization were used to improve overfitting across a wide range of resolutions. Ramachandran outliers were corrected semi-automatically in Coot, and MolProbity statistics were computed to ensure proper stereochemistry. The model of the N was validated by computing a Fourier shell correlation (FSC)slush with the density map. The revised atomic NDV N and poly-Uracils were duplicated and docked as a rigid body to the other protomers using UCSF Chimera software (Pettersen et al., 2004).

Elastase and RNase A enzymatic assay

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Elastase and RNase A were selected to test the susceptibility of the NWT and NLoop samples. A mixture of 40 μl Tris buffer at pH 8.0 (20 mM Tris-HCl, 150 mM NaCl and 2 mM DTT) containing NWT or NLoop (0.15 mg/ml) and 0.275 mg/ml of RNase A was incubated at 37°C and sampled after90 s for negative-stain EM. Forty-five images were captured at ×49,000 magnification for each grid and the number of either clam-shaped structures or filaments was counted at different digestion timepoints.

NWT or NLoop (0.15 mg/ml) in TRIS buffer was incubated with 0.1 mg/ml chymotrypsin-like elastase at 37 °C and sampled every 30 min for SDS-PAGE analysis.

Statistical analysis

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For double-headed filaments, the distance between the helix tip and the clam-shaped core were measured using ImageJ software. A total of 1371 filaments from 169 raw micrographs were statistically counted to calculate the averaged length of the filaments and the percentage of filaments with unequal length of single spiral.

In nuclease and elastase cleavage assay, the number of particles of both clam-shaped structures and filaments of NWT and NLoop were counted at different timepoints such as 0 s, 90 s and 180 s. A total of 120 micrographs were statistically counted.

MALDI-TOF-MS analysis

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The samples of NLoop and NWT after chymotrypsin-like elastase digestion were resolved by SDS PAGE. The resulting gel bands were reduced with 10 mM dithiotreitol in 25 mM NH4HCO3 at 56°C for 60 min and alkylated by 55 mM iodacetamide in 25 mM NH4HCO3 in the dark for 45 min at room temperature. The gel pieces were washed with 40 μl of 25 mM NH4HCO3 for 5 min following the addition of 40 μl acetonitrile and then incubated for 15 min. After the gel pieces were dried in Speedvac for 15 min, proteins were digested with trypsin (100 ng for each band) in 25 mM NH4HCO3 overnight at 37°C. The samples of NLoop and NWT after trypsin treatment were excised for Ultraflextreme matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometer (MALDI-TOF/TOF-MS) assay. MALDI data processing was performed by the Peptide Mass Fingerprint method (www.matrixscience.com) using the SwissProt database.

NDV minigenome assay for the assembly mechanism of the N–RNA complex in vivo

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NDV minigenome p-LGT and three helper plasmids pCI-N, pCI-P and pCI-L from the NDV strain ZJ1 were constructed by Zhang et al. (2005). BSR-T7/5 cells stably expressing the phage T7 RNA polymerase, which were developed by Buchholz et al. (1999), were donated by Dr. Zhigao Bu (Harbin Veterinary Institute, China). The cells were maintained in DMEM (Gibco) supplemented with 10% fetal calf serum (FCS) and 1 mg/ml G418, as previously reported (Peeters et al., 2000).

Different mutant and truncated versions of N were cloned into the pCI-neo plasmid (Promega) and designated the names NLoop, N∆N-arm, N∆C-tail, N∆C-arm∆C-tail and N∆N-arm∆C-arm∆C-tail, respectively. The co-transfection was performed with minigenome and helper plasmids as reported previously (Peeters et al., 2000; Zhang et al., 2005). Briefly, the minigenome p-LGT (3 μg), pCI-P (1.5 μg), pCI-L (1.5 μg), with each different N expression plasmid (3 μg), including wild type pCI-N and pCI-N mutants, were cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase. One co-transfection, in which the N expression plasmid was replaced by vector pCI-neo was also conducted as the negative control. The transfection reagent was PolyJet reagent and the transfection procedure was carried out according to the manufacturer’s protocol. At 96 hr posttransfection, the GFP fluorescence of different samples was observed by fluorescence microscopy.

Ribonucleoprotein complex isolation from NDV strain LaSota

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NDV strain LaSota was propagated in 9-day-old specific-pathogen-free (SPF) embryonated chicken eggs at 37°C for 96 hr. The infected allantoic fluid was collected and centrifuged at 4320 g for 30 min to remove the cell debris. The supernatants were then subjected to pelleting through a 20% sucrose cushion at 38,900 g for 2 hr at 4°C. The pellets were resuspended in PBS buffer (pH 7.4) in the presence of the EDTA-free protease inhibitor cocktail complete from Roche Diagnostics, and lysed by five cycles of freezing and thawing (in liquid nitrogen and at 37°C, respectively) (Schoehn et al., 2004). The NDV lysate was loaded onto the top of a continuous 10% to 30% (w/v) sucrose gradient in PBS buffer (pH 7.4) and centrifuged for 6 hr at 16 × 104 g and 4°C with the SW40 rotor (Beckman). The samples were collected by puncturing the tube and dialyzing in PBS buffer. The presence of the N-RNA complex was verified by negative-stain EM.

Data availability

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The cryo-EM density map of clam-shaped structure was deposited in the Electron Microscopy Data Bank (EMDB) with the accession number EMD-9793. The atom coordinates of the single N subunit with six uracils were deposited in the Protein Data Bank (PDB) with the PDB ID 6JC3.

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Decision letter

  1. David M Knipe
    Reviewing Editor; Harvard Medical School, United States
  2. Detlef Weigel
    Senior Editor; Max Planck Institute for Developmental Biology, Germany
  3. Z Hong Zhou
    Reviewer; University of California, Los Angeles, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Self-capping of nucleoprotein filaments protects Newcastle Disease Virus genome" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Detlef Weigel as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Z Hong Zhou (Reviewer #1).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

This manuscript reports the first high-resolution cryoEM structure of the nucleoprotein (N) protein of Newcastle disease virus (NDV), a negative strand RNA virus. They found that recombinantly expressed N protein forms clam-shaped double-headed filaments that could serve as "caps" or nucleation sites on nucleocapsids for viral RNA replication and/or encapsidation. This novel architecture is potentially of great importance for RNA virus replication. Mutagenesis studies are also provided that are interpreted to show the biological importance of this structure for viral replication. It is not clear how specific the mutational alterations are, i.e., whether the alteration specifically affects formation of the clam-shaped structures or alters the protein more generally. Therefore, there was some concern that these functional studies were over-interpreted. There was also a request from all three reviewers for improved images and analysis of the native RNP structures.

Essential revisions:

1) The essential revision is to provide more convincing native RNP images and analysis of those structures.

2) The functional studies are over-interpreted, so those studies should be removed from the manuscript. The least would be to tone down the conclusions because of the reasons outlined below.

The reviewers have made a number of suggestions in their individual reviews that I think will improve the manuscript. Therefore, I am including those important points below:

Reviewer #1:

In this paper, the groups led by Liu, Shen and Ouyang reports the first high-resolution cryoEM structure of the nucleoprotein (N) of Newcastle disease virus (NDV). NDV belongs to a group of negative-sense RNA viruses that also include such medically important viruses as Ebola and Measles. The authors found that recombinantly expressed N forms clam-shaped double-headed filaments with a clam-like core at the middle of the filament. Isolates have flexible structures, ranging from long to short filaments and spirals, as well as a single species containing double spirals. They used the sample fraction containing primarily the double spiral core to determine the structure at 4.8Å resolution by single-particle cryoEM. Since NDV N has 40% sequence identity to the known crystal structure Parainfluenza virus 5 (PIV5) (Alayyoubi et al., 2015), atomic model of NDV N was built needing only minor modifications and shares great similarity to known N structures of other negatively strand RNA viruses (e.g., Figure 1—figure supplement 3B showing nearly identical backbone trace). Placement of this atomic model of N to the clam-shaped reconstruction shows two spirals self-capping each other in a back-to-back manner, revealing a loop (residues 114-120) that is not involved in formation the single-headed helix but responsible for the formation of the clam in the double-headed filament. The authors also carried out cryoEM analyses on the filament fraction and obtained low resolution reconstructions (about 15Å) for both the double heads and the helical segment, which are consistent with the single-particle cryoEM reconstruction of the double-headed clam-like core at higher resolution.

The authors supplement the structural analyses with a series of functional studies with constructs harboring wild-type N, loop 114-120-mutated N (named Nloop), and several other truncation mutants. Most significantly, using mini-genome constructs, the authors demonstrated that minigenome replication was dependent on wild-type, presumably clam-shaped, nucleocapsid. Protease treatment experiments revealed different protection patterns between the double-headed and single-headed helices, yielding 40 kDa verses 30 kDa protein fragment. The clam-shaped filaments were also resistant to nuclease treatment while single-headed filaments formed by Nloop mutant were less resistant to such treatment.

Taking these results together, the authors posit a self-capping mechanism by which the clam-shaped structure forms double-headed filaments to protect the NDV viral genome in vivo. Overall, I think the studies are well designed and the authors uncovered a novel architecture of N that has not been described before for any negative-sense RNA viruses. This model has the potential to stimulate further future studies.

Reviewer #2:

This study reports the roughly 5Å structure of a "clam shaped" Newcastle disease virus nucleocapsid by cryo-EM. The clam shape derives from the juxtaposition of 2 NP-RNA complexes packing in a "back to back" manner. This arrangement would juxtapose the 5' end of the 2 encapsidated RNAs. The structure reveals that a loop comprising residues 114-120 participates in hydrogen bonding interactions with the equivalent loop in the back to back arrangement of the two N-RNA spirals. Mutation of all of the residues in that loop results in a defect in a cell based minigenome assay of RNA synthesis, and the bacterial expressed mutant appears distinct by negative-stain EM forming more filaments and less rings. Proteolysis and RNase sensitivity assays indicate the loop mutant produces a distinct pattern of cleavage and the RNA is more sensitive to digestion.

The structural observations of this study are new, and differ from prior work on other nonsegmented negative-sense RNA virus nucleocapsids. The significance of the structural observations is not clear from the manuscript. The manuscript also does an inadequate job of connecting the structures with the biology of negative-strand RNA virus nucleocapsids.

1) The conclusion that NDV N assembles into a clam shaped dimer is based on the isolation of 200Å diameter round-shaped particles. The dimensions of those particles are similar to the ring-like N-RNA structures attained for several of the Mononegavirales. What fraction of the round-shaped particles are closed discs as seen for other viruses vs the clam shaped structures?

2) The initial clam shaped structure was at 6.4Å resolution with obvious C2 rotational symmetry. Into this structure the authors dock an NP protomer structure. The NP structure was based on homology modeling with that of PIV5 NP which shares 40% sequence identity. Docking of that protomer into the density map shows that the protomers either side of the "seam" have the lowest resolution. This arrangement leads the authors to a model that the junction comprises two NP molecules that are each bound to the 5' end of an RNA. The best evidence for this arrangement is provided by protomers that are, however, furthest away from the seam. What is the evidence that the RNA is fully coated by NP? Could this arrangement reflect interactions of unencapsidated RNA that helps bring the two molecules together? The authors must determine the length of RNAs that are fully coated directly.

3) Have the authors separately fitted the two lobes of NP into their density map? This would be of particular interest around the "seam" where the resolution is lowest.

4) The conclusion that viral genome replication is dependent on a clam-shaped nucleocapsid reflects a significant over-interpretation of the data at hand.

In Figure 3, the authors perform a minigenome assay to determine whether there are functional consequences for RNA synthesis for the loop mutant. The readout of this assay is GFP expression which indirectly reflects transcription of mRNA from the minigenome, which indirectly reflects encapsidation of the RNA. The mutant could however, encapsidate the template perfectly well, but be defective in recruitment of the polymerase to the template, or could alter polymerase processivity – both of which would affect reporter gene activity. However, the authors interpret this to claim that the clam shaped nucleocapsid is required for genome replication.

A second issue relates to the evidence that the mutation of the loop disrupts the production of "clam-shaped" structures. The negative-stain EM of Figure 3E is offered as evidence that the loop mutant can form filaments, but we have no idea whether those structures bind RNA.

A more rigorous analysis of the RNA bound, and dissection of the step at which the NP mutant affects gene expression is needed to support the conclusion that the clam-shape is required for replication.

5) In Figure 4E the authors provide evidence that the NLoop mutant is more sensitive to nuclease attack. It is curious that the extent of NP-RNA disassembly is unaltered at 90s but is affected at 180s. What is the authors explanation for this? Importantly, the values are expressed as% of remaining particles. How where these particles quantified? Were the equivalent number of like particles tested for Nloop vs Nwt? The description in the Materials and methods is inadequate.

Reviewer #3:

In this manuscript, authors use cryoEM to characterize the N protein from NDV. negative sense RNA viral nucleoproteins (N or NP) are critical for several different steps in viral replication cycle and the current structure at 4.8 A (nominal resolution) provides structural insights into the role of NP-NP interactions. Structure derived mutations show loss of function.

Overall the study provides important structural insights. That said, there are many questions that are not addressed in the manuscript with regard to biology. The structure is derived from in vitro purified samples. Loss of function is easier to generate, but hard to interpret. Thus, the authors could/should think about how best to position their work in the context of the biological processes that N protein plays roles in. For example, in Figure 3 D vs E, no biochemial difference, some difference in the micrographs and a large different in the minigenome. Why? what are the biological consequences.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Self-capping of nucleoprotein filaments protects Newcastle Disease Virus genome" for further consideration at eLife. Your revised article has been favorably evaluated by Detlef Weigel (Senior Editor), a Reviewing Editor, and two reviewers.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

There are a few areas where the interpretation, conclusions and writing can be improved as outlined in reviewer 2's comments. We feel that this will significantly improve the manuscript.

Reviewer #1:

I am satisfied by the authors' responses and the revised manuscript. In particularly, the authors have made an effort to isolate native NPs and revised Figure 1—figure supplement 4. It is understandable that such effort is very laborious and not very fruitful, as also documented in the old literature of this and related viruses.

Reviewer #2:

This is a revised manuscript that reports the structure of a clam-shaped NDV nucleocapsid. The functional significance of the clam-shape remains to be formally demonstrated, but the structure itself is interesting and suggests a potential – although untested – model for the process of nucleocapsid assembly.

In revising this work the authors provide new images of the native RNPs – which responds to one of the two main criticisms. The authors state "it is extremely time challenging and time consuming to obtain the nucleoprotein with clam-like structure from the Newcastle disease virus because such kind of nucleoprotein is in very low abundance in the virion." This should also further caution the authors to not over interpret the functional relevance of the structure – who focus a good deal of their response and discussion on polyploid virions. This reviewer found the structure provocative and wondered whether the true relevance of the structure relates to "seeding" of the correct assembly of the nucleocapsid during RNA replication with the net result that on occasion 2 copies of the genome end up in the virion.

There remain some serious overstatements of the results in the title and Abstract and some other inaccuracies in the text.

1) The evidence that the self-capping protects the genome from RNAse is weak. As pointed out in the first review a more rigorous analysis of the bound RNA is needed to make such a conclusion. As it stands, the present data do not tell us anything about the bound RNA itself. Absent this knowledge it is a major over-interpretation that the clam protects the RNA from nuclease digestion. The data of Figure 4E do not look at the RNA, they look only at the presence of clam shapes. Thus it is a significant over interpretation of the data.

2) The statement in the Abstract "Uncovering the helical assembly mechanism of the negative-strand RNA virus will help the development of new antiviral therapies" is unclear and is entirely speculation and should be removed. Similarly the last sentence of the Abstract.

3) Throughout the text the authors correctly state that the nucleocapsid is used for replication and transcription. The polymerase displaces the nucleocapsid protein transiently during transcription and replication. Transcription precedes replication and is hence usually referred to first. It is incorrect to state that the RdRP moves across the nucleocapsid for viral translation (Introduction first paragraph).

4) What is "frontal attack" in the final paragraph of the Introduction? The authors should include some explanation.

https://doi.org/10.7554/eLife.45057.029

Author response

Essential revisions:

1) The essential revision is to provide more convincing native RNP images and analysis of those structures.

We thank the reviewers and editors for the opportunity to resubmit a revised version of our manuscript by Song et al. entitled “Self-capping of nucleoprotein filaments protects Newcastle Disease Virus genome”. All authors of this manuscript have carefully discussed the questions raised by the reviewers, and we are trying to address all the questions and hope that our revised manuscript will be accepted for publication at eLife.

We agree that the most convincing way is to show the clam-like structure of nucleoprotein complex from the native virus. So, we have attempted to obtain more electron microscopic images of the nucleoprotein-RNA complex purified from the newcastle disease virus propagated in 9-day-old embryonated chicken eggs. We have also obtained more negative stain EM images of the nucleoprotein-RNA complex and added them to Figure 1—figure supplement 4. Please note that it is extremely challenging and time consuming to obtain the nucleoprotein with clam-like structure from the newcastle disease virus because such kind of nucleoprotein is in very low abundance in the virion. We should also point out that several previous reports showed that the nucleocapsids in newcastle disease virus are more prone to be disrupted during purification than those of Hemagglutinating virus of Japan (Hosaka et al., J.Mol.Biol., 1968). This fact added extra hurdles to our effort on obtaining enough samples for the cryo-electron microscopy analysis.

In summary, we managed to obtain the negative-stain images of nucleoprotein filaments from the newcastle disease virus and we could identify the self-capping filaments which is in similar shape as that of overexpressed nucleoprotein filaments observed by electron microscopy. The new negative-stain images have been added into Figure 1—figure supplement 4.

2) The functional studies are over-interpreted, so those studies should be removed from the manuscript. The least would be to tone down the conclusions because of the reasons outlined below.

We have modified and toned down the interpretation of the minigenome studies for verifying the function of the clam-like structure in minigenome description and discussion sections of the manuscript.

Reviewer #2:

[…] The structural observations of this study are new, and differ from prior work on other nonsegmented negative-sense RNA virus nucleocapsids. The significance of the structural observations is not clear from the manuscript. The manuscript also does an inadequate job of connecting the structures with the biology of negative-strand RNA virus nucleocapsids.

We have added the importance and implications of our work to further understanding the function of the NP in the first three paragraphs of the Discussion section of the manuscript as suggested.

1) The conclusion that NDV N assembles into a clam shaped dimer is based on the isolation of 200Å diameter round-shaped particles. The dimensions of those particles are similar to the ring-like N-RNA structures attained for several of the Mononegavirales. What fraction of the round-shaped particles are closed discs as seen for other viruses vs the clam shaped structures?

Many crystal structures of nucleoprotein-RNA complex, such as rabies virus, Respiratory Syncytial Virus, Vesicular stomatitis virus and paramyxovirus parainfluenza virus 5 are ring-like structures with around 200 Å diameter. There are both round shaped and filament shaped nucleocapsids in our NDV N samples after the sucrose gradient centrifugation separation. We managed to determine both shaped components. However, the structure of round shaped sample is the clam shaped nucleoprotein-RNA complex, while the structure of the filament sample is the clam shaped structure with the helical nucleoprotein-RNA complex extending out from the clam shaped structure. There is no closed ring like structure observed in our study.

2) The initial clam shaped structure was at 6.4Å resolution with obvious C2 rotational symmetry. Into this structure the authors dock an NP protomer structure. The NP structure was based on homology modeling with that of PIV5 NP which shares 40% sequence identity. Docking of that protomer into the density map shows that the protomers either side of the "seam" have the lowest resolution. This arrangement leads the authors to a model that the junction comprises two NP molecules that are each bound to the 5' end of an RNA. The best evidence for this arrangement is provided by protomers that are, however, furthest away from the seam. What is the evidence that the RNA is fully coated by NP? Could this arrangement reflect interactions of unencapsidated RNA that helps bring the two molecules together? The authors must determine the length of RNAs that are fully coated directly.

The density map of the RNA strand can be recognized and traced clearly in the clam shaped map (Figure 1—figure supplement 3), whose EMDB ID is EMD-9793.

The seam between the two single spirals is about 6 nm and there is no density for the unencapsidated RNA. Thus we are unable to conclude if unencapsidated RNAs bring the two molecules together, unfortunately.

The clam like structures is not homogeneous in length and as result, the density at far end of filament is smeared (so a soft mask was applied to get a high resolution by averting the influence of the smear density), so it is almost impossible to determine the 3’ end of the RNA strand.

3) Have the authors separately fitted the two lobes of NP into their density map? This would be of particular interest around the "seam" where the resolution is lowest.

Yes, we fitted the nucleoprotein subunit structure of the parainfluenza virus 5 (PDB ID 4XJN) to the clam shaped structure of the NDV separately. We can recognize the subunit of the clam shaped structure clearly even at the regions near the “seam” of the nucleoprotein-RNA complex.

4) The conclusion that viral genome replication is dependent on a clam-shaped nucleocapsid reflects a significant over-interpretation of the data at hand.

Agreed, we have modified and toned down the interpretation of the minigenome studies for verifying the function of the clam-like structure in the minigenome description section and discussion section of the manuscript.

In Figure 3, the authors perform a minigenome assay to determine whether there are functional consequences for RNA synthesis for the loop mutant. The readout of this assay is GFP expression which indirectly reflects transcription of mRNA from the minigenome, which indirectly reflects encapsidation of the RNA. The mutant could however, encapsidate the template perfectly well, but be defective in recruitment of the polymerase to the template, or could alter polymerase processivity – both of which would affect reporter gene activity. However, the authors interpret this to claim that the clam shaped nucleocapsid is required for genome replication.

Agreed, we have modified the interpretation of the minigenome studies as suggested.

A second issue relates to the evidence that the mutation of the loop disrupts the production of "clam-shaped" structures. The negative-stain EM of Figure 3E is offered as evidence that the loop mutant can form filaments, but we have no idea whether those structures bind RNA.

The single headed filament with the loop mutant contains RNA because of the A260/A280 value of which is about 1.05, which is measured by the Nanodrop instrument.

A more rigorous analysis of the RNA bound, and dissection of the step at which the NP mutant affects gene expression is needed to support the conclusion that the clam-shape is required for replication.

Yes, agreed. However, we can’t finish the experiments of dissection of the NP mutants affecting gene expression in cell level within two months, which means that we are not sure if the NP mutant affected replication or transcription, so we toned down the interpretation of the minigenome assay by stating that the NP mutant affected the function of nucleoprotein-RNA complex.

5) In Figure 4E the authors provide evidence that the NLoop mutant is more sensitive to nuclease attack. It is curious that the extent of NP-RNA disassembly is unaltered at 90s but is affected at 180s. What is the authors explanation for this? Importantly, the values are expressed as% of remaining particles. How where these particles quantified? Were the equivalent number of like particles tested for Nloop vs Nwt? The description in the Materials and methods is inadequate.

Both the wild type and NLoop mutant are about 25% remained at the 90s point after RNase digestion, while the NLoop mutant samples are almost 100% disassembled and 25% of wild type samples remained intact, which means that the samples were digested gradually by the RNase and the wild type samples are more stable than NLoop mutant samples

Both the clam shaped particles (rounded shape particles) and the filament samples were counted. we described this in Materials and methods section “In nuclease cleavage assay, the number of particles of both clam-shaped structures and filaments of NWT and NLoop were counted at different timepoints such as 0s, 90s and 180s.”

Reviewer #3:

In this manuscript, authors use cryoEM to characterize the N protein from NDV. negative sense RNA viral nucleoproteins (N or NP) are critical for several different steps in viral replication cycle and the current structure at 4.8 A (nominal resolution) provides structural insights into the role of NP-NP interactions. Structure derived mutations show loss of function.

Overall the study provides important structural insights. That said, there are many questions that are not addressed in the manuscript with regard to biology. The structure is derived from in vitro purified samples. Loss of function is easier to generate, but hard to interpret. Thus, the authors could/should think about how best to position their work in the context of the biological processes that N protein plays roles in. For example, in Figure 3 D vs E, no biochemial difference, some difference in the micrographs and a large different in the minigenome. Why? what are the biological consequences.

For the Figure 3D and E, though both the NWT and NLoop are eluted similarly in the void volume of the size exclusion chromatography, yet their three dimensional structures of the NWT and NLoop are different. The NWT has clam-like structure while the NLoop shows single-headed spiral as shown in the Cryo-EM images (Figure 2A and Figure 3C). Thus, NWT and NLoop may function differently in the minigenome assay. The NWT succeeds in the transcription and translation for the target GFP gene, while the NLoop fails to transcribe or translate the GFP gene, for reasons, such as the NLoop can’t keep the genome integrity, or the NLoop affects the formation of N-RNA-RdRp complex by RdRp binding to the nucleocapsid to start the expression of target GFP. But the elaborate mechanism of the NLoop above remains to be illuminated. We also added this relative issue in the discussion section of this manuscript.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Reviewer #2:

This is a revised manuscript that reports the structure of a clam-shaped NDV nucleocapsid. The functional significance of the clam-shape remains to be formally demonstrated, but the structure itself is interesting and suggests a potential – although untested – model for the process of nucleocapsid assembly.

In revising this work the authors provide new images of the native RNPs – which responds to one of the two main criticisms. The authors state "it is extremely time challenging and time consuming to obtain the nucleoprotein with clam-like structure from the Newcastle disease virus because such kind of nucleoprotein is in very low abundance in the virion." This should also further caution the authors to not over interpret the functional relevance of the structure – who focus a good deal of their response and discussion on polyploid virions. This reviewer found the structure provocative and wondered whether the true relevance of the structure relates to "seeding" of the correct assembly of the nucleocapsid during RNA replication with the net result that on occasion 2 copies of the genome end up in the virion.

Agreed, the clam shaped structure of the nucleoprotein-RNA complex has never been reported before, as far as we know, thus we know very little about its functional indications. Due to the limitation of the scope of this manuscript, we have to make extra effort to address the new emerging questions, including the reviewer’s concerns, and hopefully, we can publish the follow up results in the future. However, we’d like to share the current discoveries with the community and speculate the functional influence of the clam shaped structure of the nucleoprotein.

There remain some serious overstatements of the results in the title and Abstract and some other inaccuracies in the text.

1) The evidence that the self-capping protects the genome from RNAse is weak. As pointed out in the first review a more rigorous analysis of the bound RNA is needed to make such a conclusion. As it stands, the present data do not tell us anything about the bound RNA itself. Absent this knowledge it is a major over-interpretation that the clam protects the RNA from nuclease digestion. The data of Figure 4E do not look at the RNA, they look only at the presence of clam shapes. Thus it is a significant over interpretation of the data.

Disagree, Figure 4E shows the obvious difference on RNase A digesting NWT and NLoop at different timepoints, where NWT rather than NLoop protects the clam-shaped structure after the RNase A digestion. Also, as matter of fact, the NWT contained RNA because of the A260/A280 value was about 0.9, while the NLoop didn’t contain RNA due to its low A260/A280 value of about 0.6 after RNase A digestion. In order to make it more clear, we added the following sentence "Meanwhile, the NWT rather than the NLoop contained RNA with an absorbance of A260/A280 ∼0.9, while that of NLoop was ∼0.6".

2) The statement in the Abstract "Uncovering the helical assembly mechanism of the negative-strand RNA virus will help the development of new antiviral therapies" is unclear and is entirely speculation and should be removed. Similarly the last sentence of the Abstract.

We deleted the sentence "Uncovering the helical assembly mechanism of the negative-strand RNA virus will help the development of new antiviral therapies" and the last sentence of the Abstract.

3) Throughout the text the authors correctly state that the nucleocapsid is used for replication and transcription. The polymerase displaces the nucleocapsid protein transiently during transcription and replication. Transcription precedes replication and is hence usually referred to first. It is incorrect to state that the RdRP moves across the nucleocapsid for viral translation (Introduction first paragraph).

We deleted the word " translation " in the sentence " …the RdRp moves across the nucleocapsid for viral transcription and translation ".

4) What is "frontal attack" in the final paragraph of the Introduction? The authors should include some explanation.

We changed the "frontal attack" to "digestion by proteases".

https://doi.org/10.7554/eLife.45057.030

Article and author information

Author details

  1. Xiyong Song

    1. Institute of Molecular and Clinical Medicine, Kunming Medical University, Kunming, China
    2. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
    3. College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China
    Contribution
    Conceptualization, Software, Formal analysis, Validation, Investigation, Methodology, Writing—original draft, Writing—review and editing
    Contributed equally with
    Hong Shan and Yanping Zhu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5088-3783
  2. Hong Shan

    iHuman Institute, ShanghaiTech University, Shanghai, China
    Contribution
    Software, Formal analysis, Validation, Methodology, Writing—original draft
    Contributed equally with
    Xiyong Song and Yanping Zhu
    Competing interests
    No competing interests declared
  3. Yanping Zhu

    National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
    Contribution
    Conceptualization, Formal analysis, Validation, Investigation, Methodology
    Contributed equally with
    Xiyong Song and Hong Shan
    Competing interests
    No competing interests declared
  4. Shunlin Hu

    College of Veterinary Medicine, Yangzhou University, Yangzhou, China
    Contribution
    Formal analysis, Validation, Investigation, Methodology
    Competing interests
    No competing interests declared
  5. Ling Xue

    College of Veterinary Medicine, Yangzhou University, Yangzhou, China
    Contribution
    Validation, Investigation, Methodology
    Competing interests
    No competing interests declared
  6. Yong Chen

    1. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
    2. College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  7. Wei Ding

    Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
    Contribution
    Software, Formal analysis, Validation
    Competing interests
    No competing interests declared
  8. Tongxin Niu

    Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
    Contribution
    Software
    Competing interests
    No competing interests declared
  9. Jian Gu

    College of Veterinary Medicine, Yangzhou University, Yangzhou, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  10. Songying Ouyang

    1. The Key Laboratory of Innate Immune Biology of Fujian Province, Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, China
    2. Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, College of Life Sciences, Fujian Normal University, Fuzhou, China
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Project administration
    For correspondence
    ouyangsy@fjnu.edu.cn
    Competing interests
    No competing interests declared
  11. Qing-Tao Shen

    iHuman Institute, ShanghaiTech University, Shanghai, China
    Contribution
    Software, Formal analysis, Funding acquisition, Validation, Methodology, Writing—original draft
    For correspondence
    shenqt@shanghaitech.edu.cn
    Competing interests
    No competing interests declared
  12. Zhi-Jie Liu

    1. Institute of Molecular and Clinical Medicine, Kunming Medical University, Kunming, China
    2. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
    3. iHuman Institute, ShanghaiTech University, Shanghai, China
    Contribution
    Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Methodology, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    liuzhj@shanghaiTech.edu.cn
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7279-2893

Funding

National Natural Science Foundation of China (31330019)

  • Zhi-Jie Liu

National Natural Science Foundation of China (31770948)

  • Songying Ouyang

National Natural Science Foundation of China (31570875)

  • Songying Ouyang

National Natural Science Foundation of China (81590761)

  • Songying Ouyang

Yunnan Provincial Science and Technology Department (2016FC007)

  • Zhi-Jie Liu

Pujiang Talent Program (17PJ1406700)

  • Qing-Tao Shen

National Key R&D Programme of China (2017YFA0504800)

  • Qing-Tao Shen

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was supported by the National Nature Science Foundation of China (grants 31330019 (ZJL), 31770948, 31570875 and 81590761 (SO)), the National Key R&D program of China (2017YFA0504800 (QS)), Yunnan Provincial Science and Technology Department Project (2016FC007) and the Pujiang Talent program (17PJ1406700 (QS)). The Cryo-EM work was performed at the Center for Biological Imaging (CBI), Institute of Biophysics, Chinese Academy of Sciences and the EM facility of the National Center for Protein Science Shanghai (NCPSS). We would like to thank Fei Sun, Gang Ji, Xiaojun Huang, Zhenxi Guo, Deyin Fan, Bolin Zhu, and Shuoguo Li from the CBI, Institute of Biophysics, Chinese Academy of Science (CAS) for helping with EM sample preparation and data collection. We are also grateful to Mi Cao, Liangliang Kong and Junrui Li from the EM facility of NCPSS for cryo-EM data collection. We also thank Baidong Hou at the Institute of Biophysics, CAS for sharing Qβ virus-like particles.

Senior Editor

  1. Detlef Weigel, Max Planck Institute for Developmental Biology, Germany

Reviewing Editor

  1. David M Knipe, Harvard Medical School, United States

Reviewer

  1. Z Hong Zhou, University of California, Los Angeles, United States

Publication history

  1. Received: January 10, 2019
  2. Accepted: July 9, 2019
  3. Accepted Manuscript published: July 10, 2019 (version 1)
  4. Version of Record published: August 1, 2019 (version 2)

Copyright

© 2019, Song et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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