Abstract
How antiviral immunity first arose in animals is a central question in evolutionary biology. Here, using the sea anemone Nematostella vectensis, we identify CARDIB, a previously uncharacterized gene located next to RLRb—a cnidarian homologue of the vertebrate RIG-I-like receptor family. This conserved genomic linkage across Anthozoa reveals an ancient coupling between immune sensing and regulation. Despite sequence similarity to vertebrate MAVS, CARDIB performs an opposing function: it represses immune genes under basal conditions yet is essential for activation upon viral challenge. CARDIB binds RLRb through a single CARD domain, forming a repressive complex. Loss of either gene abolishes antiviral transcription, disrupts apoptosis and elevates viral load under laboratory conditions. Both genes, as well as the RLRb paralogue RLRa, are essential for antiviral defence under native conditions. Phylogeny places the cnidarian CARDs distinctly from the vertebrate RLR–MAVS families, revealing an ancient mechanism that regulates the antiviral response through CARD-based signalling.
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Data availability
All sequencing data generated in this work are publicly available under accessions PRJNA1250240 and PRJNA1262874 at the SRA database of the National Center for Biotechnology Information (NCBI) under BioProject: an ancient anthozoan protein reveals an alternative evolutionary path of antiviral signalling and BioProject: N. vectensis Total RNA-seq from a mesocosm study conducted in a South Carolina estuary. The scRNA-seq data analysed in this study were generated previously and are available in the corresponding publication31 and associated repositories. Processed data used for correlation analyses are provided in Supplementary Table 6. Source data are provided with this paper.
Code availability
Bioinformatic processing scripts and figure generation are available via GitHub at https://github.com/sydneybirch/Nematostella_virome_Mesocosm_2023, https://github.com/adrianjaimes/Cnidarian-immune-system.
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Acknowledgements
We would like to thank M. Bronstein and A. Turjeman for their help with sequencing. We also thank T. Hagai for helpful discussions about the results and their interpretation.
Funding
Y.M. discloses support for the research of this work from the European Research Council (grant no. 863809), Y.M. and A.M.R. disclose support for the research of this work from US–Israel Binational Science Foundation joint Grant with the National Science Foundation (grant no. 2020669).
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Authors and Affiliations
Contributions
T.S. and Y.M. conceived the study. D.A., S.B., H.-J.K., T.S., R.A., Y.M. and A.M.R. designed the experiments. T.S., D.A. and H.-J.K. performed the microinjections. T.S. and H.-J.K. generated the CRISPR–Cas9 mutant lines and maintained the animals for experiments. R.A. performed the western blotting and immunoprecipitation experiments. T.S. conducted the viral load assays under laboratory conditions. S.B., H.-J.K. and H.J. conducted the mesocosm assays for viral load analysis. D.A. conducted the PLA assays. A.J.-B. performed the AlphaFold2 prediction of the CARD domains, the scRNA-seq correlation analysis and the CARD phylogenetic and clustering analyses. J.M.S. analysed the genomic organization of CARDIB and RLRb in anthozoa. T.S. and M.L. conducted the quantification of gene expression. T.S. and A.J.-B. conducted the bulk RNA-seq analysis. H.K. conducted the apoptotic assays. All authors contributed to revising the manuscript and approved the final version.
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Extended data
Extended Data Fig. 1 Basal expression of immune-related proteins in CARDIB KO adult animals and CARDIB over-expression after 28 hrs of mRNA injection.
a, Western blot images showing the basal expression of six immune-related proteins and GAPDH as loading control, in wild-type adult animals compared to CARDIB mutants of two different CRISPR KO strains. Protein sizes in KD are indicated. All western blot results are based on at least four independent biological repeats for each mutant line and wild type. b, Western blot images showing the expression of six immune-related proteins, GAPDH as a loading control, and CARDIB in CARDIB mRNA-injected animals compared to mCherry mRNA-injected animals as a control at 28 hrs post injection. Protein sizes in KD are indicated. All western blot results are based on at least four independent biological repeats of CARDIB mRNA-injected animals against control mRNA-injected animals.
Extended Data Fig. 2 Functional and genetic characterization of viral communities following mesocosm exposure.
Viral communities associated with wild-type and immune mutant N. vectensis lines (RLRa, RLRb, and CARDIB) following 96 h of exposure to natural estuarine water in a mesocosm experiment (T96). a, Genetic composition of viral communities based on genome type (dsDNA, dsRNA, ssDNA, ssRNA, and unclassified viruses). Relative abundances (left) and absolute counts (right) are shown for each genotype. Viral genome classifications were assigned using the ICTV Virus Metadata Resource (VMR). b, Functional annotation of viral open reading frames using the Virus Orthologous Groups (VOG) database. Bars represent the relative abundance (left) and total counts (right) of VOG functional categories, including viral replication, structure, and host–virus interaction functions. c, Functional annotation of viral genes using KEGG Orthology (KO). Bars depict the relative abundance (left) and total counts (right) of KEGG functional categories associated with viral and host-related biological processes.
Extended Data Fig. 3 Schematic representation of CRISPR/Cas9-generated knockout alleles in N. vectensis.
Schematic depictions of the wild-type (WT) loci and CRISPR/Cas9-generated knockout alleles for RLRa a, RLRb b, and CARDIB c. For RLRa, two independent knockout lines are shown, carrying a 4-nucleotide deletion (Δ4 bp) or a 2-nucleotide deletion at the guide RNA target site. For RLRb, a single knockout allele harboring two distinct CRISPR-induced modifications on the same allele is shown, consisting of a 5-nucleotide insertion and a large 676-nucleotide deletion. For CARDIB, two independent knockout lines are shown, one carrying a 5-nucleotide deletion and the other carrying a 22-nucleotide insertion at the target site. Exons are represented as boxes and introns as lines; the scissors symbol marks the Cas9 cleavage site.
Extended Data Fig. 4 The sequence and the design of the eGFP Nve gBlock. C-terminal CARDIB-FLAGx3-eGFP Nve gBlock, CARDIB-mCherry Nve gBlock and mCherry gBlock.
Complete sequence of the synthetic gBlock template encoding the C-terminal CARDIB-3xFLAG-eGFP construct. From 5′ to 3′, the sequence includes a T7 promoter for in vitro transcription, an ef1a Kozak consensus sequence to enhance translation initiation, the Nematostella vectensis CARDIB coding region, a P2A self-cleaving peptide sequence enabling bicistronic expression, mCherry as a fluorescent reporter, a 3xFLAG epitope tag, eGFP, a complexity-fix sequence to improve cloning and transcription stability, and a 3′ untranslated region (3′ UTR) to support transcript stability. The entire sequence shown represents the transcriptional template used for mRNA synthesis.
Extended Data Fig. 5 Co-immunoprecipitation of CARDIB and RLRb measured by western blot.
Western blot image showing CARDIB presence, after Co-Immunoprecipitation of RLRb with CARDIB from N. vectensis 24-h embryos as described in the materials and methods section. Membrane was incubated with αCARDIB antibody as primary antibody and Peroxidase conjugated antibody against rabbit as secondary antibody and finally detected with a CCD camera of the Odyssey Fc imaging system (Li-COR Biosciences) using Clarity™ ECL and Clarity™ max kits (Biorad) as described in detail. Co-IP Sample name and biological repeat number are indicated above the blot image. The upper numbers below the blot image show the serial numbers of the bands on the blot. The lower numbers below the blot image indicate band intensities as were measured by the Image Studio software (Li-COR Biosciences) and used for the fold change calculations between RLRb and NeNaC2 samples in each repeat. CoIP experiment was done on four independent biological repeats.
Extended Data Fig. 6 Co-immunoprecipitation of CARDIB and RLRb measured by western blot.
c Zygotes were injected with shRNA against each of the target genes in addition to poly (I:C), and the gene expression was tested at the protein level. Scramble shRNA with poly (I:C) injected as a control. a, extracted protein from planulae that were injected with shRNA with poly (I:C) against CARDIB and scramble shRNA with poly (I:C) as a control. b, extracted protein from planulae that were injected with shRNA with poly (I:C) against GBP and scramble shRNA with poly (I:C) as a control. c, extracted protein from planulae that were injected with shRNA with poly (I:C) against OAS and scramble shRNA with poly (I:C) as a control. For the normalization of the amount of protein in the western blot, GAPDH was used for the samples of αCARDIB and αGBP, for the samples of αOAS, AGO1 was used for the normalization of the amount of protein to avoid size similarity (which was shown not to respond to poly (I:C) or be involved in the N. vectensis immunity1) used for the normalization. All antibodies validations were done on at least three independent biological repeats. Additional information on the shRNA sequences is provided in Supplementary File 1, Table 23.
Extended Data Fig. 7 Phylogenetic relationship of metazoan RLRs.
Maximum likelihood consensus phylogenetic tree of representative RLR sequences, numbers in parentheses are SH-aLRT support (%) / aBayes support / ultrafast bootstrap support (%). Amu, Acropora muricata; Aqu, Amphimedon queenslandica; Bbe, Branchiostoma belcheri; Cte, Capitella teleta; Cgi, Crassostrea gigas; Cel, Caenorhabditis elegans; Dre, Danio rerio; Edia, Exaiptasia diaphana; Hsa, Homo sapiens; Lan, Lingula anatina; Mli, Macrostomum lignano; Nve, Nematostella vectensis; Pda, Pocillopora damicronis; Spi, Stylophora pistillata; Xsp, Xenia spp; Xtr, Xenopus tropicalis. anatina; Mli, Macrostomum lignano; Nve, Nematostella vectensis; Pda, Pocillopora damicronis; Spi, Stylophora pistillata; Xsp, Xenia spp; Xtr, Xenopus tropicalis.
Extended Data Fig. 8 Mesocosm Field Design.
a, Cartoon depiction of the mesocosm samples used in this study. Each Mesocosm (plastic bin) is a replicate. Animal container placements were randomized in each replicate. b, The five mesocosm replicates in the field at Belle W. Baruch Marine Field Laboratory (Georgetown, South Carolina, US). c, A close-up image of the inside of an animal container. Each animal container contained one N. vectensis strain and began with six individuals to be sampled at different time points. d, An above view of the inside of one mesocosm. e, A side view of the mesocosms to demonstrate the water line. Natural estuary water was added to submerge half of the animal containers to prevent animals from escaping. Panel a created in BioRender; Reitzel, A. https://biorender.com/zot2t2l (2026).
Extended Data Fig. 9 Heatmap of T96 Mesocosm N. vectensis immune gene expression.
Gene expression of the same 56 immune-related genes was interrogated in Fig. 4g. The heatmap shows expression of N.vectensis KOs at the T96 timepoint (96 h after mesocosm exposure). Each row represents a gene in the 56 immune-related gene set, with yellow indicating high expression and dark blue indicating low expression.
Extended Data Fig. 10 Bioinformatic pipeline to assess viral read counts.
N. vectensis samples were collected at two timepoints during the mesocosm experiment: the initial timepoint (T0), prior to exposure, and after 96 h of exposure to natural estuary water (T96). Four strains were examined with 3-4 biological replicates each, yielding a total of 31 samples (that is, four replicates for each T0 and T96 strain, except for CARDIB T96, which had three replicates). RNA was extracted from each sample and used to generate RNA libraries for total RNA sequencing. In the bioinformatics pipeline, the first step after adapter trimming and quality control (FastQC) was host read removal. Trimmed reads were iteratively aligned to the N. vectensis genome until no reads mapped, leaving viral and microbial reads. An rRNA filtration step was then performed by aligning these unmapped reads to an rRNA database to remove prokaryotic and eukaryotic rRNA sequences. The resulting rRNA-filtered dataset was designated as ’presumably viral reads.’. These reads were used to assemble transcriptomes. Two assemblies were generated: the Total-T0 assembly, constructed from all 16 T0 samples, and the Total-T96 assembly, constructed from all 15 T96 samples. Viral reads from each sample were then mapped back to the appropriate assembly, and normalization factors were calculated. Statistical analyses were subsequently performed. Figure created in BioRender; Reitzel, A. https://biorender.com/co5tt71 (2026).
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Sharoni, T., Jaimes-Becerra, A., Birch, S. et al. An ancient anthozoan protein reveals an alternative evolutionary path of antiviral signalling.
Nat Ecol Evol (2026). https://doi.org/10.1038/s41559-026-03112-3
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DOI: https://doi.org/10.1038/s41559-026-03112-3
Source: Ecology - nature.com
