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. 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)  mediates the ability of RdRp to access nucleoprotein, and the RdRp moves across the nucleocapsid for viral transcription (Members of the order us (RSV) . Mononeggenicity . One remgenicity . During genicity . In Paracription . RNA is cription .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)  . RNP canex (RNP) . In RNP ex (RNP) .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.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 . The mecEscherichia 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  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 . Furtheri 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  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  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  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 Glysinresidues . The Loofication . Direct ormation .114–120 is functionally relevant in vivo, minigenome analyses of several N mutants , 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  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  is not easily recognized in the EM map because of the long, intrinsically flexible domain reported in other structures (3 at the threshold of 0.0054) of the density map, which are apparently from the C-tail  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 . The traEscherichia 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  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 ) 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  preequilibrated with TRIS buffer at pH 8.0 . The target proteins with endogenous RNA were collected for the following experiments.NDV N and its derived mutants were expressed in 4 g and 4°C with an SW40 rotor (Beckman). The samples were collected by puncturing the tube and dialyzing in the TRIS buffer.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 × 10Grids of N or its mutants for negative-stain EM were prepared as described previously . SpecifiTo 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  with a thin layer of continuous carbon film. The grids were blotted using a Vitrobot Mark IV  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.2 electron microscope equipped with Cs corrector  and a K2 Summit direct electron detector , 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  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.Elastase and RNase A were selected to test the susceptibility of the 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.NFor 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.WT and NLoop were counted at different timepoints such as 0 s, 90 s and 180 s. A total of 120 micrographs were statistically counted.In nuclease and elastase cleavage assay, the number of particles of both clam-shaped structures and filaments 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.The samples of NNDV minigenome p-LGT and three helper plasmids pCI-N, pCI-P and pCI-L from the NDV strain ZJ1 were constructed by Loop, 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 (Different mutant and truncated versions of N were cloned into the pCI-neo plasmid (Promega) and designated the names Neviously . Briefly4 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.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  . The NDVThe 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. 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.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).Thank you for submitting your article "Self-capping of nucleoprotein filaments protects Newcastle Disease Virus genome" for consideration by 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) , 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 . 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.Mononegavirales. What fraction of the round-shaped particles are closed discs as seen for other viruses vs the clam shaped structures?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 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  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.eLife. Your revised article has been favorably evaluated by Detlef Weigel (Senior Editor), a Reviewing Editor, and two reviewers.Thank you for resubmitting your work entitled "Self-capping of nucleoprotein filaments protects Newcastle Disease Virus genome" for further consideration at 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. Essential revisions:1) The essential revision is to provide more convincing native RNP images and analysis of those structures.eLife.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 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 . 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 , 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.260/A280 value of which is about 1.05, which is measured by the Nanodrop instrument.The single headed filament with the loop mutant contains RNA because of the AA 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.Loop 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 samplesBoth the wild type and NWT and NLoop were counted at different timepoints such as 0s, 90s and 180s.”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 NReviewer #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  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.WT 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 . 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.For the Figure 3D and E, though both the NReviewer #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.WT 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".Disagree, Figure 4E shows the obvious difference on RNase A digesting NWT and NLoop at different timepoints, where N2) 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".


Bivalve mollusks thrive in environments rich in microorganisms, such as estuarine and coastal waters, and they tend to accumulate various particles, including viruses. However, the current knowledge on mollusk viruses is mainly centered on few pathogenic viruses, whereas a general view of bivalve-associated viromes is lacking. This study was designed to explore the viral abundance and diversity in bivalve mollusks using transcriptomic datasets. From analyzing RNA-seq data of 58 bivalve species, we have reconstructed 26 nearly complete and over 413 partial RNA virus genomes. Although 96.4% of the predicted viral proteins refer to new viruses, some sequences belong to viruses associated with bivalve species or other marine invertebrates. We considered short non-coding RNAs (sncRNA) and post-transcriptional modifications occurring specifically on viral RNAs as tools for virus host-assignment. We could not identify virus-derived small RNAs in sncRNA reads obtained from the oyster sample richest in viral reads. Single Nucleotide Polymorphism (SNP) analysis revealed 938 A-to-G substitutions occurring on the 26 identified RNA viruses, preferentially impacting the AA di-nucleotide motif. Under-representation analysis revealed that the AA motif is under-represented in these bivalve-associated viruses. These findings improve our understanding of bivalve viromes, and set the stage for targeted investigations on the specificity and dynamics of identified viruses. Mimiviruses genomes being in the order of megabases and far exceeding the few kilobases of circular single stranded DNA genomes of cress viruses [Viruses are the most abundant biological entity on the Earth, likely outnumbering bacteria and eukaryotic cells , with th viruses ,8. The p viruses , althoug viruses . In fact viruses  as they  viruses . The gen viruses . Convers viruses ,15,16,17 viruses . Thanks  viruses ,20, alth viruses . Neverth viruses .Ostreid herpesvirus-1, OsHV-1) associated with temperature-related oyster mortalities [Papovaviridae and Iridoviridae families have been associated with bivalve diseases, whereas a few members of the Togaviridae, Reoviridae, Birnaviridae, and Picornaviridae virus families have been reported without evidence of associated disease [Although it is steadily expanding, virus discovery and the study of antiviral immunity in invertebrates is biased towards a few model organisms and arthropods of economic and medical importance . In thistalities  has becotalities ,36,37,38 disease ,40. Unti disease .Crassostrea gigas and Mytilus galloprovincialis growing up in association [Virome discovery through RNA HTS is challenging when applied to bivalve samples. According to the ability to detect minute quantities of viral nucleic acids, HTS also catches sequences that possibly derive from tissue surface contamination, or from the simple transit of another virus host in bivalve tissues ,43. The ociation , exempliThe overall analysis pipeline is summarized in Bathymodiolus platifrons, C. virginica, Mizuhopecten yessoensi, Modiolus philippinarum, and M. galloprovincialis) were downloaded from the NCBI WGS database, while C. gigas and Pinctada fucata genomes were obtained from EnsembleMetazoa release35 and from [Cytochrome C Oxidase Subunit I (COI) sequences were downloaded from the NCBI nucleotide archive, and their redundancies were reduced using cd-hit-est [C. gigas sample .Public sequence datasets were retrieved from NCBI databases in April 2017. A total of 7125 viral genomes, including 3008 RNA viruses, were downloaded from the NCBI Genome database. Additionally 1102 invertebrate-associated RNA virus genomes were downloaded from the NCBI nucleotide database ,47 for aand from , respect-hit-est , applyinE-value of 10−5.RNA-seq reads were trimmed for the presence of adaptor sequences, and for quality, using TrimGalore! , allowinE-value 10−50). Moreover, all the bivalve-derived protein sequences encoding a viral RNA-dependent RNA-polymerase (vRdRp) domain were selected. A vRdRp was identified by using six different PFAM Hidden Markov Models, corresponding to IDs: PF00680, PF00978, PF00998, PF02123, PF07925, and PF04197. Bivalve genome scaffolds were used to compose a genomic database to discriminate between host-encoded and viral sequences. Sequences identified from the redundant protein database, and from the search of the vRdRp domains, and showing no matches against bivalve genomes, were further processed to reduce the redundancy, applying a cut-off of 90% of similarity (cd-hit). The resulting protein sequences were used to recover the corresponding nucleotidic contigs from the initial transcriptome assemblies and they were considered as complete or partial genomes of RNA viruses. For the purposes of this paper, a viral genome was considered to be “nearly complete” if it was composed of a unique contig that was longer than 5 kb and encoding at least one complete ORF.A redundant BLAST database  was builC. gigas gene models [The amount of reads mapping to the “nearly complete” viral genomes in selected RNA-seq samples was determined by stringent mapping of the trimmed reads on the viral genome sequences . For a selection of informative RNA-seq samples, the total read counts were used to calculate the percentage of reads mapping to each virus over the total numbers of reads of the sample, thus providing a comparison of the amount of viral RNA between RNA-seq samples that was not biased by different sequencing depths or read layouts. To obtain the expression profiles of selected oyster RNAi-related genes, 183 RNA-seq datasets  were mape models  and usede models . RNA-seqTo provide an estimation of the fraction of reads that were not related to the declared biological sample (as indicated in the SRA details), we mapped the RNA-seq reads onto a collection of 205,357 non-redundant COI sequences. Reads were mapped applying a similarity fraction of 0.8, over 0.8 of the read length (CLC mapper tool) and the TPM values were computed. Similarly, to estimate the presence of known viruses among the RNA-seq datasets, the amount of reads mapping to the 7125 virus genomes obtained from NCBI was also computed .C. gigas sample that was used to prepare the paired polyA and ribo-depleted libraries were extracted using the Mirvana kit . RNA was quantified by using a Qubit fluorimeter instrument, and the RNA size profile was determined with an Agilent small RNA chip . Library preparation and sequencing (PE150) was outsourced and carried out on an HiSeq Illumina platform , and submitted to the NCBI SRA archive, under the accession ID SRR8587800. The paired reads were trimmed for quality, and for the presence of adaptors, as described for mRNA reads, and the correctly paired reads were joined into fragments. The resulting clean fragments, in a length range of 15–50 nt, were used for the detection of viral-derived small RNAs (vsRNAs) by direct mapping on the identified viral contigs or by using the VirusDetect pipeline [The fraction of small RNAs (<200 bp) of the pipeline . To discp-value. A sequence with a p-value of <0.05 is said to be under-represented in that motif metric, whereas a p-value of >0.95 is said to be over-represented in that motif metric . To look for these adaptive genome changes, we used the cytidine deaminase under-representation reporter (CDUR) . Given ture 2 in ).A particularly interesting case is when, for a given motif, a sequence is under-represented in the “below” metric, and is over-represented in the “repTrFrac” metric for that motif. This suggests that this sequence has maximally tried to reduce the number of occurrences of that motif, as any further reduction would result in amino acid changes, which may negatively impact that coding sequence. Recent studies have shown that certain gammaherpesvirus may be under such pressures . In thisProtein sequences referring to vRdRP domains were aligned using MUSCLE , and treRNA-dependent RNA polymerase (vRdRp). In the absence of a conserved viral gene, we used these domains to identify RNA viruses, since vRdRp is needed for the transcription of the viral genome during productive virus replication [E-value of 10−50, and to further limit false positive results, we discharged the BLAST matches with less than five hits. Moreover, we screened all of the positive hits against a database composed of available genomic scaffolds of bivalve species, to remove genomically encoded sequences , or obtained from geographically unrelated samples of the same species, although few exceptions are present, and they are discussed below. Despite most sequences being retrieved by BLAST searches using viral sequences as queries, they showed a limited similarity to known viruses , namely, six bivalve-associated RNA viruses from the lagoon of Goro , two viruses associated with marine invertebrates from China, plus three other RNA viruses, the algal Heterosigma akashiwo RNA virus, the plant virus Zygocactus virus X, and the Sacbrood virus . To identify the putative virus sequences associated with the analyzed RNA-seq samples from different bivalve species, we queried biv_aa with 9376 protein sequences belonging to 4110 known RNA virus genomes . We extrlication ,64. For equences . As a re domains . The rem viruses . We coulod virus .C. gigas specimen naturally infected with OsHV-1, using the same two RNA selection methods (sample ID: SAMN09760011). The analysis of the four meta-transcriptomic samples showed that, despite a considerable variability in the numbers of raw reads, the assembled contigs, as well as the number of predicted proteins yielded somewhat comparable values, except for SRR3401755, for which only few proteins could be predicted. For these datasets, polyA-selection allowed for the recovery of a higher ratio of viral to total proteins, particularly for the bivalve samples . We ana samples . On the C. gigas, M. galloprovincialis, Ruditapes philippinarum, and M. edulis), whereas the unique viral contig found in the Elliptio complanata transcriptome , E. complanata (1), M. galloprovincialis (1), Mizuhopecten yessoensis (1) and R. philippinarum (1)). In three viral genomes  we could identify a polyA tail at the 3’ end of the sequence  with the median lengths of the known RNA virus genomes (4.8 kb), a realistic assumption is that most of these represent incomplete genomes. For the purpose of this paper, we considered “nearly full-length viral genomes” as only being contigs that are longer than 5 kb and encoding at least one complete ORF. Therefore, we identified 26 contigs ranging in length between 5.4 and 9.7 kb as being “nearly complete viral genomes”, with 12 contigs encoding two ORFs corresponding to one replicative and one structural protein, while the other contigs (14) encoded a single ORF. These “nearly complete” viruses were named according to the species from which they were assembled  RNAs, we included additional 24 RNA-seq samples in the distribution analysis, for a total of 250 datasets  and Bivalve RNA virus G4  were mapped to this virus, whereas, only 22 viral reads (<0.00001‰) were detected in the paired control , a “mix of gastropods” (Ribo-0 and polyA), one P. olseni, four R. decussatus, and seven R. philippinarum samples, since they included reads of multi-host viruses , where we detected several contaminant species . We noted the presence of a known bivalve-associated tunicate (Diplosoma listerianum) in 10 of the tested RNA-seq samples. Although the D. listerianum COI value is equal to 100% in the P. olseni sample, due to the absence of the Alveolata entries in the COI dataset, our analysis confirmed the absence of clam RNAs in the Perkinsus samples, including a high level of Rudphi_virus3. Intriguingly, both the Ribo-0 and polyA S15 datasets showed a low contamination of Lacconectus peguensis (Coleoptera).We mapped the reads of 16 selected RNA-seq samples to a collection of COI gene sequences, namely,  viruses . We calc viruses . ObviousOur analysis further demonstrated that most of the transcriptome-derived viruses could be only tentatively assigned to a specific host, due to their occurrence in samples of even phylogenetically distant species. Under this context, the application of coverage cut-offs appeared to be an unreliable approach for host-assignment. Therefore, we investigated the feasibility of two alternative approaches for the host-assignment of bivalve-associated viruses obtained from transcriptomic data, as follows.DICER, DROSHA, ARGONAUTE, PIWI, and RNA-dependent RNA polymerase) in 184 C. gigas RNA-seq samples, including some samples that were very rich in viral reads (RNA-dependent RNA polymerase transcript (EKC38952), belonging to a gene family typically expressed in the digestive gland, showed considerable expression levels in a few other samples, namely two out of three biological replicates of oysters infected with OsHV-1  and a spat sample highly infected by the same virus , suggesting that the reads mostly originated from RNA degradation products, instead of being genuine vsRNAs. We further analyzed the coverage of the sncRNA reads along the five “nearly complete viral genomes” originating from the S15 oyster ribo-depleted data. To do this, we mapped to the viral genomes the sncRNA reads that did not match to the oyster genome (1.436 M reads), and we calculated the size profiles of each of the mapped subsets (C. gigas sncRNA reads peaked at 21 nt (microRNAs), whereas the unmapped sncRNA reads peaked at 30 nt, indicative of their Piwi-interacting RNA (piRNA) nature. However, only 199 sncRNA reads mapped to one of the five viral genomes reconstructed by using the paired Ribo-depleted RNA-seq reads, and the size profiles showed a low enrichment of 29–30 nt reads with a distribution over the whole viral genome (The first approach investigates the presence of virus-derived RNAi products (vsRNAs), and it is used to reconstruct full-length genomes of viruses infecting arthropods ,66,67. Sal reads , to corral reads . We showal reads , and PIWal reads . Apart fal reads . Althougme virus . Taken tuses see . Small n subsets . Notablyl genome .Moreover, we subjected the sncRNA reads to VirusDetect, a bioinformatics pipeline that is designed for the identification and reconstruction of viral genomes starting from short reads . Althougadenosine deaminase acting on dsRNA (ADAR), which is assumed to specifically modify viral dsRNAs through A-to-I editing [The second approach that we tested leveraged on the identification of single-nucleotide modifications (SNPs) occurring specifically on viral transcripts produced by the action of host enzymes acting as antiviral defenses. Therefore, we attempted to select and count the subset of total SNPs generated by the host double-stranded RNA (dsRNA) editor enzyme  editing . For eac editing  . None of our viruses grouped in clusters were characterized by the presence of abundant vertebrate viruses, while Mytcor virus1 was grouped with plant viruses, supporting its BLASY similarity to Pitaya virus X.To better contextualize our results, and to assign the 26 “nearly complete viral genomes” to a viral group, we attempted a phylogenetic analysis based on the regions corresponding to the vRdRP domains . AlthouPicornavirales), although we could find evidence of the presence of polyA-tails only in three out of 26 viruses. Overall, our result strongly enforced the use of ribo-depletion for the preparation of RNA-seq libraries targeting viral discovery.Viruses can infect almost every living organism, and viral nucleic acids, either DNA or RNA, are often found when the host sequences are analyzed, making host RNA-seq samples suitable targets for viral discovery ,70. In tHeterosigma akashiwo RNA virus, which we traced in RNA-seq samples of co-cultured C. gigas and M. galloprovincialis, and even in an unrelated R. philippinarum sample. In this study, we exploited the COI reads to identify possible co-occurring organisms of the RNA-seq samples rich in viral reads. Although COI is not a universal gene marker, such an analysis can provide an immediate view of the purity of the samples [D. listerianum RNA present in several bivalve RNA-seq samples confirmed the wide distribution of this fouling tunicate, but it could represent only a partial explanation for the multi-species distribution of Rudphi_virus3. The R. philippinarum sample, including 3% of Rudphi_virus4 reads was shown to be heavily contaminated by this tunicate, and by other non-bivalve species, but none of these species correlated with the distribution of this virus.In agreement with a recent study reporting a wide host distribution of invertebrate viruses , we trac samples . In our C. elegans, it functions at nano-molar concentrations thanks to the amplification of vsRNA signals using RNA-dependent RNA polymerase [Drosophila, although the underpinning genetic mechanism is still unknown [Nucella lapillus) opens intriguing questions about the phylogenetic distribution of the antiviral defense system, and about the mechanism itself [C. gigas PIWI1 is induced in RNA-seq samples containing RNA viruses and a more limited induction of one oyster RDR gene was consistent with the active transcription of OsHV-1, we were not able to clearly identify vsRNAs among sncRNA reads obtained from the same oyster sample, including abundant reads of RNA viruses and of OsHV-1 (dsDNA virus). Considering all of the viral contigs obtained from this RNA-seq dataset, our analyses strongly suggest that the sncRNA reads that mapped on these viral contigs are due to RNA degradation. Differently, looking only at the five complete viral genomes, we could not exclude that weak RNAi activity generated few vsRNAs. Improving the power of our analysis by increasing the coverage or by using chemical treatments to specifically enrich the sncRNA fraction [The determination of the host of meta-transcriptomics-derived viruses is likely one of the main challenges of viromics based on high-throughput data . At the lymerase . Recentl unknown . At leas unknown  that rec unknown . Recentlm itself . In insem itself ,66,67 bum itself , the antm itself . Our anafraction , it woulfraction ,71.apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC), enzymatically mutate viral transcripts and genomes [Antarctic picorna-like virus 1 and 3, Acute bee paralysis virus and Aphid lethal paralysis virus, which represented Picornavirales with a phylogenetic vicinity with some bivalve-derived viruses [In vertebrates, the antiviral role of RNAi is superseded by the interferon pathway, which through the activation of interferon-stimulated genes (ISGs), promotes the recognition of viral-derived products and inhibits viral propagation. Among ISGs, powerful sequence editors like ADAR and  genomes ,82 by ac genomes ,83,84,85 genomes ,86,87. S viruses . SNP anaOverall, This study underlines the heterogeneity and variability of RNA viruses that are associated with marine mollusks, and the limited data that is available on environmental RNA viruses. While the simultaneous analysis of viral products, antiviral host defense processes, and products in the RNA-seq samples could support host assignment, this alone is not enough when dealing with suspension-feeders that are able to accumulate environmental microbes, and their viral symbionts. Given the growing body of knowledge on the role of viruses in host fitness, targeted investigations aimed at unraveling the diversity of “genuine” bivalve viruses are needed for a better understanding of factors affecting the health and well-being of these ecologically- and economically-important species.


Comparison of the RNA radiocarbon signature to those of potential carbon pools in the aquifer indicated that at least 51% of the RNA was derived from autotrophy, in close agreement with the RNA-Seq data, which documented the prevalence of autotrophic taxa, such as Thiobacillus and Gallionellaceae. Overall, this hybrid method for RNA analysis provided cultivation-independent information on the in-situ carbon sources of active subsurface microbes and reinforced the importance of autotrophy and the preferential utilization of dissolved over sedimentary organic matter in alluvial aquifers.Determining the carbon sources for active microbial populations in the subsurface is a challenging but highly informative component of subsurface microbial ecology. This work developed a method to provide ecological insights into groundwater microbial communities by characterizing community RNA through its radiocarbon and ribosomal RNA (rRNA) signatures. RNA was chosen as the biomolecule of interest because rRNA constitutes the majority of RNA in prokaryotes, represents recently active organisms, and yields detailed taxonomic information. The method was applied to a groundwater filter collected from a shallow alluvial aquifer in Colorado. RNA was extracted, radiometrically dated, and the 16S rRNA was analyzed by RNA-Seq. The RNA had a radiocarbon signature (Δ Determining the source of allochthonous and/or autochthonous carbon utilized by microbes can be difficult in aquifer systems. Potential carbon sources include dissolved organic carbon (DOC) transported from the surface5, dissolved inorganic carbon (DIC) from recharge, respiration, or mineral dissolution6, carbon associated with mineral grains (sediment organic carbon and adsorbed organic carbon)8, and carbon-rich material deposited with sediment 12. Understanding these sources is critical for constraining the biogeochemical reactions that can control water quality and biogeochemical feedbacks to the atmosphere15.Shallow aquifers are often characterized as oligotrophic environments in which a heterotrophic microbial lifestyle is supported by surface-derived allochthonous organic matterGallionella)16 or targeting genes/transcripts for diagnostic metabolic pathways, for example, for RubisCO of the Calvin-Benson-Bassham cycle18 or for the reductive tricarboxylic acid cycle6. In addition to such targeted  studies, metagenomic and metatranscriptomic studies have also revealed information about heterotrophic vs. autotrophic lifestyles in the subsurface. For example, genome reconstruction based upon metagenomic data from aquifer sediment has strongly indicated an autotrophic lifestyle for prominent but uncultivated subsurface microorganisms19. Metatranscriptomic (RNA-Seq) and metagenomic analysis of an alluvial aquifer near Rifle, CO, highlighted the unexpected abundance and activity of a small group of autotrophic taxa both before and during injection of nitrate into a suboxic aquifer2. Still, comparatively few studies have sequenced RNA from the subsurface to better understand the importance of autotrophy vs. heterotrophy among uncultivated subsurface microbes.RNA profiling with next-generation sequencing technologies (RNA-Seq) and radiocarbon analysis of RNA offer insights to constrain carbon utilization by microbes in a shallow aquifer, but to our knowledge these techniques have not yet been used in combination. DNA- or RNA-sequence-based techniques have been used to infer heterotrophic vs. autotrophic lifestyles. Examples include targeting genes for diagnostic taxa -oxidizing 23. Such radiocarbon techniques are most powerful when different carbon sources  have distinct radiocarbon signatures. PLFA are components of the cell walls of active microbial populations and can provide information on bulk microbial populations in addition to microbial functional groups. PLFA are typically analyzed in sediment samples25 but have also been analyzed in aqueous samples27. Radiocarbon analysis of DNA represents carbon utilized for cell division but has the potential to represent both active and non-viable cells28 and also potentially long-lived ‘relic-DNA’29. RNA is conventionally thought to offer advantages over DNA for representing active, or recently active, microbes, although there are caveats for equating rRNA with actively growing or metabolizing cells30. In aquifer systems, radiocarbon analysis has indicated the importance of allochthonous carbon, such as petroleum products in contaminated aquifers31, and advected DOC in alluvial aquifers with geogenic arsenic32. In the deep subsurface, radiocarbon analysis of PLFA has been able to identify autotrophy27. Stable isotope analysis, but not radiocarbon analysis, has been performed on environmental RNA samples34.Radiocarbon analysis is a sequence-independent method that has been used to determine carbon sources in surface waters and in the subsurface, based on the study of targeted biomolecules, such as phospholipid fatty acids (PLFA) and DNAunamplified RNA from the same environmental sample, and that collectively characterizes community carbon utilization under in situ conditions. The method was applied to a filtered groundwater sample from a geochemically reduced zone of a shallow alluvial aquifer near Rifle, CO, where multiple potential carbon sources co-occur. The radiocarbon and RNA-Seq results, while addressing diverse sample properties, indicated a strikingly similar conclusion about the quantitative role of autotrophy in the subsurface microbial community.The goal of this work was to develop and test a cultivation-independent method that analyzes both the radiocarbon and rRNA signature of 14C) and a comparatively old C age. In practice, a sample with little to no radiocarbon has a Δ14C value approaching −1000‰ and contains carbon more than 50,000 years old. In contrast, LB and dextrose are derived from recent photosynthetically derived material and have a contemporary signature with a positive Δ14C value and a modern age , and dextrose] were utilized to validate the RNA radiocarbon purification method. Commercially available acetate is derived mostly from petroleum and thus possesses a highly negative radiocarbon signature Δ4C and a E. coli grown with the different media were analyzed for their radiocarbon signatures to develop the current method , so CTAB (hexadecyltrimethyl ammonium bromide) was used instead (Table 36. E. coli grown with acetate had an RNA radiocarbon signature close to that of the pure acetate medium (Table E. coli controls grown with LB. Seven samples extracted using water-saturated phenol (pH 6.6) all produced non-modern, more negative RNA signatures than the cells  Table . When us14C value that was more negative than the LB and the washed cells . For this medium, the Δ14C value of E. coli RNA was 37.8 ± 4.2‰, compared to the dextrose medium value of 55.6 ± 3.5‰, a difference of 17.8‰. Whole E. coli cells grown with dextrose were not analyzed, but given the single carbon source, we assume that the signature of E. coli cells would have been similar to the RNA and dextrose. It is not clear if the consistent 17–20‰ difference between the radiocarbon signatures of RNA vs. the growth medium (for LB or dextrose) was due to petroleum-derived contaminants in the RNA or isotopic fractionation during growth. For example, there could be relic carbon from the extraction buffers or carbon leaching from plastic or the Milli-Q system that was not removed. An extraction without cells did not have enough carbon for radiometric analysis and could not be used to trace exogenous carbon sources. Regardless, the radiocarbon signatures of the RNA and the medium/cells were consistent and comparable, and indicated that the method could be used to assess a range of carbon sources in the environment.The final method still produced a Δ38. The well was sampled to determine if radiocarbon and RNA-Seq analyses could be combined to better understand carbon sources for microbes in aquifer systems. Approximately 23,520 L of groundwater were filtered (0.2-μm) and the RNA was extracted from the filter for radiocarbon and sequence analysis.Monitoring well LQ107 is located within a so-called “naturally reduced zone” of a shallow alluvial aquifer near Rifle, CO14C value of the RNA was −193.4 ± 5.6‰. The Δ14C values of the DIC and DOC were −157.7 ± 1.7‰ and −231.0 ± 1.7‰, respectively. In the aquifer, the average Δ14C value of the sediment organic carbon (SOC) and plant material were −504.4 ± 173.0‰ (n = 20) and −55.1 ± 61.8‰ (n = 8), respectively12. The range in radiocarbon values for potential carbon sources is much larger than the variability in radiocarbon signatures associated with the E. coli controls, indicating the method can be utilized to determine in situ carbon sources. Assuming that the RNA represents carbon from a linear combination of autotrophy and heterotrophy, the percent of RNA carbon from DIC was calculated with a DOC or SOC end-member. The DIC-DOC pair indicated 51.2% autotrophy and the DIC-SOC pair indicated 89.7% autotrophy. This assumes no contribution from buried plant material, as this plant material could not have been the primary source of carbon for the RNA, given the radiocarbon signatures  among the most abundant taxa  and/or Fe(II) as electron donors, including the following , Sideroxydans and/or other Gallionellaceae family members (e.g.43), and Sulfuricella (e.g.45). The close phylogenetic relationships among the five most abundant taxa and experimentally characterized lithoautotrophic, β-proteobacterial isolates are depicted in Fig. Sulfurimonas spp., which belong to the ε-proteobacteria (e.g.46) - and S-oxidizing lithoautotrophic bacterial strains belonging to Sulfurimonas and the Gallionellaceae were found to be highly active, collectively accounting for ca. 80% of the metatranscriptome (community gene expression). Further, a different Rifle aquifer study indicated the predominance of another S-oxidizing lithoautotrophic bacterium, Candidatus Sulfuricurvum sp. RIFRC-1, that constituted ca. 47% of the aquifer sediment community based upon metagenomic data19.Thus, RNA-Seq analysis provided strong evidence of lithoautotrophic bacterial activity in the Rifle aquifer. Notably, these results are very consistent with a recent study of the Rifle aquifer47.Radiocarbon and RNA-Seq analysis of RNA are both resource- and time-intensive methods that can currently only be performed on a small number of well-constrained samples, but together can offer novel insights. Targeting RNA in environmental samples makes processing more difficult than DNA analyses, but ensures a focus on predominantly active microbes. While PLFA analysis also targets active microbes, it provides much less detailed taxonomic information than rRNA sequencing. In this study, two methods that target RNA but independently provide estimates of carbon sources through either a geochemical measurement or a taxonomic assessment indicated the important role of autotrophy in a subsurface microbial community. This mirrors recent findings at Rifle and other sites32. It is not clear why SOC is not utilized by the microbes in alluvial aquifers, but multiple ecosystem properties might account for this stabilization39, even though sediment organic matter could be bioavailable once it is released from the sediment48. In this shallow alluvial aquifer, it appears that heterotrophic redox reactions are supported by DOC, while reduced inorganic compounds mix with oxidants delivered to the aquifer during seasonal hydrologic fluctuations49 to support a thriving and diverse chemolithoautotrophic community that accounts for approximately half of the active, planktonic, microbial biomass. The combination of next-generation sequencing of unamplified 16 S rRNA with radiocarbon analysis of RNA could be a powerful, cultivation-independent method for understanding subsurface carbon dynamics under in situ conditions.The data also provided information on the probable source of the organic matter used by heterotrophs. In order for the radiocarbon-based estimates of autotrophy  and sulfate reduction at the site have contributed to the presence of reduced Fe- and S-containing compounds in the aquifer, including Fe-sulfide minerals, such as FeS and FeS2. DIC and DOC concentrations are relatively constant over time. The alluvial Rifle aquifer is underlain by the impermeable Wasatch Formation at a depth of 5.5 meters at well LQ107.Groundwater samples were collected in August, 2011 from monitoring well LQ107, located in a shallow alluvial aquifer near Rifle, Colorado. The well is 6.1 meters deep and screened over the bottommost 3 meters. The water table varies from approximately 1.8 to 4.1 meters below the land surface, depending on river stage in the Colorado River, which adjoins the site to the south. An extensive overview of the site was presented by Williams Groundwater samples were collected from monitoring well LQ107 in August 2011 using a peristaltic pump; groundwater was pumped through a 0.2-μm nylon 10-inch filter cartridge  for 4 days at 3.8 liters per min for a total volume of 23,520 L. The high flow rate through the filter maintained the temperature of the filtered residuum at an aquifer temperature of ca. 13 °C over the sampling duration. The filter was kept in the dark during filtering to limit microbial activity, and then immediately frozen at −20 °C and shipped frozen to Barnard College, where it was stored at −80 °C until processing.51. The first round used 50-mL PLG tubes and the second round used either 50-mL or 1.5-mL PLG tubes . In addition, the phenol was tested as both water-saturated (pH 6.6) and Tris-saturated (pH 7.9), and again, this was an important variable. The extracts were precipitated with isopropanol, washed with 70% ethanol, and resuspended in DNA-grade sterile water. The RNA was further purified with LiCl and/or CsCl ultracentrifugation (see SI). DNase treatment was not utilized at this point to avoid adding any excess carbon to the sample that could impact radiocarbon dating; however, DNase was used prior to RNA-Seq analysis, as described below. The purity and amounts of RNA were assessed with a NanoDrop Spectrophotometer  and verified with a Qubit Fluorometer (Thermo Fisher) using both RNA and DNA kits. RNA was stored in DNA-grade sterile water at −80 °C until RNA-Seq or radiocarbon analysis.The complete RNA extraction and purification procedure is presented in the SI. An abbreviated version that highlights less conventional portions is presented here. The frozen filter was cut into ~1-inch rings with a sterilized electric saw, then cut into small sections and the outer-shiny layer was placed in a 50-mL tube. The RNA extraction buffer was 2% CTAB, 1.4M NaCl, 20 mM EDTA (pH 8), and 100 mM Tris-Cl (pH 8), with 64 mg lysozyme and 16 mg proteinase-K added along with 1.7 g of 3-mm sterile beads. The mixture was vortexed, incubated for 30 min at 55 °C, decanted, and washed a second time with 1x Tris-EDTA (TE). The extract was purified using phenol:chloroform in two rounds with Phase Lock Gel (PLG) tubes , which were made with a silicone lubricant. E. coli  was grown with either sodium acetate  or dextrose (Fisher) with M9 medium52 or lysogeny broth (LB) (Fisher). Acetate is mostly petroleum-derived and is an older end-member, whereas LB contains yeast extract as a carbon source and provides a modern radiocarbon end-member along with dextrose. Cells were grown and diluted multiple times (to dilute out previous carbon signatures), pelleted, resuspended in 5 mL of DNA-grade sterile water, and stored at −80 °C until 1 mL was used for extractions.Two end-member cases were used to test the RNA extraction procedure2/O2 torch. The sealed tubes were placed in a furnace set at 900 °C for 3.5 h to oxidize all carbon to CO2. The evolved CO2 was purified, trapped, and reduced to graphite using standard techniques54. Graphite samples were measured at the Center for AMS at LLNL using the HVEE FN AMS system operating similarly to our previous analyses of DNA21. Most samples were too small to obtain CO2 splits for δ13C analyses. Based on a few large sample splits, a fractionation correction of δ13C = −20 ± 2‰ was used for RNA samples. The measurement error was determined for each sample and ranged between ±4‰ and 11‰ (1SD) for samples with greater than 25 μg carbon. All data was reported as decay-corrected ∆14C55 and post-bomb F14C56.Purified RNA samples suspended in water and process controls were shipped to Lawrence Livermore National Laboratory (LLNL) for accelerator mass spectrometry (AMS) sample processing and analyses through the natural carbon preparation laboratory. RNA storage is not critical for radiocarbon analysis, as storage at room temperature results in RNA degradation, but not mineralization. Samples were transferred to quartz combustion tubes and lyophilized to dryness. Isotopic standards NIST SM 4990C Oxalic Acid II and IAEA C-6 sucrose were dissolved in water to produce secondary standards with similar masses as the RNA and lyophilized with the samples. After removal from the lyophilizer, all dry samples, controls, and standards received excess CuO and were promptly evacuated and sealed with a Het al.2. Briefly, we used a TruSeq RNA Sample Preparation Kit v1 (Illumina), with a single modification: the protocol began with the “Elute, Prime, and Fragment” step by adding 13 μL of the “Elute, Prime, and Fragment Mix” to 5 μL of purified RNA. The cDNA library concentration was assessed using the KAPA SYBR FAST qPCR Kit following the manufacturer’s instructions . Quality was assessed with a Bioanalyzer using a DNA 1000 chip (Agilent Technologies). The sample was sequenced by the Vincent J. Coates Genomics Sequencing Laboratory at U.C. Berkeley on an Illumina HiSeq4000 with paired-end, 100-bp reads.RNA purification, RNA-Seq library construction, and sequencing: Samples for molecular analysis were kept at −80 °C and shipped on dry ice to Lawrence Berkeley National Laboratory. Total isolated RNA was further purified using the Qiagen RNeasy cleanup kit following the manufacturer’s instructions with a modification for DNase digestion: after the first wash of the RNeasy mini spin column with 250 μL buffer RW1, 80 μL of DNase I incubation mix (10 μL DNase I stock and 70 μL buffer RDD) was directly loaded onto the column and incubated at room temperature for 15 min. The purified sample was analyzed for quality with a Bioanalyzer 2100, using RNA 6000 Pico Chips  and was quantified with a Qubit fluorometer, using a Broad Range RNA kit (Agilent Technologies). The cDNA library was prepared with 60 ng total RNA and was constructed as described by Jewell, 57). EMIRGE reference databases were constructed using the SILVA SSU SEED database (release 123)58 and 16S rRNA sequences from metagenome-assembled genomes59, resulting in a final candidate database of 13,386 sequences with taxonomic labels. EMIRGE was parameterized to run in single-end mode to iterate 40 times, to merge 97% identical sequences, and to accept the reference sequences that were at least 30% covered lengthwise. 16S rRNA gene relative abundances were estimated based on normalized priors from the final EMIRGE iteration. The data is available as a fasta file in the supplemental material and as GenBank SRA BioProject PRJNA483050.Ribosomal 16S rRNA sequence assembly: Near-full-length ribosomal 16S rRNA sequences were reconstructed from total RNA reads using a large database of candidate rRNA genes with a templated assembly approach (EMIRGEn = 5) were searched against SILVA SSU Reference NR sequences using the SINA 1.2.11 “search tool”60. For each Rifle sequence, sequences of neighbors with >95% sequence identity were collected. The reconstructed sequences and their close relatives were merged into a single file, sequence headers were annotated with the taxonomic labels, and aligned to SILVA seed alignment, and a maximum-likelihood phylogeny was inferred with RAxML61 (GTRGAMMA model of rate heterogeneity) using Rhodopseudomonas palustris HaA2 as an outgroup with 1000 bootstrapped trees to estimate node confidences (“raxmlHPC-SSE3 -f a -m GTRGAMMA -# 1000”). The resulting tree was visualized with Dendroscope62.For Fig. Supplementary InformationDataset 1Dataset 2


Wave-driven flooding is a serious hazard on coral reef-fringed coastlines that will be exacerbated by global sea-level rise. Despite the global awareness of atoll island vulnerability, little is known about the physical processes that control wave induced flooding on reef environments. To resolve the primary controls on wave-driven flooding at present and future sea levels, we present a globally applicable method for calculating wave overtopping thresholds on reef coastlines. A unique dataset of 60,000 fully nonlinear wave transformation simulations representing a wide range of wave energy, morphology and sea levels conditions was analysed to develop a tool for exploring the future trajectory of atoll island vulnerability to sea-level rise. The proposed reef-island overtopping threshold (RIOT) provides a widely applicable first-order assessment of reef-coast vulnerability to wave hazards with sea-level. Future overtopping thresholds identified for different atoll islands reveal marked spatial variability and highlight distinct morphological characteristics that enhance coastal resilience. Sea-level rise will exacerbate wave overtopping on low-lying coral reef islands. Here the authors present a novel method that quantifies wave overtopping thresholds and associated reef-island vulnerability trajectories based on differences in local wave climate, reef morphology and island height. This ‘natural breakwater’ effect has enabled dense development on coastal margins and complete urbanisation on a number of atoll islands that are now highly vulnerable to impacts of future sea-level rise (SLR)4. Small and low-lying atoll islands are prone to flooding from spring tides5, storm surge6 and wave overtopping7, with marine inundation predicted to become increasingly costly in the near future8. At present sea-level (SL), wave overtopping occurs every 2–3 years8, with destructive flooding events every 10–30 years10 allowing sufficient recovery time for agriculture, fresh-water resources and infrastructure. Global SL is predicted to rise by 0.44 ± 0.17 m (RCP2.6) to 0.74 ± 0.23 m (RPC8.5) by 210011, with a continued increase predicted for several centuries regardless of emission mitigation efforts13. SLR is a serious climate change hazard that has already impacted some reef-island systems14 and is predicted to severely impact all reef-fringed and atoll island shorelines in the coming decades16, affecting an estimated global population of 197 million people4. Unmitigated, SLR will increase the frequency and magnitude of wave-driven flooding events8, eventually reaching a threshold when compounding events prevent recovery and compromise permanent habitation on coastal margins or entire islands17. Atoll-populations  have limited land available for in-country relocation and may become climate change refugees when local adaptation efforts are exceeded18. However, future responses are unlikely to be uniform as there is considerable heterogeneity in island physical structure and energy exposure19. To assist local adaptation and timelines for future overtopping, there is a pressing need to resolve how the physical drivers of wave-driven flooding on reef coastlines vary spatially due to differences in wave climate exposure and island morphology. Here, we present a new model of wave overtopping that can be applied to predict SL and (or) wave height thresholds for overtopping on reef-fringed coastlines globally. The model is applied to a number of different atoll islands to understand how future inundation trajectories vary according to local morphology, wave climate exposure and potential geomorphic feedbacks.Coral reefs effectively dissipate incident wave energy through a combination of breaking and friction20. Wave breaking on coral reefs creates a setup of mean water level across the reef flat21 and the dissipation of incident wave groups drives a transfer of energy to long-period infragravity wave motions that surge across the reef flat22. Wave setup and infragravity waves are dynamically influenced by incident wave conditions and reef morphology and both significantly influence runup and overtopping on reef coastlines23. It is, therefore, essential that the nonlinear interactions between incident wave dissipation and these dynamic surf-zone processes are represented accurately when investigating runup and wave-driven flooding on reef coastlines22.Overtopping occurs when wave runup surges over the beach crest and across the island surface Fig. . Wave ru25 that are not globally applicable, neglect overtopping magnitudes26 or use simplified hydrodynamic models that do not resolve nonlinear surf-zone processes28. Our research advances previous work by applying a comprehensively evaluated physics-based numerical model29 to directly simulate wave overtopping motions resulting from incident wave dissipation and nonlinear surf-zone processes. Our approach builds on previous studies that utilised a wide range of idealised bathymetries and wave conditions to investigate the impacts of SLR on reef coastlines30. Consequently, we present the first analysis of wave overtopping that is applicable to reef coastlines globally, under present and future SLs by representing 60,000 unique morphology, wave energy and SL scenarios.Previous attempts to understand the physical drivers of future wave overtopping on reef coastlines focus on single case study islandshr, below still water level), reef width (Wr), significant incident wave height (Hs), wave period (Ts) and island elevation  used in this analysis  at the beach crest ranging between 0.01 and 2.5 m , when all other factors were consistent  quantifies the dissipative capacity of a reef and relative island elevation (Zi/Hs) represents the potential for an island to withstand overtopping during a given event. RRW decreases with increasing SL and therefore represents how reefs will become less effective at dissipating incident wave energy, resulting in elevated runup levels at the shoreline. Low-relative island elevation (RIE) values (<0.6) indicate that an island is likely to be overtopped by a given event, unless RRW is high and the reef is extremely dissipative. At present SL, overtopping occurs because high-wave events coincide with high tide, resulting in a low-RIE value. As SL increases, the associated decrease in Zi will lower RIE and therefore the incident wave energy required for overtopping will decrease. Across the spectrum of forcing and morphology conditions used in this analysis  ranged between 0.1 m (Takuu) and 1.22 m (Fatato) above present SHT Fig. . This eqmur Fig. . The pre−1)32. Using Eq. (hr = 0) and catch-up reef (Δhr = 0.5ΔSL) responses (Methods) identifies that future reef growth will delay the onset of flooding by raising SLR thresholds for overtopping by an average of 0.11 ± 0.01 m for keep-up reefs and 0.08 ± 0.01 m for catch-up reef responses  to maintain a shoreline elevation at equilibrium with the overtopping threshold associated with a give-up reef response  that are predicted to occur before 210034 provides a fully nonlinear, but weakly dispersive solution for the Boussinesq equations that is applicable to large amplitude waves interacting with complex morphologies, such as coral reefs. A shock-capturing nonlinear shallow water solution is applied in areas of wave breaking and runup to ensure areas of rapid dissipation are handled with stability and accuracy35. The phase-resolving model we apply here has been comprehensively evaluated for accurately simulating wave dissipation, infragravity wave behaviour, wave setup, runup and overtopping on coral reef environments, using field data20 and wave flume29 experiments. The documented open-source model is available online36, with existing literature providing more information on the numerical scheme34 and its application to simulate waves on coral reefs25.A previously evaluated fully nonlinear numerical model was used to simulate wave shoaling, transformation, runup, overtopping and inundation for this research. The open-source Green–Naghdi solverHs variations and 6 Ts variations) and 1000 morphology variations  that collectively represent a global spectrum of atoll islands and energy settings. Consistent values were used for beach slope (0.1) and reef slope (0.14) because these variables are known to have a deterministic but secondary influence on wave runup elevation27. Friction is known to vary on coral reefs, depending on ecological and sedimentary characteristics. We account for friction using an implicit quadratic bottom friction equation with a coefficient of Cf = 0.04, representative of a moderately rough reef environment20. We do not account for variability in roughness by adjusting Cf because this has a deterministic influence on runup and is of secondary importance compared to changes in water depth26. The contribution of possible future reductions in friction associated with declining structural reef complexity to overtopping scenarios is a subject recommended for future study. To represent wave breaking in the numerical model, a threshold free-surface slope was used to split between dispersive and dissipative equations, with a slope threshold of 1 based on previous work20. Modelled wave conditions were generated with a JONSWAP spectrum, using γ = 3.3 and each simulation was run for 35 min of wave activity to ensure that a saturated wave field developed on the reef platform, allowing a number of wave groups to potentially overwash the island. Waves released at the offshore boundary propagated across a 60 m deep shelf for 200 m before interacting with the moderately steep reef slope, reef flat and island  remained constant for 50 m before a leeward beach slope decreased island elevation to zero . When calculating RRW, a minimum reef depth of 0.1 was used to avoid dividing by zero for dry reef scenarios.Wave overwash was identified by extracting time-series data at 10 Hz at a location on the island berm positioned 70 m landward of the beach toe  result in reduced overwash magnitudes compared to moderate (8–12 s) and long (14–16 s) period waves. Overall, the influence of wave period on overwash occurrence and magnitude was of a secondary order compared to wave height and island elevation, which became the focus of subsequent analysis , Roi-Namur , Majuro , Takuu , and Fares-Maathoda . Time-series wave data were filtered to only include wave events where peak wave direction was travelling within 90 degrees of shore-normal to the island. Directionally filtered wave heights were used to calculate 50, 99, 99.9 and 99.99 percentile wave heights, and extreme wave heights, calculated as the average of the top 10 wave heights over the 30 years. Wave height statistics for each example island were used to calculate inundation trajectories and future SL thresholds for wave inundation (Supplementary Table Hourly wave climate data for 30 years 1980–2009), from the CSIRO Wave Watch 3 hindcast database009, fromZi and hr at small (0.001 m) steps that represent increases in SL. Thresholds were identified by iterating the increase in SL until the calculated Hs threshold for overtopping (using Eq. (hr) was equal to the increase in SL (ΔSL). To represent a catch-up reef response, where the rate of reef accretion is less than the rate of SLR, the increase in reef depth was equal to half the increase in SL at each increment. To represent a keep-up reef response, where carbonate accumulation processes allow a reef to accrete vertically at the same rate as SLR, the change in reef depth was zero. Given the significant uncertainty and regional variability regarding the rate and magnitude of future SLR12, we chose not to convert our identified SLR thresholds into temporal dates.SLR thresholds for wave inundation were calculated for each island and wave statistic by incrementally adjusting mall 0.00 m steps Simulations used to develop the RIOT were undertaken using a fully nonlinear Boussinesq-type model that has been comprehensively evaluated for simulating wave processes on coral reefs. However, there are limitations associated with numerical simulations and data synthesis that need to be understood before Eq.  can be aDespite these limitations, the placement of RRW and RIE combinations for 23 known inundation, overtopping and runup events  and the software is freely available to install and use. Linux shell scripts that were used to pre-process model runs and Matlab codes that were used to process model outputs are available from the corresponding author (e.beetham@auckland.ac.nz) on request.The Green–Naghdi solver used in this research is part of the open-source Basilisk software package. The documented source code is available online (Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2