Lophelia pertusa. Measurements revealed how the presence of coral framework increased current speed and turbulence above the frontal part of the reef patch, while conditions immediately behind it were characterised by an almost stagnant flow and reduced turbulence. Owing to the higher current speeds that likely promoted a higher food encounter rate and intake of ions involved in the calcification process, the coral nubbins located on the upstream part of the reef presented a significantly enhanced average growth and a lower expression of stress-related enzymes than the downstream ones. Yet, further experiments would be needed to fully quantify how the variations in water hydrodynamics modify particle encounter and ion intake rates by coral nubbins located in different parts of a reef, and how such discrepancies may ultimately affect coral growth. Nonetheless, the results acquired here denote that a reef influenced by a unidirectional water flow would grow into the current: a pattern of reef development that coincides with that of actual coral reefs located in similar water flow settings. Ultimately, the results of this study suggest that at the local scale coral reef morphology has a direct effect on coral growth thus, indicating that the spatial patterns of living CWC colonies in reef patches are the result of spatial self-organisation.Despite cold-water coral (CWC) reefs being considered biodiversity hotspots, very little is known about the main processes driving their morphological development. Indeed, there is a considerable knowledge gap in quantitative experimental studies that help understand the interaction between reef morphology, near-bed hydrodynamics, coral growth, and (food) particle transport processes. In the present study, we performed a 2-month long flume experiment in which living coral nubbins were placed on a reef patch to determine the effect of a unidirectional flow on the growth and physiological condition of  Consequently, CWC assemblages are increasingly known to be biodiversity and biomass hotspots comparable to their shallow and tropical counterparts6.The intricate structure of reef-building cold-water coral (hereafter referred as CWC) frameworks and the complex morphology of the thickets they form, promote an increase in the spatial heterogeneity of environmental conditions providing a wide range of habitats for a highly diverse and abundant associated fauna9, at some sites promoting the formation of coral mounds that can be over 300 m in height12. Unlike their warm-water counterparts, CWC reefs are commonly formed by a limited number of species, with the most well-known one being Lophelia pertusa (syn. Desmophyllum pertusum13). Although knowledge on the physiology and ecology of CWCs has considerably improved over the last decades16, the dynamic processes controlling reef growth and morphology in relation to environmental variables are still poorly understood17. Traditionally, the morphology of CWC reefs has been hypothesised to be driven by the underlying geological features  and by the hydrodynamics of the region . In regions where hard substrate is not limiting and bottom currents are mainly unidirectional, most coral reefs/mounds present a cigar- or teardrop-shaped morphology . In such environmental settings, living coral is almost exclusively reported on the upstream side of the reef/mound structures, whereas the coral rubble on their tail is progressively older, indicating that CWC reefs/mounds grow into the flow29. In the strait of Florida, besides growing into the current, coral reefs also form semi-parallel bands that are orientated perpendicularly to the N–S dominant water flow25, suggesting that under certain environmental conditions CWCs might present a self-organising behaviour30.With the presence of favourable conditions, colonial scleractinian CWCs can grow and form three-dimensional complex reefs that entrap the by-passing hemipelagic sediments31. As the colonies develop into reefs, the coral framework starts to modify the local physical conditions and food availability33, ultimately initiating a series of positive and negative feedbacks that promote suitable and unsuitable environments for coral growth34. As a consequence, CWC reefs tend to display well-developed topographic highs with living colonies, alternated with low relief areas covered by coral rubble and fine sediments36. Although the interactions between extrinsic  and intrinsic factors  play an important role in the distribution and growth of coral colonies along a reef, and ultimately in its morphology, these are yet to be properly described and quantified.CWCs tend to grow upwards into the water column seeking for water currents delivering food38, it is challenging to elucidate and quantify the main environmental variables and feedbacks that regulate their geomorphological development. So far, studies that relate the variations in water flow dynamics caused by reef morphology with growth patterns of the reef itself are still scarce, and only based on numerical simulations34. Nevertheless, L. pertusa growth rates are fast enough  to perform relatively short-term growth experiments . In fact, many aquaria experimental studies have determined the presence of differential CWC growth rates under contrasting treatments of several environmental variables  in relatively short periods of time46. According to experimental and numerical simulation studies, coral patches/reefs promote an alteration of the water flow and sediment transport in their vicinity47, enhancing particulate matter residence time and deposition, and ultimately affecting its availability within the reef itself48. Indeed, Mienis et al.33 demonstrated that, under realistic current speeds, reef patches promote higher flow velocities over the patch, whereas its wake is characterised by near-stagnant flows with reduced turbulence and vertical mixing. Similarly, water refreshment rates are drastically reduced, potentially affecting the oxygen exchange rates in and around the patch. However, all these studies do not take into account the response of living coral specimens. Therefore, there is a significant knowledge gap in quantitative experimental studies that help to understand how hydrodynamics and particle transport processes interact with reef/thicket morphology and affect coral growth, to ultimately regulate the geomorphological development of CWC reefs.As CWC reefs form over geological timescales33, we hypothesised that:Corals on the upstream side will grow faster (i.e. will accumulate more skeleton mass over the same time frame) than those on the downstream side.Corals on the downstream side will experience less suitable conditions than those on the upstream side, thus promoting a higher expression of stress-related enzymes.At a certain distance behind the reef, the water flow will be re-established to pre-reef conditions, allowing settled larvae to experience suitable living conditions and potentially give rise, with time, to the formation of a new coral reef patch.Based on previous studiesu  and w  showed considerable variations between upstream and downstream sides of the reef patch, which occupied the whole width of the flume, extended for a length of 60 cm and had a maximum height of 15 cm  ranged from 7 to 18 cm s–1 at the near-bottom and water surface, respectively, thus exposing the first set of coral nubbins to an average horizontal velocity of ~ 7–8 cm s–1 , u velocities were reduced and became almost stagnant, with values between – 1 and 4 cm s–1  were nearly 0, only to become slightly positive (i.e. flowing upwards at < 3 cm s–1) at the frontal part of the reef patch, coinciding with the first two sets of coral nubbins  from ~ 0.35 to ~ 2 m behind the structure  values also displayed considerable variations between upstream and downstream regions , was exposed to k values close to 0. A first peak of k = 2.7·10–3 m2 s–2 was observed above the frontal part of the reef , coinciding with the second set of coral nubbins  in growth rates were observed among the coral nubbin sets Fig. . Nonethe49. The coral nubbins used in this experiment displayed overall CAT activity values ranging from 0.86 to 16.74 U mg–1 of protein, with a mean value of 3.74 ± 3.93 U mg–1 of protein. The first two sets of nubbins showed activities ranging between 0.98 and 4.61 U mg–1 of protein, with average activities of 2.91 ± 1.19 and 1.46 ± 0.35 U mg–1 of protein, respectively  and Superoxide dismutase (SOD) are known to be general antioxidant alarm indicators, with higher activities of these enzymes representing greater oxidative alarms in the organism50. The whole set of coral nubbins presented MDA concentrations between 5.13 and 88.75 µmol mg–1, with an average concentration of 21.59 ± 20.89 µmol mg–1. The two coral nubbin sets located at the frontal part of the reef patch and the third set of nubbins showed intermediate MDA concentrations, ranging from 9.04 to 53.28 µmol mg–1. The average values for these three sets were 17.21 ± 4.95, 17.05 ± 12.19 and 21.78 ± 18.68 µmol mg–1, respectively , defined by the malondialdehyde (MDA) concentration, indicates the level of cell damage51. Hence, when combined with other stress-related enzymes, it can provide information on the physiological condition of the coral nubbins. The activity values of the nubbins used in this experiment ranged between 0.29 and 9.45, with an average activity of 3.28 ± 2.89 nmol min–1 mg–1. For this enzyme, the first two nubbin sets showed the lowest activity ratios, varying from 0.29 to 7.31 nmol min–1 mg–1 and with mean activities of 1.41 ± 1.35 and 2.26 ± 3.37 nmol min–1 mg–1  also has an antioxidant role in preventing oxidative stress damage through the action of oxaloacetateDespite the data presented for stress-related protein activity and/or concentration seems to show that the coral nubbins from sets 3–5 are in a worse physiological condition, no statistically significant differences (p > 0.05) were observed.L. pertusa nubbins and flow conditions similar to those occurring in actual CWC reef settings, the present flume experiment provides novel empirical data on the effects of geomorphologically steered water flow distortions on coral growth, physiological condition and its subsequent influence on reef formation along a scaled artificial CWC reef patch.By using living 33, which demonstrates the reproducibility and robustness of the experiment. For similar pre-patch u velocities (10–15 cm s–1), the coral patch/reef used in the present study  promoted variations to the hydrodynamics of the flume similar to those caused by 10 and 25 cm tall coral patches in Mienis et al.33, with u velocities increasing up to 21 cm s–1 above the reef and near-stagnant water observed at the seafloor just behind the coral structure. This region of reduced u velocities could be observed up to 2 m behind the reef both in the present study and in Mienis et al.33. In terms of turbulence, a high k region was observed, in the present experiment, above the upstream part of the reef patch with values of up to 2.7·10–3 m2 s–2, which likely promote enhanced mixing within the benthic boundary layer, thus replenishing the reef with particles52 and increasing their encounter rates with the coral nubbins. With regards to the downstream off-reef region, the most turbulent area was observed at a distance between ~ 0.4 and 2 m behind the reef patch and from the flume bottom up to 0.2 m into the water column. Nonetheless, the k values measured here (i.e. 1.4–3.9 × 10–3 m2 s–2) were considerably higher than those observed in Mienis et al.33, for both the 10 and 25 cm tall patches (i.e. ~ 0.4–2 × 10–3 m2 s–2).In terms of hydrodynamics, the results acquired here match with those of Mienis et al.–1) and low to moderate turbulence, displayed a significantly higher skeletal growth than those subjected to a near-stagnant water flow at the leeside of the reef  are compared, by means of the conversion factors presented by Büscher et al.42, to in situ linear extension rates measured in corals from environmentally contrasting regions 55, it can be observed that our results cover the whole growth rate range of L. pertusa colonies. Hence, on top of being statistically significant, the variations in growth rates among nubbin sets also seem to be biologically relevant. Indeed, the first two sets of nubbins likely benefited, from the higher turbulence occurring above the reef front and their direct exposure to the incoming nauplii-rich waters. In addition, high flow speeds considerably enhance particle encounter rate and thus, the intense currents going through the first two nubbin sets might have helped increasing their zooplankton capture rates57. The fast growth of these nubbins contrasts with the results of Purser et al.58, where the authors observed that L. pertusa displayed higher zooplankton capture rates under reduced flow speeds (i.e. < 5 cm s–1). However, the latter experiment consisted of small coral nubbins with a reduced potential to modify water hydrodynamics, whereas the larger reef patch used here affected water flow direction and speed, ultimately benefiting the upstream nubbins. In addition, Orejas et al.59 witnessed that there were no significant differences in terms of polyp expansion for colonies exposed to flow speeds ranging from 0.5 to 15 cm s–1, which combined with the growth rates acquired here, suggests that flow speeds > 5 cm s–1 might still be suitable for the feeding of L. pertusa. Indeed, long-term in situ measurements from coral reefs show average flow speeds of 10 cm s–1, albeit with generally high variability in terms of flow direction and velocity along time61. This was also discussed by Duineveld et al.60, as in situ video observations from the Mingulay Reef showed that at current speeds of ~ 40 cm s–1 the percentage of extended polyps was at its maximum. Besides the role current velocity plays in food supply, it also affects the thickness of the diffusive boundary layer (DBL), which is the region of water closest to the corals, where molecular diffusion dominates the transport between the water column and the organism64. Higher current velocities generally reduce the thickness of the DBL, thus enhancing the rate of diffusion of inorganic nutrients and dissolved gasses between the water column and coral tissues66. In this sense, the corals located on the upstream side of the reef were exposed to higher current velocities , which promote particle deposition. Although several studies have observed that L. pertusa colonies may survive under suboptimal feeding conditions using their energetic reserves over a period of months69, if this scenario is maintained for longer periods, it can potentially cause the demise of the coral colonies due to starvation17. In addition to the low food conditions, the increased residence time at the wake of the reef patch might have reduced the oxygen renewal of the water surrounding the nubbins. This combined with a thicker DBL, caused by the low water velocity66, likely reduced the oxygen, Ca2+, CO32− and HCO3– intake of the coral nubbins. Indeed, the slower growth rates of the downstream nubbins coincided with a higher activity of antioxidant enzymes (CAT and SOD), a greater cellular damage (MDA) and an increased demand for MDH activity to compensate stress51, which suggest these corals were in worse physiological conditions than the upstream ones 31. Both the data acquired here and in situ observations27 contrast with the results of Hennige et al.17, where a simulated colony subjected to a unidirectional water flow developed into an hemispherical coral patch, with living coral on its outer rim and dead coral framework in the inner parts of the patch. However, it is important to note that the theoretical results in Hennige et al.17 do not consider the distribution of particulate organic matter or 3-Dimensional flow in the experimental setting, which are affected by the varying size and morphology of the growing patch, and as demonstrated in this study are two critical variables regulating reef development.As the reef grows towards the current, it would establish a positive feedback at the reef front involving intense hydrodynamics that would promote a potential increase in oxygenation, food supply and ion intake, ultimately endorsing coral growth. Conversely, the reef back would be subjected to a negative feedback of almost stagnant water promoting a potentially lower degree of oxygenation and ion intake, enhanced sedimentation, decreased food supply and increased metabolic stress, possibly causing coral demise Fig. . This wo52, potentially providing the 5th and 6th nubbin sets with a higher availability of food particles than those located at the immediate downstream side of the reef patch (i.e. sets 3 and 4). Indeed, when comparing the growth rates of downstream reef nubbins and off-reef ones (i.e. sets 5 and 6), a tendency towards greater skeletal growth was observed in the latter, albeit with no statistically significant differences  than the downstream ones (i.e. sets 3 and 4), the environmental conditions were likely not sufficient to provide a suitable particle encounter rate that would promote a differential coral growth. Besides the potentially low food supply that the 5th set of nubbins received, the high turbulence to which they were subjected to might have also been a factor of stress. Indeed, the 5th nubbin set displayed some of the highest activities and concentrations of enzymes related with oxidative stress, cellular damage and energy expenditure , albeit with higher turbulence . Indeed, we hypothesize that if the experiment had lasted longer (4–6 months), more significant differences in terms of coral growth and stress-related enzyme expression would have been observed between the downstream nubbins (i.e. sets 3 and 4) and the set of nubbins located 3 m behind the reef (i.e. set 6). While the results acquired here are promising, they are not entirely conclusive and they should be interpreted with caution.In contrast, the 6th set of nubbins was subjected to a flow speed similar to those experienced by the upstream ones . Although at different scales, these results are in accordance with our experiment, as hydrodynamic data and coral growth measurements suggest that with a reef of 15 cm in heigh, suitable conditions for the formation of a potential new coral patch may occur at a distance of ~ 250–350 cm downstream of the reef (15–20 times the reefs’ height). Nonetheless, this distance is probably dependent on bottom current speeds, since mounds influenced by stronger currents would be expected to cast larger water flow shadows. Lastly, if all the feedbacks are considered, faster growing reefs will also expand the area influenced by near-stagnant waters and decreased food supply, hampering the development of other reefs located directly in their wake. These underdeveloped reefs could eventually be absorbed by the growing ones or buried under layers of sediments75.Although our third hypothesis cannot be confirmed, the hydrodynamic, growth rate and physiological data acquired for the off-reef nubbins suggest that at some distance behind the reef, the environmental setting could become favourable enough to promote the rise of a potential new coral patch Fig. . If thisThe present study provides empirical data that reveal the presence of local-scale positive and negative feedbacks for coral reef growth, mediated by the framework geomorphology itself. This suggests that spatial patterns of living CWC colonies in reef patches are the result of spatial self-organisation. Nonetheless, the specific role of all the factors driving these patterns is yet to be properly described. Therefore, future studies should focus on the variables that might be affected by the interaction between reef morphology and hydrodynamics , to better understand live coral distribution on growing reefs and its potential implications for reef/mound formation over long time spans.Similar to what is naturally observed in unidirectional current settings, the results acquired in this experiment confirm that a potential colony/reef would slowly grow into the current, whereas coral colonies or polyps from the same colony living on the leeside of the coral structure would most likely die due to the absence of a sufficient food supply.Local-scale positive and negative feedbacks regulated by the reef geomorphology itself occur in different parts of the reef, even affecting the physiological condition of living coral nubbins.The water flow reestablishment and food particle distribution several meters behind the reef structure (≥ 3 m) could allow the formation of a new reef patch, thus giving rise to a potential spatial pattern of successive parallel reefs developing in a cyclic manner.The results obtained here suggest that scleractinian CWC reefs exhibit spatial self-organisation, which might help towards better understanding living coral distribution on reefs, as well as reef morphology and distribution along the seafloor. However, the changes in food supply and ion intake rates related to morphology-derived hydrodynamic variations, that potentially promote such self-organisation, are yet to be properly quantified. Hence, future experiments should be directed towards improving our knowledge on the actual role of these two factors on coral growth.L. pertusa, approximately 10–30 cm in size, were collected by means of the ROV Max-Rover  from four different locations of the Cabliers Coral Mound Province  and during the SHAKE cruise, conducted on board the R/V Sarmiento de Gamboa in April–May 2015. Collection from the Coral Mound Province was at ~ 300 m water depth in the frame of the IRIS EU FP7 Eurofleets2 project (EF2-EE-003). Coral fragments were kept alive on board and then transferred to the Institut de Ciències del Mar (ICM-CSIC) in Barcelona (Spain), where they were maintained in a 200 L water tank. The water was kept at ~ 12 °C and continuously renewed with Mediterranean Sea water pumped from 15 m depth and filtered at 50 μm (Polypropylene Net Filter)76. A submersible pump (Sicce Nano 2000) with a flow rate of 2000 L h−1 provided continuous water movement within the tank. While in the maintenance tank, corals were fed 5 times per week with frozen Mysis and Artemia salina nauplii. Prior to the start of the experiment, 30 coral nubbins containing between 3 and 5 polyps were cut from the original colonies and mounted on labelled bases using epoxy resin.Living fragments of −1 for the entire duration of the experiment, which was chosen according to the average in situ flow speed observed in thriving CWC habitats78. Flume water was continuously renewed with Mediterranean seawater pumped from 15 m depth at a rate of 120 L h–1 and filtered by 5 µm (Polypropylene Net Filter). Constant water temperature of ~ 12 °C was maintained with two chillers (Teco TK 2000).The experiment was conducted in a 3150 L flume (7 m long × 0.9 m wide × 0.5 m high) equipped with a motor-controlled propeller with a variable rotational speed , collected from coral rubble deposits of CWC reefs. Larger coral branches (up to 25 cm long) were assembled together (glued with silicone) on top of the base until the desired reef patch dimensions and shape were achieved. Size and shape of the coral structure were scaled to real reef/mound dimensions36. The reef occupied the whole width of the flume , had a length of x = 60 cm, and a maximum height of z = 15 cm at x = 20–30 cm , with a mean NO3− + NO2− concentration of 4.8 ± 1.4 µmol l–1, NH4+ of 2.26 ± 0.41 µmol l–1, SiO42− of 1.69 ± 0.61 µmol l−1 and PO43− of 0.19 ± 0.04 µmol l−1. Particulate organic carbon (POC) was measured once a week before and after feeding the corals by filtering 2 l of flume water through a GFF (0.7 µm) glass fibre filter . Mean POC concentration was 0.10 ± 0.02 mg C l−1 before, and 0.29 ± 0.12 mg C l−1 just after feeding the corals.During the experiment, coral nubbins were fed once a day with freshly hatched u) was modified by the interaction between the reef structure and the downstream flow. This allowed to define the locations of the living coral nubbins within the reef structure and along the flume, in order to expose them to contrasting current regimes and turbulence conditions. The above-mentioned vertical profiles, were carried out every 10 cm over the coral reef structure and every 40 cm behind it. Finally, the last set of measurements, performed at the end of the experiment, consisted of vertical profiles collected from 2.0 to 4.9 m along the flume length. The profiles were separated by 10 cm from 2.0 to 2.9 and by 40 cm beyond that point. In order to obtain highly detailed profiles of horizontal (u) and vertical (w) velocity components together with turbulence kinetic energy (k), the number of vertical measurements was increased to acquire data at 2, 4, 8, 12, 16, 20, 24, 28 and 32 cm above the flume bed  and vertical (w) components were calculated by computing the average of all measurements obtained for each sampling point. Outliers that indicated unreliable data  were filtered and removed from the measurements. Turbulent kinetic energy (k) was calculated in order to determine the amount of vertical mixing occurring along the flume, which has an effect on the food distribution across the water column. Data of downstream velocity (u), vertical velocity (w) and turbulence (k) were gridded using the software SURFER 11 by means of a kriging method with a linear variogram model. Within the linear variogram model, anisotropy ratio was set to 4 in order to give more importance to horizontal data similarities and avoid the creation of artefacts on the plot. Dye trials were also performed at the end of the experiment to observe how the reef deflected the flow and affected food distribution in the water column behind it  in freestream conditions prior to the placement of the coral reef structure, 2) after the placement of the reef structure but before placing the living coral nubbins in the flume, and 3) at the end of the experiment, after removing the nubbins from the flume. All water flow measurements were performed without any living corals in the flume to previously investigate which were the best locations to place the reef structure and the nubbin sets, in order to properly test our hypotheses. Before the coral reef structure was placed in the flume, 8 vertical profiles with measurements at 2, 4, 8, 16 and 32 cm above the flume bed were carried out every 50 cm along the flume. Based on these measurements, the position of the coral reef structure within the flume was established at 2.25 m from the flume start, where the flow showed a well-developed boundary layer. This also left sufficient space behind the reef structure for the water flow to potentially recover pre-reef conditions. The second set of measurements, taken at a higher horizontal resolution on and behind the reef structure, allowed to assess in detail how the horizontal velocity component of the current  in Azores , for protein expression analyses. Coral nubbin growth was expressed in percentage of weight per day40. Normality of the data and variance homogeneity were tested by means of a Shapiro–Wilks and Bartlett tests, prior to the analyses. As the data did not follow a normal distribution and neither displayed variance homogeneity, growth rates of the coral nubbins from different positions along the flume were compared by means of a non-parametric Kruskal–Wallis test. A Pairwise Wilcoxon Rank Sum test was used to determine which sets of coral nubbins presented differences. All statistical analyses were carried out by means of the statistical software R 3.2.081.The weight of the 30 living coral nubbins used in the experiment was measured at the start  and end  of the experiment by means of the buoyant weight methodg and 4 °C for 30 min. All enzyme assays were tested with commercial enzymes obtained from Sigma®. Each sample was run in triplicate . Total soluble proteins content was quantified according to Bradford method82, adapted from Bio-Rad Bradford microassay set up in a 96-well microplate. Absorbance was read at 595 nm in a microplate reader (Thermo Scientific™). A calibration curve was created using bovine serum albumin  standards (0–1 mg ml−1).Coral nubbins were crushed in a mortar and pestle with liquid nitrogen to extract 1 g of sample. All samples were homogenized at a 1:5 w/v ratio in an ice-cold 10 mM phosphate-buffered saline solution (PBS pH 7.4) containing 5 mM EDTA and 1% (v/v) ProteaseArrest™ (G-Biosciences®) cocktail. An Ultra Turrax  was used to slowly increase the rotational velocity from 8000 to 20,000 rpm during the ~ 2 min extraction time. Enzyme activities were measured in the supernatant fraction after the homogenate was centrifuged at 16,000×83 based on the competition of SOD with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (I.N.T) for dismutation of the superoxide anion (O2−). In this method, O2− radicals that quantitatively react with I.N.T to form a red formazan dye were generated by means of xanthine and xanthine oxidase. Absorbance was measured at 505 nm and 25 °C, 30 s after the addition of xanthine oxidase as start reagent across a 180 s incubation period . One unit of SOD is defined as the amount of enzyme needed to inhibit the rate of formazan dye formation by 50%. Subsequently, a SOD standard curve was employed to correlate percent inhibition of samples with SOD activity. The following equation was used to calculate the percent inhibition of standards and samples: 100 − [[ΔA505/min]/[ΔAblank/min]] × 100. The overall SOD activity in all tissues is expressed as Units per milligram of protein (U mg−1) in the sample wet weight.Total superoxide dismutase (SOD) activity was characterised spectrophotometrically through an indirect method2O2 disappearance at 240 nm  and 25 °C during a 180 s incubation period84. A total reaction volume of 2.7 ml was obtained through the combination of 50 mM potassium phosphate buffer (pH 7.0) and 13.5 mM H2O2 as a substrate, initiated by the addition of sample into quartz cuvettes with a path length of 10 mm. In order to validate the assay, catalase from bovine liver (Sigma®) was used as a positive control (1524 U ml−1). The following equation was used to calculate Catalase activity: [ΔA240/min/0.04] × [total volume/sample volume]. CAT enzymatic activity in all tissues is expressed as Units per milligram of protein (nmol min−1 mg−1) in the sample wet weight.Catalase (CAT) activity was measured spectrophotometrically by measuring the rate of H85. A colorimetric reaction which uses 1-methyl-2-phenylindole (MPI) as chromogen (Randox Ltd.) was used to measure concentrations of MDA. A chromophore with an absorbance maximum at 586 nm is generated from the condensation of one molecule of MDA with 2 molecules of MPI under acidic conditions. Concentrations of MDA in tissue samples were calculated using a standard curve prepared with freshly prepared solutions of malondialdehyde bis [dimethyl acetal] (ACROS Organics™) and values were displayed as nmol mg−1 of total protein in the sample wet weight.Lipid peroxidation (LPO) values were acquired through the quantification of a specific end-product  of the oxidative degradation process of lipidsg, 4 °C for 30 min). Activity was measured by adding 2 µl of supernatant to 1 ml of the reaction mixture containing 50 mM imidazole buffer (pH 7.4), 0.5 mM oxaloacetate, and 0.15 mM NADH at room temperature. The enzyme activity was measured by the decrease in absorbance at 340 nm, and was expressed as nmol NAD(P)+ formed per milligram of tissue protein per minute.The activity of the Malate dehydrogenase (MDH) was performed according to protocol. Nubbins samples were homogenized at a 1:5 w/v ratio in ice-cold 50 mM imidazole buffer (pH 7.4) containing 5 mM EDTA and 1% (v/v) ProteaseArrestTM (G-Biosciences®) and centrifuged (10,000×Normality of the data and variance homogeneity were tested by means of a Shapiro-Wilks and Bartlett tests, prior to the statistical analyses. Since the data did not follow a normal distribution nor displayed homogeneity of variance, the activity and/or concentration of stress-related proteins in coral nubbins from different positions along the flume were compared by means of a non-parametric Kruskal–Wallis test.


Correction: Trials 23, 459 (2022)https://doi.org/10.1186/s13063-022-06377-8Following publication of the original article , the aut1 Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, 110 Ganhe Road, Shanghai 200437, ChinaThe affiliation of Jing Zhao was incorrect upon publication. Jing Zhao is affiliated to Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, 110 Ganhe Road, Shanghai 200437, China.Further to this, the funding section has been updated as follows:This project is funded by Shanghai '13th five-year plan' key clinical specialty (TCM gynecology department) (shslczdzk04501) and General program of clinical research of Shanghai Municipal Health Commission (202140173).The original article  has been


Reoviridae and Hypoviridae, and negative and positive single-stranded RNA viruses, with relatively high representations of Rhabdoviridae and Leviviridae, respectively. Sequences corresponding to potential plant and human pathogens were also detected. The detected RNA viruses primarily targeted dominant eukaryotic taxa in the samples  and the viral community structures were significantly associated with predicted host populations. These results indicate that RNA viruses are linked to eukaryotic host dynamics. Several of the RNA viral sequences contained auxiliary metabolic genes encoding proteins involved in carbon utilization , implying their potential roles in carbon cycling in thawed permafrost.Arctic permafrost is thawing due to global warming, with unknown consequences on the microbial inhabitants or associated viruses. DNA viruses have previously been shown to be abundant and active in thawing permafrost, but little is known about RNA viruses in these systems. To address this knowledge gap, we assessed the composition of RNA viruses in thawed permafrost samples that were incubated for 97 days at 4°C to simulate thaw conditions. A diverse RNA viral community was assembled from metatranscriptome data including double-stranded RNA viruses, dominated by IMPORTANCE Permafrost is thawing at a rapid pace in the Arctic with largely unknown consequences on ecological processes that are fundamental to Arctic ecosystems. This is the first study to determine the composition of RNA viruses in thawed permafrost. Other recent studies have characterized DNA viruses in thawing permafrost, but the majority of DNA viruses are bacteriophages that target bacterial hosts. By contrast RNA viruses primarily target eukaryotic hosts and thus represent potential pathogenic threats to humans, animals, and plants. Here, we find that RNA viruses in permafrost are novel and distinct from those in other habitats studied to date. The COVID-19 pandemic has heightened awareness of the importance of potential environmental reservoirs of emerging RNA viral pathogens. We demonstrate that some potential pathogens were detected after an experimental thawing regime. These results are important for understanding critical viral-host interactions and provide a better understanding of the ecological roles that RNA viruses play as permafrost thaws. Rhabdoviridae was the most abundant (-) ssRNA viral family detected   ssRNA viruses that had the highest representation of members of Leviviridae, Closteroviridae, and Picornaviridae  . Guampa diseases . Given thabitats , the finaviridae  to f. Todetected . During Arthopoda)   Fig. 2,thopoda)    , California grassland (nodes in brown), Kansas grassland (nodes in yellow) and invertebrates (nodes in red) were clustered and non-singleton clusters are visualized in the network. Download https://doi.org/10.1128/AuthorWarrantyLicense.v1This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.The California and Kansas grassland RdRp sequences grouped into 1387 and 189 clusters and shared the most RNA viral clusters with each other  . The majMitoviridae and Narna-Levi were the most diverse phylogenetic groups with relatively more RNA viral clusters assigned  detected from the four biomes (‘Ecosystem’) were then mapped to the assigned phylogenetic groups (‘Phylogenetic group’) . The claassigned , and the10.1128/msystems.00582-22.5FIG S2FIG S2, PDF file, 1.6 MB.Phylogenetic assignments of the RNA viral clusters detected from each ecosystem. The alluvial plot shows the pairings of the RNA viral clusters  detected from each ecosystem (‘Ecosystem’) to the phylogenetic group assignments (‘Phylogenetic group’). The alluvia were colored by the phylogenetic assignment of each detected permafrost RNA viral cluster. Download https://doi.org/10.1128/AuthorWarrantyLicense.v1This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.Viridiplantae, fungi, and the SAR supergroup . The potential hosts of the detected RNA viruses were also linked to the dominant groups within the eukaryotic communities . Another potential limitation of using existing databases to predict hosts of the detected RNA viruses is that our current knowledge of plant viruses is more complete relative to that of fungal viruses. This may partially contribute to the higher representation of plant viruses detected in our data set.To identify the potential hosts of the RNA viruses in thawed permafrost, we first established viral-host linkages by retrieving host assignments of the detected RNA viral families from the Virus-Host database (DB) . The majmunities . One limMetazoa, Viridiplantae, SAR supergroup , fungi, and Discoba (Excavata is a clade of Discoba) were detected as the main eukaryotic groups , independently. Eukaryotic community composition best explained viral community composition and supported a final model with four variables  fitting the data with an adjusted R2 = 0.71. The analysis linking environmental variables to viral community composition supported a final model with one variable (oxygen concentration post-incubation) fitting the data with an adjusted R2 = 0.14. Spatial variation based on PCNMs original permafrost sampling location alone was not significantly associated with the RNA viral communities  further supported a close linkage between the detected RNA viruses and eukaryotes in thawed permafrost. Forward stepwise model building based on adjusted Rmunities .10.1128/msystems.00582-22.6DATA SET S1Data Set S1, DOCX file, 0.02 MB.Eukaryotic hosts influence RNA viral community assemblages in thawed permafrost. The RDA modeling results are shown for each analysis. Download https://doi.org/10.1128/AuthorWarrantyLicense.v1This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.2 = 0.29. Oxygen concentration as an emergent condition post-thaw has also been shown to impact the bioavailability of organic matter  viruses  and to an avian infectious bronchitis virus . No evidence to date shows that the two detected strains can infect humans based on the Immune Epitope Database and Analysis Resource (http://www.iedb.org/home_v3.php). However, based on these data, it is still not possible to determine if there are other unclassified viral sequences that could represent infectious human viral pathogens. By contrast, known bacterial pathogens  have emerged from thawed permafrost (Aspergillus)  . Fragmenrgillus) . Our resPartitiviridae, Reoviridae, Totiviridae, Hypoviridae, Mitoviridae, and Narnaviridae (Partitiviridae and Totiviridae have co-evolved with their respective hosts and evidence suggests they may have been integrated into host genomes (Partitiviridae) are a family of cryptic viruses  infections , 53, weraviridae  and c. M genomes . For exa viruses , 56 whos viruses . Thus, tFlaviviridae and Totiviridae)  in RNA viruses , 59. Theviridae) , 60 wereviridae) . A rangeviridae) . These cviridae) . Exampleviridae) .10.1128/msystems.00582-22.3TABLE S3Table S3, XLSX file, 0.06 MB.Functional annotation of detected RNA viral contigs. Information for each predicted protein from RNA viral contigs and their functional annotations with statistics. Download https://doi.org/10.1128/AuthorWarrantyLicense.v1This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.Rhabdoviridae and Phasmaviridae, with Eukaryotes as hosts. These RNA viruses, unlike the ones that are passed via host meiosis, are known to replicate cytoplasmically and release from hosts via budding  and cell wall hydrolase . To our  budding , which m budding . Alterna10.1128/msystems.00582-22.7DATA SET S2Data Set S2, DOCX file, 0.02 MB.Conserved regions of functional genes across taxa. Download https://doi.org/10.1128/AuthorWarrantyLicense.v1This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.Leviviridae- and Reoviridae-like RNA viruses , and the samples were immediately flash frozen in liquid nitrogen.Permafrost cores were collected from the Caribou Poker Creek Research Watershed near Fairbanks, AK in August 2015. A total of 33 permafrost samples along four parallel transacts were collected and preserved frozen on dry ice during shipment to the Pacific Northwest National Laboratory and subsequently stored at −20°C as intact cores . Duplicate extractions were done strictly to get enough RNA for sequencing, and these duplicate extractions were performed consistently for each sample to ensure the RNA pools represented extracts from the same amount of total soil for each sample . To guard against contamination, all manipulations of frozen samples were performed using flame-sterilized steel implements, on heat sterilized aluminum foil-covered surfaces (450°C for 8 h) in a biological safety cabinet. Samples were homogenized in purpose-built stainless steel sample homogenizers, which were alcohol washed, flame-sterilized, and cooled on dry ice. Samples were poured directly from the homogenizers into sterile Falcon tubes and stored at −80°C until extraction. Samples were transferred from Falcon tubes into extraction tubes using a flame-sterilized scoopula. Individual RNA extractions yielded between 800 ng and 6500 ng of RNA per extraction, and pools of duplicate extractions contained between 1900 ng and 11970 ng RNA total, as determined by Qubit RNA HS kit assays (Thermo Fisher Scientific), which provided sufficient RNA for sequencing.de novo assembled by rnaSPAdes (v3.13.0) using default settings.Metatranscriptomic sequencing was completed on Illumina HiSeq2500 sequencing platform at Argonne National Laboratory (Lemont) , complemented with the whole sequence similarity searches against NCBI RNA viral genomes . When the assembly was too short to detect a RdRp gene, the identified RNA viral contig inherited the taxonomy assignment of the qualified hit in NCBI RNA viral genomes and the RdRp gene from the qualified hit genome was extracted to represent the phylogenetic placement of the short RNA viral assembly. Taxonomy assignment of the identified RNA viral contigs was based on RdRp gene detected or assigned. As the RNA viral contigs were classified based on RdRp, RNA viruses without the RdRp gene, such as retroviruses and plant viroids  genomes vary in size by 1 order of magnitude , we did  viroids , were noAlphaproteobacteria (WP_012231479.1) as an outgroup using MAFFT . The multiple sequence alignment (MSA) was manually inspected and adjusted in Jalview (v2.11.1.4). The MSA was then used to construct a phylogenetic tree for each group of RNA viruses that was rooted by the outgroup using FastTree (v2.1.10).All the retrieved RdRp amino acid sequences were de-replicated at 99% amino acid identity using CD-hit . The lonhttps://www.genome.jp/virushostdb/) with the virus-host pairing information manually curated from NCBI Virus, Refseq, UniProt, and related literature. For a relatively conservative prediction, host lineage was assigned to Phylum or Superphylum level.As the majority of detected RNA viral contigs were classified and assigned to a Family based on RdRp, we can predict their hosts by leveraging the Virus-Host DB . We then used samtools  to calculate the reads coverage per base for each assembled contig. The abundances of the identified RNA viral contigs were estimated by the average base-coverage of the assembly after normalizing by the total quality-filtered forward reads across all the samples.The average read coverages of the detected RNA viral contigs were calculated to represent their genomic coverage. To avoid double counting, only the quality-filtered forward metatranscriptomic reads were mapped to the To compare RNA viruses detected in thawed permafrost to those previously detected in other terrestrial habitat types, we collected the RdRp protein sequences from metatranscriptome data from three published studies; one from invertebrates  and two hmmsearch (HMMER 3.3) with the parameters as previously described . The protein sequences were then annotated by a collection of databases using escribed . In brieDue to their relatively high prevalence in our samples, polygalacturonase and cell wall hydrolase were selected to construct protein trees and to assess their sequence relatedness to the close hits in the public database. The protein sequences were first dereplicated at 99% identity using CD-HIT  and searhttps://github.com/Ecogenomics/BamM) and filtered by percent identity higher than 0.95 and percent alignment greater than 0.80 . The transcript abundances of members of the Eukarya were estimated by the average base coverage of the mapped 18S rRNA reference sequences  normalized by the total counts of reads per sample.The 18S rRNA transcript reads that were extracted from the metatranscriptomes were analyzed to determine eukaryotic community compositions. The quality-filtered forward metatranscriptomic reads were first mapped to a well-curated set of reference sequences in the Silva Database (silva-euk-18s-id95) using BamM  transformed values of viral community composition using three data sets independently as variables: (i) log (x + 1) transformed values for eukaryotic community composition, (ii) standardized environmental variables, and (iii) PCNMs derived from GPS coordinates of original sampling locations. The same modeling approach was used to determine relationships between log (x + 1) transformed values of eukaryotic community composition and standardized environmental variables. All models were run with 200 permutations.An RDA modeling approach was used to evaluate how much variation in the thawed permafrost RNA viral communities could be explained by eukaryotic community composition, environmental variation, and spatial variables (PCNMs) using the PRJNA852283. The RdRP profile HMMs and the R code used for statistical analysis and data visualization are available at https://github.com/Ruonan0101/PermafrostVirome_PNNL.The raw metatranscriptome data were deposited to NCBI with BioProject ID


Ulcerative colitis (UC) is an inflammatory disease of the intestinal tract with unknown etiology. Both genetic and environmental factors are involved in the occurrence and development of UC. Understanding changes in the microbiome and metabolome of the intestinal tract is crucial for the clinical management and treatment of UC.Here, we performed metabolomic and metagenomic profiling of fecal samples from healthy control mice (HC group), DSS  -induced UC mice (DSS group), and KT2-treated UC mice (KT2 group).Bacteroides, Odoribacter, and Burkholderiales, which were correlated with aggravated UC, and Anaerotruncus, Lachnospiraceae, which were correlated with alleviated UC. We also identified a disease-associated network connecting the above bacterial species with UC-associated metabolites, including palmitoyl sphingomyelin, deoxycholic acid, biliverdin, and palmitoleic acid. In conclusion, our results indicated that Anaerotruncus, Lachnospiraceae, and Mucispirillum were protective species against DSS-induced UC in mice. The fecal microbiomes and metabolomes differed significantly among the UC mice and KT2-treated and healthy-control mice, providing potential evidence for the discovery of biomarkers of UC.In total, 51 metabolites were identified after UC induction, enriched in phenylalanine metabolism, while 27 metabolites were identified after KT2 treatment, enriched in histidine metabolism and bile acid biosynthesis. Fecal microbiome analysis revealed significant differences in nine bacterial species associated with the course of UC, including  ComplexChanges in the intestinal microbiota are a crucial environmental factor in the development of IBD . Animal Antimicrobial peptides (AMPs) are short amino acids with innate immune defense functions  that regKT2 is a cationic, amphipathic, ultra-short AMP, which shows antibacterial activity against both gram-negative and gram-positive bacteria but low toxicity toward normal cells . We prevBacteroides, Odoribacter, Anaerotruncus, and Lachnospiraceae, were similar between the heathy control (HC) and KT2-treated UC mice, but not with the non-treated UC mice. This study identified several metabolites as potential biomarkers for UC, which may help guide the application of specific probiotics in UC treatment.We hypothesized that KT2 alters the composition of the intestinal microbiota, and thus the metabolites in UC patients. Therefore, in the present study, we characterized the changes in the intestinal microbiota and metabolites in UC mice with and without KT2 treatment. The bacterial abundances of several species, including 2.2.1.Twelve C57BL/6 male mice  were bought from the Animal Experiment Center of Kunming Medical University . All mice were randomly divided into three groups and kept in cages at 25 ± 2°C. To avoid cage and maternal effects on the gut microbiome , the micThe KT2 peptide (purity >95%) was provided by Nanjing Jiepeptide Biotechnology Co., Ltd. DSS was provided by the MP Corporation (USA). The KT2 was prepared as 2 mg/mL solution in phosphate-buffered saline (PBS) and stored at −80°C. The UC model was established according to prior research  and KT2 All animal assays were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of Kunming Medical University, China (No. kmmu20211575).2.2.Each fecal sample (200 mg) from HC, UC and KT2 groups was suspended in 250 μl of guanidine thiocyanate with 0.1 M Tris (pH 7.5) and 40 μl of 10% N-lauroyl sarcosine. DNA was extracted using a Qiagen QIAamp DNA Stool Mini Kit . DNA concentration was estimated using a NanoDrop instrument , and DNA molecular weight was estimated by agarose gel electrophoresis.A DNA library was constructed according to previous study . Paired-2.3.Clean reads of metagenome sequencing were processed with MetaPhlAn2  to obtai2.4.Metabolites were profiled in fecal samples. Briefly, the fecal samples (50 mg) were completely homogenized in a 2-mL tube, followed by the addition of 800 μL of 80% methanol. After vortexing for 90 s at 65 Hz, the mixture was ultrasonically treated for 30 min, then maintained at −20°C for 1 h. The mixture was then centrifuged at 12,000 rpm for 15 min at 4°C. The resulting supernatant  was filtered through a 0.22 μM membrane, with the filtrate (200 μL) then mixed with 5 μL of lysophosphatidylcholine  and transferred to a 1.5-mL microcentrifuge tube.Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) was performed with a Waters ACQUITY UPLC Scientific Q-Exactive high-resolution mass spectrometer  and an ACQUITY UPLC HSS T3 column (2.1 × 100 mm and 1.7 μm). The column temperature was set to 40°C and the flow rate was set to 0.3 mL/min. Mobile phase A consisted of water and 0.05% formic acid, and mobile phase B consisted of acetonitrile. The injection volume was 6 μL at 4°C.The positive-ion conditions were as follows: Heater temperature, 300°C; sheath gas flow rate, 45 arbs; aux gas flow rate, 15 arbs; sweep gas flow rate, 1 arb; spray voltage, 3.0 kV; capillary temperature, 350°C; and S-lens RF level, 30%. The negative-ion conditions were as follows: Heater temperature, sheath gas flow rate, aux gas flow rate, sweep gas flow rate, and capillary temperature consistent with positive-ion conditions; spray voltage, 3.2 kV; and S-lens RF level, 60%. The MS analysis was performed in full-scan dd-MS2 (TopN = 10) mode with a scan range of 70 to 1,000 m/z.2.5.p < 0.05 (Student’s t-test), log2FC > 1, and Variable Importance in Projection (VIP) > 1.Multivariate analysis was used to analyze differences in metabolites among the three groups. Differential metabolites (DMs) were selected based on 2.6.To investigate the similarity in DMs functions, we performed hierarchical cluster analysis using complete-linkage and counting the Euclidean distance matrix. The results were visualized as a heatmap.2.7.MetaboAnalyst2.8.r > 0.05 and p < 0.05 were considered statistically significant.Correlations between DMs and differential microbes were analyzed by calculating Pearson correlation coefficients. Correlations with Spearman’s 2.9.Colon tissue samples were kept in 10% formaldehyde  for 1 d. After dehydration and paraffin infiltration, the tissues were embedded in paraffin and cut into 5-μm thick sections. The slices were then stained with H&E  and imaged under a light microscope  for histological evaluation based on previously described parameters .2.10.Transforming growth factor β (TGF-β) concentrations in the colon calprotectin (FC) concentrations in the feces were detected using ELISA kits  in accordance with the manufacturer’s instructions. Optical density (OD) was measured at 450 nm and the corresponding concentration was calculated.2.11.TNF-α), Interleukin-6 (IL-6), and Interleukin-1 beta (IL-1β) in colon tissues were analyzed by comparing threshold period (Ct) analysis of the data with GAPDH as an internal reference.RNA-Easy™ Isolation Reagent  was used to extract RNA from the colon. A Maxima First-Strand cDNA Synthesis Kit  was used for reverse transcription synthesis of cDNA. The mRNA level of individual genes was measured by RT-PCR with a fluorescent quantitative reagent. The relative expression levels of inflammation-related genes Tumor necrosis factor ligand member 2 . Spearman correlation analysis was performed between microbiota and metabolites. Significant differences were defined at 3.3.1.TNF-α, IL-6, IL-1β, and fecal calprotectin were increased in the DSS group but decreased in the KT2-treated group to levels similar to those in the HC group , but no significant differences between the DSS and KT2-treated groups. The Shannon and Chao 1 indices were similar in the HC, DSS, and KT2 groups. There was no significant differences in the Shannon indices among the HC, DSS, and KT2 groups  and Firmicutes (phyla) were dominant in the HC  and KT2  groups. However, in the DSS group, Anaerotruncus and Firmicutes abundance was low , with Bacteroidetes (phyla) and Alistipes (genus) found to be most dominant . In contrast, the HC and KT2 groups showed a low abundance of Bacteroidetes  and Alistipes .The proportions of dominant taxa at the phylum and genus level were assessed among the different groups. We observed considerable variation in the gut microbiota of each group. The top 10 differentially enriched phyla and genera in the different groups were identified ,D. AnaerLachnospiraceae, Ruminococcaceae, Oscillospiraceae, Eubacteriaceae, and Deferribacteraceae were the most abundant microbiota in the HC group, with LDA scores (log10) > 3.6, whereas Porphyromonadaceae, Rikenellaceae, Bacteroidaceae, Sutterellaceae, and Enterobacteriaceae were significantly over-represented in the DSS group, with LDA scores (log10) > 3.5  > 3.5  effect size (LEfSe) was used to generate a cladogram to identify specific microbes associated with DSS and KT2 treatment . Lachnos0) > 3.5 ,B. Simil0) > 3.5 ,D. LEfSeA effect 0) > 3.5 . These d3.3.Given the association between intestinal microbes and UC, we performed metabolomic analysis of fecal samples. The differentially changed metabolites in the HC, DSS, and KT2 groups were shown in We also identified several metabolites changed in both the DSS and KT2 groups , which m3.4.Bacteroides and Lachnospiraceae were associated with most DMs between the HC and KT2 groups, with opposite correlation relationships  and DMs . ResultsBacteroides and a group of 12 metabolites (three enriched and nine depleted in the DSS group), associations between Odoribacter and a group of 12 metabolites (two enriched and 10 depleted in the DSS group), and associations between Anaerotruncus and a group of 17 metabolites , associations between Lachnospiraceae and a group of five metabolites (two enriched and three depleted in the KT2 group), and associations between Burkholderiales and a group of three metabolites , indicating an association between lymphocyte infiltration and Lachnospiraceae family enrichment in the gut microbiome  are protective against serovar Typhimurium-induced colitis in mice by interfering with pathogen invasion and virulence factor expression  and immunoglobulin G (IgG) responses, suggesting that these species may exhibit efficient immune priming  cell function upon activation during DSS colitis  can be found in the article/The animal study was reviewed and approved by the Animal Ethics Committee of Kunming Medical University (No. kmmu20211575).JM, YM, and JJ conceived and designed the study. QN and YY performed study subject recruitment, oversaw sample collection, metagenomic sequencing, and metabolite analysis. XJ performed hematoxylin–eosin (H&E) staining and enzymelinked immunosorbent assay. JJ and YT contributed to data analysis. JM and QN contributed to manuscript preparation. All authors reviewed the draft version and approved the final submission. All authors contributed to the article and approved the submitted version.This work was partly supported by the National Natural Science Foundation of China , Medicine Leading Talent of Health and Family Planning Commission of Yunnan Province (L-201607), and Yunnan Health Training Project of High Level Talents (H-2018040). The “Rejuvenating Yunnan Talents Support Plan” for Prestigious Doctors (RLMY20220010).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.https://www.frontiersin.org/articles/10.3389/fmicb.2023.1027658/full#supplementary-materialThe Supplementary material for this article can be found online at: Click here for additional data file.


These vRNPs depend on regions of N protein that promote protein–RNA and protein–protein interactions. Phosphorylation of N protein in its disordered serine/arginine region weakens these interactions to generate less compact vRNPs. We propose that unmodified N protein binds structurally diverse regions in genomic RNA to form compact vRNPs within the nucleocapsid, while phosphorylation alters vRNP structure to support other N protein functions in viral transcription.The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 is responsible for compaction of the ∼30-kb RNA genome in the ∼90-nm virion. Previous studies suggest that each virion contains 35 to 40 viral ribonucleoprotein (vRNP) complexes, or ribonucleosomes, arrayed along the genome. There is, however, little mechanistic understanding of the vRNP complex. Here, we show that N protein, when combined  At different stages of the viral life cycle, viral genomes switch between two distinct structural states: a tightly packaged state inside the virion and a decondensed state that serves as a substrate for translation, transcription, or other processes in the infected cell. The mechanisms that govern the switch between these states are not well understood.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, is a highly contagious betacoronavirus . FollowiTranscription of structural protein genes by the viral RNA-dependent RNA polymerase generates negative-sense subgenomic RNAs through a template switching mechanism. These RNAs are then transcribed to positive-sense RNAs, which are translated to produce the spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins . The S, A) (A) (The 46 kDa N protein contains two globular domains flanked by three regions of intrinsic disorder A 20). T. TA) (20A) , 25, 26.A) A 21, 23, 23A (A)A) (A) , 28, 29.A) (A) , 31, 32.A) (A) , 34, 35.in vitro , indicating that N protein bound the RNAs and retarded their electrophoretic mobility.We previously observed vRNP complexes t of RNA , 44, 45.t of RNA B, indicaC). N protein mixed with 5′-800 RNA formed two broad peaks: one smaller peak that appears similar in size to the 5′-600 species (both ∼750–800 kDa) and a second larger peak roughly twice as large as the first (∼1400 kDa). This suggests that one (∼750 kDa) or two vRNPs (∼1400 kDa) can form on a single 5′-800 RNA molecule (C). The 5′-600 RNA was therefore chosen as a representative viral RNA for further study.We used mass photometry to characterize these RNA–N protein complexes. Mass photometry uses light scattering to measure the mass of single molecules in solution, resulting in a histogram of mass measurements centered around the average molecular mass of the protein complex. N protein in complex with 5′-400 RNA resulted in two mass peaks that were smaller than the single broad peak of N protein bound to 5′-600 RNA, suggesting the 5′-400 vRNP was not fully assembled and contained subcomplexes C. N protmolecule C. The 5′D, top). We observed a broad RNA–protein peak that migrated in the gel at the same size (at the 1000 nt marker) as that seen in native gel electrophoresis of unpurified complexes . Peak fractions (7 and 8) were combined for analysis by mass photometry. We observed three major peaks centered at 97 ± 2 kDa, 207 ± 6 kDa, and 766 ± 6 kDa , unbound 5′-600 RNA (predicted mass 192.5 kDa), and the vRNP complex, respectively. The presence of free N protein dimer and unbound RNA suggests that the vRNP complex dissociated upon dilution for mass photometry analysis.To purify the vRNP complex for more detailed analysis, we used velocity sedimentation on a glycerol gradient, which is commonly used for purification of nucleosomes . N proteFig. 1D, bottom). The distribution of vRNP complexes across the gradient was similar in the two conditions, although crosslinked preparations contained additional large species at the bottom of the gradient, which are likely to represent crosslinked complexes of multiple vRNPs. The GraFix-purified main peak (fractions 7 + 8) was analyzed by mass photometry, revealing one sharp peak with an approximate mass of 719 ± 7 kDa  was added to the 40% glycerol buffer, creating a gradient of glutaraldehyde to crosslink the protein complex during centrifugation  . AnalysiFig. 1F). Two-dimensional classification revealed particles with variable composition and conformation, suggesting inherent structural heterogeneity in the vRNP complex that may result from the diverse RNA secondary structures in the 600 nt RNA strand (see A). While these averages are heterogeneous, they are similar in size and shape to vRNP complexes previously observed within SARS-CoV-2 virions by cryo-electron tomography  5′-600 (nucleotides 1–600), (2) Nsp3 (nucleotides 7800–8400), (3) Nsp8/9 (nucleotides 12250–12850), and (4), Nsp10/12 (nucleotides 13200–13800). All RNAs appeared to form vRNPs (G). Each of these regions is known to contain a diverse array of secondary structure in infected cells and in the mature virion  (B and S1A). Each crosslinked complex was analyzed by mass photometry. SL4a mixtures contained three broad peaks of 515 ± 19 kDa, 739 ± 5 kDa, and 876 ± 1 kDa  B and S1AC). Analysis of peak fractions (7 + 8) by mass photometry (D) indicated that SL8 vRNPs were similar in mass to vRNPs assembled with 5′-600 RNA (E). Negative stain EM and two-dimensional class averages revealed ring structures that resemble vRNPs assembled with the 5′-600 RNA .SL8-containing vRNPs were purified by GraFix C. Analysotometry D indicat-600 RNA E. NegatiThese data suggest that ribonucleosome formation does not require 600 continuous bases of RNA but can be achieved with multiple copies of a relatively short and simple stem-loop structure. Unlike the 5′-600 RNA, the short stem-loop RNA is unlikely to serve as a platform to recruit multiple copies of N protein to assemble a vRNP. We speculate that the binding of a short RNA to N protein induces a conformational change that promotes protein–protein interactions required for vRNP formation. In the more physiologically relevant context of long RNAs, these weak protein–protein interactions are likely stabilized by multivalent interactions with an RNA molecule.A, bottom). The mSL8 RNA promoted vRNP formation across a range of N protein concentrations . Analysis by GraFix and mass photometry confirmed that mSL8 forms a heterogeneous vRNP that is similar in size to vRNPs containing SL8 (B and C). mSL8 vRNPs included some higher molecular mass species in the native gel and GraFix experiments. Thus, vRNPs can form in the presence of structured and largely unstructured RNA molecules, but vRNPs assembled with different RNAs are likely to exhibit differences in overall structure or composition. We therefore speculate that the structural heterogeneity of the viral genome results in variations in the structure of the vRNPs of the viral nucleocapsid.To test the requirement for secondary structure in vRNP formation, we generated a mutant SL8 (mSL8) carrying 12 mutations predicted to abolish the SL8 stem-loop structure . These data support the possibility that the vRNP assembled with 5′-600 RNA (E) contains 12 N proteins bound to one RNA.Analysis of noncrosslinked SL8-N protein complexes provided important clues about vRNP assembly. Mass photometry of the SL8-N sample revealed a major species at ∼110 kDa, with five evenly spaced complexes every 120 to 130 kDa thereafter up to ∼755 kDa  . Based on the similar molecular mass of the smaller peak in the crosslinked sample (737 ± 11 kDa) to the noncrosslinked sample (736 ± 27 kDa), we suspect that the larger crosslinked complex of 840 ± 12 kDa contains 14 N proteins. These results suggest that the ribonucleosome defaults to a stable complex of 12 N proteins bound to a variable number of RNA stem-loops but can adapt to accommodate fewer or more N protein dimers bound to additional RNA.In some crosslinked vRNP preparations, we observed an additional large peak in mass photometry that is likely to contain more than 12 N proteins. As mentioned above, crosslinked SL8 vRNPs contain a broad peak of 840 ± 12 kDa in addition to the 737 ± 11 kDa peak .Next, we sought to explore the regions of the N protein required for vRNP formation. We analyzed mutant proteins lacking the following regions: (1) the 44-aa NTE, a poorly conserved disordered sequence , 51; (2)Figs. 3B). All mutant N proteins, with the exception of the CTE deletion, appeared to form fully assembled vRNPs. Most mutants displayed a small population of lower bands beneath the fully shifted vRNP. These lower bands might represent subcomplexes in which the 5′-600 RNA is bound to fewer N proteins, presumably due to defects in vRNP assembly or stability. Deletion of the CTE resulted in a small shift that was considerably lower than the fully shifted vRNP. These results suggest that the ΔCTE N protein binds RNA but fails to form the fully assembled vRNP, hinting at an important role for the CTE in vRNP formation.Mutant N proteins were mixed with 5′-600 RNA and analyzed by native gel electrophoresis B. All muC and D and Studies of deletion mutants in complex with SL8 RNA, which minimizes the contribution of multivalent RNA binding, allowed us to investigate the critical protein–protein interactions that contribute to ribonucleosome formation. Mutant N proteins were mixed with SL8 RNA, crosslinked, and analyzed by native gel electrophoresis and mass photometry (C). These mutants appeared cloudy, and turbidity analysis revealed a higher absorbance at 340 nm compared to wildtype, suggesting formation of biomolecular condensates (B) , further confirming the essential role of the CTE in vRNP formation and suggesting that multimerization driven by the CTE is required for ribonucleosome formation or stability.Deletion of the CTE and LH resulted in almost complete disappearance of the vRNP when analyzed by native gel electrophoresis C. These densates B 39). M. MC). ThC and D and Deletion of the SR or CBP region also resulted in defects in vRNP assembly; both mutants exhibited a laddering of ribonucleoprotein subcomplexes when analyzed by native gel electrophoresis, as well as stepwise 120 to 130 kDa increases in molecular mass revealed by mass photometry .To test if SR deletion causes defects by reducing the length of the central disordered region, we also constructed an ‘SR linker’ mutant in which the 31-aa SR region is replaced with a random sequence of glycines, alanines, and serines. This mutant displayed defects similar to those seen with the SR deletion, confirming that specific sequence features of the SR region are required for vRNP formation C.C and S3C), suggesting reduced RNA binding in these mutants. Many of the deleted regions  have been implicated in protein–protein and protein–RNA interactions. RNA binding defects might result simply from partial loss of RNA-binding sites or they could occur because cooperative RNA binding is associated with vRNP assembly.Native gel analysis revealed an increase in free SL8 RNA in some mutant N protein samples compared to wildtype C and S3CA, left). The mutant formed an appropriately sized vRNP, as well as minor subcomplexes below the fully assembled vRNP. GraFix purification of the 10D mutant in complex with 5′-600 RNA revealed a range of ribonucleoprotein complexes similar to that seen with wildtype N protein . Mass photometry of fractions 7 + 8 confirmed a similar mass of the 10D and wildtype vRNPs, apart from a minor ∼830 kDa peak observed with the 10D mutant .The SR region of N protein is heavily phosphorylated in cells infected by SARS-CoV-2, and this modification promotes the protein’s role in viral transcription , 32, 57.Fig. 4C compared with Fig. 1F). The 10D complex appears extended and heterogeneous, unlike the compact structure of the wildtype vRNP, and does not average into discrete, recognizable two-dimensional classifications. We therefore speculate that the 600 nt RNA provides sufficient binding sites for twelve 10D N proteins, but the 10D mutant is unable to condense into the compact ring structure observed with the wildtype N protein.Negative stain EM and two-dimensional class average analysis of the GraFix-purified 10D ribonucleoprotein complex, however, revealed a markedly different structure compared to the wildtype vRNPs . Both assays revealed a laddering of vRNP complexes, consistent with an inability of the 10D mutant to form a stable, fully assembled vRNP. Purification of the 10D + SL8 complex by GraFix revealed a clear shift toward lower molecular mass species when compared to wildtype N . This result was confirmed by mass photometry analysis of fractions 19 + 20 (B). Interestingly, the minimal unit of vRNP complex assembly with the 10D mutant (like the SR deletion) is ∼230 kDa, which is consistent with an N protein tetramer bound to two SL8 RNAs. Negative stain EM and two-dimensional class averages of the GraFix-purified complex (fractions 19 + 20) revealed a smaller overall structure with an electron density distribution clearly distinct from vRNP complexes formed by wildtype N .vRNP formation with the SL8 RNA was severely reduced in the 10D mutant when analyzed by native gel electrophoresis and mass photometry . Consistent with this model, we observed maximal phosphorylation of N in the presence of all three kinases (B). Phosphorylation was greatly reduced when both SRPK priming sites were mutated to alanine (S188A + S206A mutant) (B). We mixed kinase-treated wildtype or S188A + S206A N proteins with SL8 RNA and purified the resulting vRNP complexes by GraFix (C). Wildtype phosphorylated N protein migrated as a low molecular weight ribonucleoprotein complex similar to the 10D mutant. The poorly phosphorylated S188A + S206A mutant, however, formed a vRNP similar to wildtype unphosphorylated N protein (C). Mass photometry of the GraFix-purified samples further substantiated the defect in wildtype phospho-N vRNP assembly , which is rescued by mutation of the two priming phosphorylation sites (the S188A + S206A mutant) .We next tested vRNP assembly with N protein that had been phosphorylated rylation , 57, 58.  B. We mixy GraFix C. Wildty protein C. Mass passembly D, top, w mutant) D, bottomin vitro to form ribonucleosomes. These structures, which have been observed previously in intact SARS-CoV-2 virions by cryo-electron tomography  do not fall apart as readily when diluted for mass photometry and do not require crosslinking for visualization by native gel electrophoresis. These results suggest that vRNPs assembled with 600-nt RNAs are more stable than those formed with multiple copies of a single short RNA, potentially because a single long RNA provides binding sites for all 12 N proteins in the vRNP. This multivalent RNA scaffold stabilizes low-affinity protein–protein interactions within the vRNP and reflects the more physiologically relevant state of RNA compaction by N protein in the virion.Phosphorylation of N protein in its disordered SR region results in a less compact vRNP structure, providing further mechanistic insight into the functions of N protein phosphorylation and dephosphorylation during coronavirus infection. Given the high level of N protein phosphorylation in the infected cell, mechanisms must exist to generate a poorly phosphorylated N protein population at sites of viral assembly.Coronavirus genomic RNA is structurally heterogeneous , 47, andOur studies of ribonucleosome assembly with a small stem-loop RNA demonstrate that the vRNP is compositionally adaptive; that is, it can contain a variable number of N protein dimers bound to a variable number of stem-loop RNAs, and assembles by iterative additions of N protein dimers bound to stem-loop RNAs. Our data suggest that the most stable form of the vRNP is 12 N proteins in complex with ∼600 nt of RNA, but we also observed complexes that contain fewer or more N protein dimers. Given the iterative assembly of the vRNP, the multitude of protein–protein and protein–RNA interactions, and the high concentrations of N protein and RNA in the nucleocapsid, it seems reasonable to expect that the vRNP can expand to expose binding sites that allow additional N protein dimers to insert themselves into, or dissociate from, the vRNP complex.Our results, together with data from other studies, provide insights into the general architecture of coronavirus RNA packaging. There are ∼38 vRNPs per virus , 45, witDuring viral assembly, one copy of the ∼30 kb viral genome is packaged in each virus, while cellular and subgenomic viral RNAs are excluded . In muriM protein is a 25 kDa structural protein containing three transmembrane helices followed by a ∼100 aa CTD that faces the interior of the virion and is thought to interact with the C terminus of N protein , 68, 69.et al. (A). Our results are consistent with their model. We also show that phosphorylated N protein cannot form compact ribonucleosomes and instead forms elongated, heterogeneous vRNP structures when mixed with longer viral RNA. Perhaps these heterogeneous vRNPs help maintain RNA in an uncompacted state that facilitates RNA processing at the RTC.N protein is highly phosphorylated in the cytoplasm of infected cells, and numerous kinases have been implicated in this process , 57, 58.et al.  recentlyet al. A. Our reChemical inhibition of SRPK with the FDA-approved drug Alectinib severely reduces replication of SARS-CoV-2 in multiple cell types . AdditioE. coli BL21 Star (Thermo #C601003), grown in TB–Kanamycin to absorbance 0.6, and induced with 0.4 mM IPTG. Cells were harvested, washed with PBS, snap frozen in LN2, and stored at −80 °C until use. Thawed cells were resuspended in buffer A  and lysed by sonication. The lysate was clarified by centrifugation and bound to Ni-NTA agarose beads (QIAGEN #30230) for 45 min at 4 °C. Ni-NTA beads were washed three times with ten bed volumes of buffer A and eluted with buffer B . The eluate was concentrated in centrifugal concentrators (Millipore Sigma #UFC803024), transferred to dialysis tubing (Spectrum Labs #132676), and renatured overnight by dialysis in buffer C . Recombinant Ulp1 catalytic domain (purified separately from E. coli) was added to renatured protein to cleave the 6xHis-SUMO tag, and cleaved protein was injected onto a Superdex 200 10/300 size-exclusion column equilibrated in buffer C. Peak fractions were pooled, concentrated, frozen in LN2, and stored at −80 °C.Wildtype and mutant N proteins were produced as described previously . Brieflyin vitro transcription of 5′-600 RNA was a synthetic DNA (IDT), inserted by Gibson assembly into a pUC18 vector with a 5′ T7 promoter sequence. The 5′-600 insert, including the 5′ T7 sequence, was excised by EcoR1 digestion and purified by size-exclusion chromatography on a Sephacryl 1000 column equilibrated in TE buffer . Peak fractions of the purified DNA insert were pooled and stored at −4 °C.Sequences of all RNAs used in this study are provided in http://rna.tbi.univie.ac.at/). PCR-amplified DNA was purified and concentrated by spin column (Zymo Research #D4004) before being used to generate RNA.Templates for all other long RNAs  were amplified by PCR of a plasmid containing the SARS-CoV-2 genome . All forward primers included a 5′ T7 promoter sequence. The SL8 and mSL8 templates were generated by PCR of synthetic DNA (IDT). The sequence for mutant SL8 (mSL8) was designed manually and checked for predicted secondary structure by RNAfold (in vitro synthesized RNA was purified and concentrated by spin column (Zymo Research #R1018). To promote formation of proper RNA secondary structure, all purified RNAs were heat denatured at 95 °C for 2 min in a preheated metal heat block and then removed from heat and allowed to cool slowly to room temperature over the course of ∼1 h. RNA concentration (A260) was quantified by nanodrop.RNA synthesis was performed using the HiScribe T7 High Yield RNA synthesis kit (NEB #E2040S) according to the manufacturer’s protocol. Following incubation at 37 °C for 3 h, in vitro the day of analysis, heat-denatured, and cooled slowly to allow for proper secondary structure. To assemble vRNP complexes, RNA was mixed with N protein  in a total volume of 10 μl and incubated for 10 min at 25 °C. RNA concentration was 256 ng/μl, regardless of RNA length, to ensure that all mixtures contained the same nucleotide concentration. Samples containing stem-loop RNAs  were crosslinked by addition of 0.1% glutaraldehyde for 10 min at 25 °C and then quenched with 100 mM Tris pH 7.5. Samples containing longer RNAs  were not crosslinked. After assembly, vRNP complexes were analyzed as described below.The day before each experiment, N protein was dialyzed into reaction buffer  overnight. RNA was transcribed After assembly (and crosslinking in the case of stem-loop RNAs), 10 μl vRNP mixtures were diluted 1:10 in dilution buffer . Diluted vRNP mixture (2 μl) was loaded onto a 5% polyacrylamide native TBE gel (Bio-Rad) and run at 125 V for 80 min at 4 °C. Another aliquot of diluted sample (1 μl) was denatured by addition of 4 M urea and Proteinase K , incubated for 5 min at 65 °C, loaded onto a 6% polyacrylamide TBE-Urea Gel (Thermo Fisher), and run at 160 V for 50 min at room temperature. Gels were stained with SYBR Gold (Invitrogen) and imaged on a Typhoon FLA9500 Multimode imager set to detect Cy3.Mass photometry experiments were performed using a OneMP instrument (Refeyn). A silicone gasket well sheet (Grace Bio-Labs) was placed on top of a microscope coverslip and positioned on the microscope stage. Reaction buffer (10 μl)  was first loaded into the well to focus the objective, after which 1 μl of vRNP complex sample was added to the reaction buffer, mixed, and measured immediately. Samples containing stem-loop RNA were diluted 1:10 before a second 1:10 dilution directly on the coverslip, while samples containing longer RNAs were only diluted 1:10 on the coverslip.The mass photometer was calibrated with NativeMark Unstained Protein Standard (Thermo #LC0725). Mass photometry data were acquired with AcquireMP and analyzed with DiscoverMP software (Refeyn). Mass photometry data are shown as histograms of individual mass measurements. Peaks were fitted with Gaussian curves to determine the average molecular mass of the selected distributions. Each condition was independently measured at least twice.Glycerol gradients were assembled as previously described, with slight modifications . BrieflyFor negative stain EM, 2.5 μl of vRNP samples were applied to a glow discharged Cu grid covered by continuous carbon film and stained with 0.75% (w/v) uranyl formate. A Tecnai T12 microscope (ThermoFisher FEI Company) operated at 120 kV was employed to analyze these negatively stained grids. Micrographs were recorded at a nominal magnification of 52,000× using a Gatan Rio 16 camera, corresponding to a pixel size of 1.34 Å on the specimen. All images were processed using cryoSPARC. Micrographs were processed with Patch-Based CTF Estimation, and particles were picked using the blob picker followed by the template picker. Iterations of 2D classification generated final 2D averages.Freshly prepared and renatured RNA was mixed with dialyzed N protein and incubated for 2 min at room temperature. Absorbance was measured at 260 nm and 340 nm using the Nanodrop Micro-UV/Vis Spectrophotometer. Turbidity was calculated by normalization of the 340 nm measurements to the absorbance value at 260 nm.2, 1 mM DTT, 0.5 mM ATP, and 0.001 mCi/ml 32P-γ-ATP). Reactions were quenched upon addition of SDS loading buffer for analysis by SDS-PAGE and autoradiography.Protein kinases were purchased from Promega . N protein (1.25 μM) was incubated with 80 nM kinase for 30 min at 30 °C in kinase reaction buffer  and 80 nM SRPK, GSK3, and CK1 in kinase reaction buffer. Reactions were incubated 30 min at 30 °C before addition of 5 mM EDTA. RNA was added to a final concentration of 256 ng/μl  and incubated at room temperature for 15 min. vRNP samples were analyzed by gradient centrifugation with crosslinker (GraFix) as described above.All data are included in the article or available from the corresponding author D. O. M.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


During COVID-19 pandemic, mutations of SARS-CoV-2 produce new strains that can be more infectious or evade vaccines. Viral RNA mutations can arise from misincorporation by RNA-polymerases and modification by host factors. Analysis of SARS-CoV-2 sequence from patients showed a strong bias toward C-to-U mutation, suggesting a potential mutational role by host APOBEC cytosine deaminases that possess broad anti-viral activity. We report the first experimental evidence demonstrating that APOBEC3A, APOBEC1, and APOBEC3G can edit on specific sites of SARS-CoV-2 RNA to produce C-to-U mutations. However, SARS-CoV-2 replication and viral progeny production in Caco-2 cells are not inhibited by the expression of these APOBECs. Instead, expression of wild-type APOBEC3 greatly promotes viral replication/propagation, suggesting that SARS-CoV-2 utilizes the APOBEC-mediated mutations for fitness and evolution. Unlike the random mutations, this study suggests the predictability of all possible viral genome mutations by these APOBECs based on the UC/AC motifs and the viral genomic RNA structure. Efficient Editing of SARS-CoV-2 genomic RNA by Host APOBEC deaminases and Its Potential Impacts on the Viral Replication and Emergence of New Strains in COVID-19 Pandemic The causative agent of the COVID-19 pandemic, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is a member of the enveloped Coronaviridae family that has a single-stranded positive-sense RNA genome  and refeHowever, sequencing data of SARS-CoV-2 from patients revealed persistent accumulation of new mutations over time, leading to the emergence of new viral strains that can be more transmissible or virulent , 6 or evreactive oxygen species (ROS) ((adenosine deaminases acting on RNA) and APOBECs (the apolipoprotein-B (ApoB) mRNA editing enzyme, catalytic polypeptide-like proteins) (The host responses that can cause mutations on SARS-CoV-2 include s (ROS) ( and referoteins) , 10, 12.roteins) , 14. Theroteins) , 16 and The APOBEC proteins are a family of cytosine deaminases that can deaminate cytosine to uracil (C-to-U) in single-stranded nucleic acids and function in a variety of biological processes, including innate and adaptive immune responses to viral pathogens -20 and r and r20 Our first goal is to design an experimental system to that can efficiently address whether a particular APOBEC enzyme can actually edit SARS-CoV-2 RNA. We adapted our previously reported cell-based RNA editing system  to exami−4 - 10−5 error rates) was used for the initial reverse transcription of the target SARS-CoV-2 RNA transcripts from the cells to single-stranded cDNA. The JUNC forward primer is used to ensure only mature spliced mRNA segments of SARS-CoV-2 were amplified (30), discerning each original target molecule. Third, the initial 2 cycle-PCR products were purified and then amplified with Illumina adaptors (PCR error rate is ~10−7). Finally, the high rates of errors from paired-end Illumina sequencing  are minimized by eliminating random mutations in the same UID family (see Methods in SI).To minimize the sequencing errors when evaluating the C-to-U RNA editing, we employed the SSS system, a targeted next generation deep-sequencing system, with slightly adapted protocols , 28. Thimplified . Second,−2 - 10−3 error rates) may drown out the signals of true mutations caused by APOBEC deamination. However, the SSS approach has its own drawback: the SSS sequencing costs several times more than a regular NGS sequencing. Each SSS sequencing read covers ~150-200 nt, making it too expensive for us to cover the entire 30,000 nt RNA genome of SARS-CoV-2. That is the main reason that we selected only seven viral RNA segments for our editing study.Using this SSS approach in this system to check the APOBEC-mediated mutations overcomes two major limitations from directly analyzing the patient-derived SARS-CoV-2 sequences. The first limitation is that the deposited SARS-CoV-2 sequence is “one-consensus-sequence from one-patient”, i.e. only one consensus viral sequence from the NGS sequencing data is reported from one patient sample, which neglects many of the non-consensus sequence variants that may be caused by APOBEC deamination. The second limitation is that regular NGS sequencing’s intrinsic high error rate . The C-to-U editing levels by each APOBEC are normalized by the control group (Supplementary Dataset File 2). The C-to-U editing by all three APOBECs is detected, with A1+A1CF and A3A showing much higher editing than A3G . Here, we define the significant target C site where the C-to-U editing efficiency is at least 3 times higher than control. Out of 307 total C in the selected viral RNA segments, the number of significant target sites with A1+A1CF is 135, A3A is 67, and A3G is 11 (Supplementary Dataset File 2). Analysis of the sequence contexts around the significant target C sites (with ± 5 nucleotides from target C) showed that A3A prefers for an UCa/u trinucleotide motif and A1+A1CF prefers ACu/a motif  has an average of about 130,000  from a total of ~ 484 million (paired) reads  and UC-to-UU (preferred by A3A) account for 38.23% and 31.83%, respectively, significantly higher than GC-to-GU and CC-to-CU that account for 15.44% and 14.50%, respectively , indicating the significance of APOBEC editing on the viral mutation.We also performed the same analysis of the sequence contexts with the top 30% editing efficiency by APOBECs (or hotspot editing sites), which is translated to 38 times higher than control for A1+A1CF, or 15 times higher than control for A3A, or 6 times higher than control for A3G) . It distsegments , D. InteSupplementary Dataset File 2). Furthermore, many of these AC, UC, and CC motif sites on the viral RNA have no detectable editing by A1+A1CF, A3A, and A3G, respectively, suggesting that other RNA features beyond the dinucleotide sequence motifs, such as the secondary and tertiary structures, must play a role in the editing efficiency of a particular motif site.Although each of the three APOBEC proteins showed a strong preference for specific dinucleotide sequence motifs  for editing on the viral RNA, the relative editing efficiency of these motif sites vary greatly, such as between 0.0041% to 22.15% for A1+A1CF, and between 0.0040% to 4.46% for A3A . These data indicate that the C-to-U changes on the RNA are not caused by DNA mutation, but are the result of direct RNA editing mediated by A1+A1CF and A3A in our cell-based assay system.To rule out the possibility that the RNA C-to-U mutations result from DNA C-to-U deamination instead of the direct RNA editing by APOBECs, we performed a side-by-side sequencing of the DNA on the reporter vector and its corresponding RNA transcript containing the Orf1b region of SARS-CoV-2 . The reporter DNA and the RNA transcript extracted from the cells expressing APOBECs were PCR amplified using the forward prime annealing to either the AAV-intron specific for the DNA or to the JUNC specific for the spliced RNA only. The PCR products from the DNA and mRNA were subjected to Sanger sequencing, and the C-to-U changes were analyzed. No DNA C-to-U mutation was detected, but specific RNA C-to-U changes on the mRNA transcript were present in a frequency consistent with our SSS results obtained in the presence of A1+A1CF  or A3A (C16063) . Of particular interest is the prominent mutation occurring at UC203, UC222, and UC241 in the 5’UTR region of these virus variants . These results suggest that A3A generated these mutations on the viral RNA genome, and the mutations can be maintained, likely because these three mutations generated by A3A editing are not determinantal to the virus. Two of these three mutations, UC203 and UC222, were detected in some of the SARS-CoV-2 sequences in late 2020 but are not persistently present in the main circulating strains, suggesting these two mutations may be neutral for the viral fitness. Surprisingly, the C-to-U mutation at UC241 occurred in early January 2020 and has rapidly become a signature of the dominant strains (including Delta and Omicron) that spread worldwide , strongly suggesting that this C-to-U mutation at UC241 may contribute to the better fitness for SARS-CoV-2. Although UC241 to U mutation is within 5’UTR, the correlation of this mutation with the dominant new strains is reminiscent of that of the D614G mutation of the spike protein-coding region  that survived the selection pressure for fitness. With the direct experimental evidence that APOBECs can target specific sites on SARS-CoV-2 for editing, we analyzed the publicly available SARS-CoV-2 genome sequence data , with hovariants , as thesg region . Becauseg region , 38, theFig S5A). Even the very recent omicron variants, which began to spread from Nov. 2021 rapidly, continue to show noticeable C-to-U editing pattern, including some biologically significant mutations at the A1+A1CF preferred AC motifs . Notably, the AC to AU mutation at C23525 resulted an H655Y mutation on the spike protein, and the H655Y mutation was shown to alter cell entry pathways, i.e. the mutation is responsible for the preferential usage of endosomal pathway over cell surface entry pathways , we first constructed Caco-2 stable cell lines expressing one of the three APOBEC genes. The externally inserted APOBECs are under tetracycline-controlled promoter so that their expression can be induced by doxycycline . The Caco-2-APOBEC stable cell lines were then infected by SARS-CoV-2, and the viral RNA replication and progeny yield were measured and compared with the control cell line without APOBEC expression. The viral RNA abundance as an indicator for RNA replication was measured using real-time quantitative PCR (qPCR) to detect the RNA levels using primers specific for amplifying three viral regions: the Nsp12 region, the S region, and the N region, covering the genomic and subgenomic regions. The viral progeny yield was assayed through plaque assay in Vero E6 cell line using the virions produced from the Caco-2-APOBEC stable cell lines at different time points post-infection . Again, the abundance of viral RNA based on the qPCR results of the three viral regions  all displayed significant increases in the cell lines expressing A3A-WT at 72 and 92 hours post SARS-CoV-2 infection but showed no significant difference in the control Coca-2 cells and the ΔA3A cells . Furthermore, the viral progeny yield harvested from the A3A-WT expressing cell line also was significantly higher than those from the control Caco-2 cells and the ΔA3A cells . Interestingly, A3A-E72A also showed slight enhancement of viral RNA replication  and viral progeny yield . Taken together, this result suggests that the A3A deaminase activity plays a major role for promoting the viral replication and viral progeny production. This is consistent, among other things, with the observation that the UC241 to UU241 mutation, a site highly edited by A3A in our study, is within the viral packaging signal and near the viral replication regulation area at the 5’UTR , which could explain why this mutation becomes prevalent in the widely circulating SARS-CoV-2 strains after January 2020  and an A3A catalytically inactive mutant-expressing (A3A-E72A) Caco-2 cell lines, and the SARS-CoV-2 replication and progeny yield were compared in the four different Caco-2 cell lines, i.e. original Caco-2 cell line (control), and Caco-2 with A3A-knockout (ΔA3A), and the stable cell lines expressing A3A WT or the inactive mutant A3A-E72A , which suggest that additional RNA structural features around the UC/AC motifs should play an important role in dictating whether a UC or AC site can be efficiently edited . Such expression profiles make it possible for A3A and A1+A1CF to edit the viral RNA genome in the real world. Many human cell types expressing ACE2 in multiple organs can be infected by SARS-CoV-2, including (but not limited to) the lungs, heart, small intestine, and liver . A1 and its two known cofactors, A1CF and RBM47, are not expressed in the lungs but are expressed in the small intestine or liver .While the editing of SARS-CoV-2 RNA by A3A, A1+A1CF, and A3G has been demonstrated in our cell culture system, the analysis of the expression profiles reveals that A3A and A1+A1CF, but not A3G, are expressed in the human organs and cell types infected by SARS-CoV-2  than UC motif mutations (31.2%) , as also reported in the previous bioinformatic database analysis , it indicates that the AC-to-AU editing also requires a cofactor A1CF. Given the closely overlapping RNA target and editing efficiency by another A1-cofactor RBM47  the improvement of viral RNA replication, protein expression, evasion of host immune responses, and receptor binding and cell entry.C203, UC222, UC241 , suggesting that C-to-U mutation at UC241 is selected for better viral fitness. This observation is surprising considering UC241 is in the non-coding 5’UTR region and, thus, not affecting the ORF coding of a protein, as in the case of the previously reported D614G mutation of the spike protein for viral fitness  . Thus, this UC241 to UU241 mutation may impact the viral RNA packaging and virion production, and may even impact RNA replication, sub-genomic production, or/and the translation efficiency of the downstream proteins to endow the virus with better fitness.Mutations outside protein-coding ORFs of SARS-CoV-2 were previously designated as non-functional changes . However (TRS-L)  There is also the possibility of host RNA or DNA editing by WT A3A that leads to increased viral RNA replication and viral progeny production. Given the known activity of A3A in mutating both cellular genomic DNA and RNA transcripts, the possibility of triggering certain cellular events by A3A editing/mutation activity that favors SARS-CoV-2 replication cannot be excluded and warrants future investigation.C for A3A or AC for A1 as well as certain RNA structural features around the target C. Even though some APOBECs, including A3A, are regarded as host antiviral factors, we show here that RNA editing of SARS-CoV-2 by A3A can promote viral replication/propagation. These results suggest that SARS-CoV-2 can take advantage of APOBEC-mediated mutation for their fitness and evolution. Unlike the random mutations caused by RNA replication or ROS, the presence of the finite number of the UC/AC motifs on the SARS-CoV-2 genomic RNA and the potentially correct prediction of the viral RNA structures suggest that it is possible to predict all the possible target C sites in both the coding and non-coding regions to be edited by these APOBECs. With the new selection pressure from use of vaccines and antiviral drugs and the continued large-scale circulation of SARS-CoV-2 variants among unvaccinated and vaccinated people, such prediction may be meaningful for anticipating potential new viral mutations and the emergence immune escape and drug-resistant strains.In summary, we report here the experimental evidence demonstrating that certain sites of SARS-CoV-2 genomic RNA can be directly edited with high efficiency by A3A and A1+A1CF to cause C-to-U mutation. Critical factors dictating the RNA-editing efficiency by these two APOBECs include a dinucleotide motif U