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Over 99% of ray-finned fishes (Actinopterygii) are teleosts, a clade that comprises half of all living vertebrates that have diversified across virtually all fresh and saltwater ecosystems. This ecological diversity raises the question of how the immunogenetic diversity required to persist under heterogeneous pathogen pressures evolved. The teleost genome duplication (TGD) has been hypothesized as the evolutionary event that provided the genomic substrate for rapid genomic evolution and innovation. However, studies of putative teleost-specific innate immune receptors have been largely limited to comparisons either among teleosts or between teleosts and distantly related vertebrate clades such as tetrapods. Here we describe and characterize the receptor diversity of two clustered innate immune gene families in the teleost sister lineage: Holostei (bowfin and gars). Using genomic and transcriptomic data, we provide a detailed investigation of the phylogenetic history and conserved synteny of gene clusters encoding diverse immunoglobulin domain-containing proteins (DICPs) and novel immune-type receptors (NITRs). These data demonstrate an ancient linkage of DICPs to the major histocompatibility complex (MHC) and reveal an evolutionary origin of NITR variable-joining (VJ) exons that predate the TGD by at least 50 million years. Further characterizing the receptor diversity of Holostean DICPs and NITRs illuminates a sequence diversity that rivals the diversity of these innate immune receptor families in many teleosts. Taken together, our findings provide important historical context for the evolution of these gene families that challenge prevailing expectations concerning the consequences of the TGD during actinopterygiian evolution.

Currently assessment of the potential immunotoxicity of a given agent involves a tiered approach for hazard identification and mechanistic studies, including observational studies, evaluation of immune function, and measurement of susceptibility to infectious and neoplastic diseases. These studies generally use costly low-throughput mammalian models. Zebrafish, however, offer an excellent alternative due to their rapid development, ease of maintenance, and homology to mammalian immune system function and development. Larval zebrafish also are a convenient model to study the innate immune system with no interference from the adaptive immune system. In this study, a respiratory burst assay (RBA) was utilized to measure reactive oxygen species (ROS) production after developmental xenobiotic exposure. Embryos were exposed to non-teratogenic doses of chemicals and at 96 hr post-fertilization, the ability to produce ROS was measured. Using the RBA, 12 compounds with varying immune-suppressive properties were screened. Seven compounds neither suppressed nor enhanced the respiratory burst; five reproducibly suppressed global ROS production, but with varying potencies: benzo[a]pyrene, 17β-estradiol, lead acetate, methoxychlor, and phenanthrene. These five compounds have all previously been reported as immunosuppressive in mammalian innate immunity assays. To evaluate whether the suppression of ROS by these compounds was a result of decreased immune cell numbers, flow cytometry with transgenic zebrafish larvae was used to count the numbers of neutrophils and macrophages after chemical exposure. With this assay, benzo[a]pyrene was found to be the only chemical that induced a change in the number of immune cells by increasing macrophage but not neutrophil numbers. Taken together, this work demonstrates the utility of zebrafish larvae as a vertebrate model for identifying compounds that impact innate immune function at non-teratogenic levels, and validates measuring ROS production and phagocyte numbers as metrics for monitoring how xenobiotic exposure alters the innate immune system.

The morphogenesis of left-right (LR) asymmetry is a crucial phase of organogenesis. In the digestive tract, the development of anatomical asymmetry is first evident in the leftward curvature of the stomach. To elucidate the molecular events that shape this archetypal laterality, we performed transcriptome analyses of the left versus right sides of the developing stomach in frog embryos. Besides the known LR gene pitx2, the only gene found to be expressed asymmetrically throughout all stages of curvature was single-minded 2 (sim2), a Down Syndrome-related transcription factor and homolog of a Drosophila gene (sim) required for LR asymmetric looping of the fly gut. We demonstrate that sim2 functions downstream of LR patterning cues to regulate key cellular properties and behaviors in the left stomach epithelium that drive asymmetric curvature. Our results reveal unexpected convergent cooption of single-minded genes during the evolution of LR asymmetric morphogenesis, and have implications for dose-dependent roles of laterality factors in non-laterality-related birth defects.

The bowfin (Amia calva) is a ray-finned fish that possesses a unique suite of ancestral and derived phenotypes, which are key to understanding vertebrate evolution. The phylogenetic position of bowfin as a representative of neopterygian fishes, its archetypical body plan and its unduplicated and slowly evolving genome make bowfin a central species for the genomic exploration of ray-finned fishes. Here we present a chromosome-level genome assembly for bowfin that enables gene-order analyses, settling long-debated neopterygian phylogenetic relationships. We examine chromatin accessibility and gene expression through bowfin development to investigate the evolution of immune, scale, respiratory and fin skeletal systems and identify hundreds of gene-regulatory loci conserved across vertebrates. These resources connect developmental evolution among bony fishes, further highlighting the bowfin’s importance for illuminating vertebrate biology and diversity in the genomic era., Analysis of a chromosome-level bowfin genome assembly sheds light into neopterygian fish evolution. Chromatin accessibility and gene expression profiling provides insight into bowfin embryonic development.

Background Presumed autoimmune diseases affecting the central nervous system (CNS) of dogs are common. In people, antibodies against neuronal cell surface antigens that are associated with a wide variety of neurological syndromes have been identified. The presence of cerebrospinal fluid (CSF) autoantibodies that target neuronal cell surface proteins has not been reported in dogs with neurologic disorders. Objectives Autoantibodies to neuronal cell surface antigens can be found in the CSF of dogs with inflammatory CNS disease. Our aim was to determine whether 6 neuronal cell surface autoantibodies were present in the CSF of dogs diagnosed with inflammatory and noninflammatory CNS disease. Animals Client-owned dogs with CNS disease and complete diagnostic evaluation including magnetic resonance imaging and CSF analysis were included. One healthy dog was included as a negative control. Methods Cerebrospinal fluid was tested for 6 antigenic targets with a commercially available indirect immunofluorescence assay test kit. Results There were 32 dogs with neurological disease, 19 diagnosed with inflammatory disease (encephalitis and meningitis), 10 with noninflammatory disease (neoplasia, intervertebral disk disease, degenerative myelopathy, and epilepsy), 2 with no diagnosis, and 1 with neoplasia and meningoencephalitis. Anti-N-methyl-d-aspartate receptor 1 (NMDAR1) antibodies were detected in 3 dogs (3/32; 9.38%). All 3 dogs responded to treatment of meningoencephalomyelitis of unknown etiology (MUE). Conclusions and clinical importance Further evaluation of the prevalence and clinical relevance of CSF and serum antibodies to neuronal cell surface antigens is warranted. Defining antigenic targets associated with encephalitis in dogs might allow diagnostic categorization of MUE antemortem.

The bowfin fish (Amia calva) diverged before the genome duplication in teleost fishes, and its archetypical body plan and slow rate of molecular evolution make it a key species for genomic exploration as a basal representative of the neopterygian fishes. To investigate the evolution and development of ray-finned fishes, we generated a chromosome-level genome assembly for bowfin that enables gene-order analyses which settle its long-debated, phylogenetic relationship with gars. We analyze the genomic underpinnings of the bowfin’s unique combination of derived and ancestral phenotypes involving the immune system as well as scale, respiratory organ, and skeletal development. By detailing chromatin accessibility and gene expression through bowfin development, we connect developmental gene regulatory loci across vertebrates. We illustrate the utility of these genomic resources to connect developmental evolution across bony fishes, showing the importance of bowfin in understanding vertebrate biology and diversity.

Background Advances in next-generation sequencing technologies have reduced the cost of whole transcriptome analyses, allowing characterization of non-model species at unprecedented levels. The rapid pace of transcriptomic sequencing has driven the public accumulation of a wealth of data for phylogenomic analyses, however lack of tools aimed towards phylogeneticists to efficiently identify orthologous sequences currently hinders effective harnessing of this resource. Results We introduce TOAST, an open source R software package that can utilize the ortholog searches based on the software Benchmarking Universal Single-Copy Orthologs (BUSCO) to assemble multiple sequence alignments of orthologous loci from transcriptomes for any group of organisms. By streamlining search, query, and alignment, TOAST automates the generation of locus and concatenated alignments, and also presents a series of outputs from which users can not only explore missing data patterns across their alignments, but also reassemble alignments based on user-defined acceptable missing data levels for a given research question. Conclusions TOAST provides a comprehensive set of tools for assembly of sequence alignments of orthologs for comparative transcriptomic and phylogenomic studies. This software empowers easy assembly of public and novel sequences for any target database of candidate orthologs, and fills a critically needed niche for tools that enable quantification and testing of the impact of missing data. As open-source software, TOAST is fully customizable for integration into existing or novel custom informatic pipelines for phylogenomic inference. Software, a detailed manual, and example data files are available through github carolinafishes.github.io

Upon binding to thalidomide and other immunomodulatory drugs, the E3 ligase substrate receptor cereblon (CRBN) promotes proteosomal destruction by engaging the DDB1–CUL4A–Roc1–RBX1 E3 ubiquitin ligase in human cells but not in mouse cells, suggesting that sequence variations in CRBN may cause its inactivation. Therapeutically, CRBN engagers have the potential for broad applications in cancer and immune therapy by specifically reducing protein expression through targeted ubiquitin-mediated degradation. To examine the effects of defined sequence changes on CRBN's activity, we performed a comprehensive study using complementary theoretical, biophysical, and biological assays aimed at understanding CRBN's nonprimate sequence variations. With a series of recombinant thalidomide-binding domain (TBD) proteins, we show that CRBN sequence variants retain their drug-binding properties to both classical immunomodulatory drugs and dBET1, a chemical compound and targeting ligand designed to degrade bromodomain-containing 4 (BRD4) via a CRBN-dependent mechanism. We further show that dBET1 stimulates CRBN's E3 ubiquitin–conjugating function and degrades BRD4 in both mouse and human cells. This insight paves the way for studies of CRBN-dependent proteasome-targeting molecules in nonprimate models and provides a new understanding of CRBN's substrate-recruiting function.

The vertebrate immune response comprises multiple molecular and cellular components that interface to provide defense against pathogens. Because of the dynamic complexity of the immune system and its interdependent innate and adaptive functionality, an understanding of the whole-organism response to pathogen exposure remains unresolved. Zebrafish larvae provide a unique model for overcoming this obstacle, because larvae are protected against pathogens while lacking a functional adaptive immune system during the first few weeks of life. Zebrafish larvae were exposed to immune agonists for various lengths of time, and a microarray transcriptome analysis was executed. This strategy identified known immune response genes, as well as genes with unknown immune function, including the E3 ubiquitin ligase tripartite motif-9 (Trim9). Although trim9 expression was originally described as “brain specific,” its expression has been reported in stimulated human Mϕs. In this study, we found elevated levels of trim9 transcripts in vivo in zebrafish Mϕs after immune stimulation. Trim9 has been implicated in axonal migration, and we therefore investigated the impact of Trim9 disruption on Mϕ motility and found that Mϕ chemotaxis and cellular architecture are subsequently impaired in vivo. These results demonstrate that Trim9 mediates cellular movement and migration in Mϕs as well as neurons.

The resolution of the gar genome affords an opportunity to examine the diversification and functional specialization of immune effector molecules at a distant and potentially informative point in phylogenetic development. Although innate immunity is effected by a particularly large number of different families of molecules, the focus here is to provide detailed characterization of several families of innate receptors that are encoded in large multigene families, for which orthologous forms can be identified in other species of bony fish but not in other vertebrate groups as well as those for which orthologs are present in other vertebrate species. The results indicate that although teleost fish and the gar, as a holostean reference species, share gene families thought previously to be restricted to the teleost fish, the manner in which the members of the multigene families of innate immune receptors have undergone diversification is different in these two major phylogenetic radiations. It appears that both the total genome duplication and different patterns of genetic selection have influenced the derivation and stabilization of innate immune genes in a substantial manner during the course of vertebrate evolution.

Metastasis accounts for the majority of all cancer deaths, yet the process remains poorly understood. A pivotal step in the metastasis process is the exiting of tumor cells from the circulation, a process known as extravasation. However, it is unclear how tumor cells extravasate and whether multicellular clusters of tumor cells possess the ability to exit as a whole or must first disassociate. In this study, we use in vivo zebrafish and mouse models to elucidate the mechanism tumor cells use to extravasate. We found that circulating tumor cells exit the circulation using the recently identified extravasation mechanism, angiopellosis, and do so as both clusters and individual cells. We further show that when melanoma and cervical cancer cells utilize this extravasation method to exit as clusters, they exhibit an increased ability to form tumors at distant sites through the expression of unique genetic profiles. Collectively, we present a new model for tumor cell extravasation of both individual and multicellular circulating tumor cells. This article has an associated First Person interview with the first author of the paper.

The regulation of caspase-3 enzyme activity is a vital process in cell fate decisions leading to cell differentiation and tissue development or to apoptosis. The zebrafish, Danio rerio, has become an increasingly popular animal model to study several human diseases because of their transparent embryos, short reproductive cycles, and ease of drug administration. While apoptosis is an evolutionarily conserved process in metazoans, little is known about caspases from zebrafish, particularly regarding substrate specificity and allosteric regulation compared to the human caspases. We cloned zebrafish caspase-3a (casp3a) and examined substrate specificity of the recombinant protein, Casp3a, compared to human caspase-3 (CASP3) by utilizing M13 bacteriophage substrate libraries that incorporated either random amino acids at P5-P1′ or aspartate fixed at P1. The results show a preference for the tetrapeptide sequence DNLD for both enzymes, but the P4 position of zebrafish Casp3a also accommodates valine equally well. We determined the structure of zebrafish Casp3a to 2.28A resolution by X-ray crystallography, and when combined with molecular dynamics simulations, the results suggest that a limited number of amino acid substitutions near the active site result in plasticity of the S4 sub-site by increasing flexibility of one active site loop and by affecting hydrogen-bonding with substrate. The data show that zebrafish Casp3a exhibits a broader substrate portfolio, suggesting overlap with the functions of caspase-6 in zebrafish development.

Stem cells possess the ability to home in and travel to damaged tissue when injected intravenously. For the cells to exert their therapeutic effect, they must cross the blood vessel wall and enter the surrounding tissues. The mechanism of extravasation injected stem cells employ for exit has yet to be characterized. Using intravital microscopy and a transgenic zebrafish line Tg(fli1a:egpf) with GFP-expressing vasculature, we documented the detailed extravasation processes in vivo for injected stem cells in comparison to white blood cells (WBCs). While WBCs left the blood vessels by the standard diapedesis process, injected cardiac and mesenchymal stem cells underwent a distinct method of extravasation that was markedly different from diapedesis. Here, the vascular wall undergoes an extensive remodeling to allow the cell to exit the lumen, while the injected cell remains distinctively passive in activity. We termed this process Angio-pello-sis, which represents an alternative mechanism of cell extravasation to the prevailing theory of diapedesis. Video Highlight: https://youtu.be/i5EI-ZvhBps

Moore et al. reports the first single-cell gene expression analysis in zebrafish blood to distinguish major blood lineages, identify new cell types, and delineate heterogeneity in T cell leukemia., Hematopoiesis culminates in the production of functionally heterogeneous blood cell types. In zebrafish, the lack of cell surface antibodies has compelled researchers to use fluorescent transgenic reporter lines to label specific blood cell fractions. However, these approaches are limited by the availability of transgenic lines and fluorescent protein combinations that can be distinguished. Here, we have transcriptionally profiled single hematopoietic cells from zebrafish to define erythroid, myeloid, B, and T cell lineages. We also used our approach to identify hematopoietic stem and progenitor cells and a novel NK-lysin 4+ cell type, representing a putative cytotoxic T/NK cell. Our platform also quantified hematopoietic defects in rag2E450fs mutant fish and showed that these fish have reduced T cells with a subsequent expansion of NK-lysin 4+ cells and myeloid cells. These data suggest compensatory regulation of the innate immune system in rag2E450fs mutant zebrafish. Finally, analysis of Myc-induced T cell acute lymphoblastic leukemia showed that cells are arrested at the CD4+/CD8+ cortical thymocyte stage and that a subset of leukemia cells inappropriately reexpress stem cell genes, including bmi1 and cmyb. In total, our experiments provide new tools and biological insights into single-cell heterogeneity found in zebrafish blood and leukemia.

Three sequence lineages of MHC class I genes have been described in zebrafish (Danio rerio): U, Z, and L. The U lineage genes encoded on zebrafish chromosome 19 are predicted to provide the classical function of antigen presentation. This MHC class I locus displays significant haplotypic variation and is the only MHC class I locus in zebrafish that shares conserved synteny with the core mammalian MHC. Here we describe two MHC class I U lineage genes, mhc1ula and mhc1uma, that map to chromosome 22. Unlike the U lineage proteins encoded on chromosome 19, Ula and Uma likely play a nonclassical role as they lack conservation of key peptide binding residues, display limited polymorphic variation, and exhibit tissue-specific expression. We also describe a null haplotype at this chromosome 22 locus in which the mhc1ula and mhc1uma genes are absent due to a ∼30 kb deletion with no other MHC class I sequences present. Functional and non-functional transcripts of mhc1ula and mhc1uma were identified; however, mhc1uma transcripts were often not amplified or amplified at low levels from individuals possessing an apparently bona fide gene. These distinct U lineage genes may be restricted to the superorder Ostariophysi as similar sequences only could be identified from the blind cavefish (Astyanyx mexicanus), fathead minnow (Pimephales promelas), goldfish (Carassius auratus), and grass carp (Ctenopharyngodon idellus).

Mitochondria are known primarily as the location of the electron transport chain and energy production in cells. More recently, mitochondria have been shown to be signaling centers for apoptosis and inflammation. Reactive oxygen species (ROS) generated as by-products of the electron transport chain within mitochondria significantly impact cellular signaling pathways. Because of the toxic nature of ROS, mitochondria possess an antioxidant enzyme, superoxide dismutase 2 (SOD2), to neutralize ROS. If mitochondrial antioxidant enzymes are overwhelmed during severe infections, mitochondrial dysfunction can occur and lead to multiorgan failure or death. Pseudomonas aeruginosa is an opportunistic pathogen that can infect immunocompromised patients. Infochemicals and exotoxins associated with P. aeruginosa are capable of causing mitochondrial dysfunction. In this work, we describe the roles of SOD2 and mitochondrial ROS regulation in the zebrafish innate immune response to P. aeruginosa infection. sod2 is upregulated in mammalian macrophages and neutrophils in response to lipopolysaccharide in vitro , and sod2 knockdown in zebrafish results in an increased bacterial burden. Further investigation revealed that phagocyte numbers are compromised in Sod2-deficient zebrafish. Addition of the mitochondrion-targeted ROS-scavenging chemical MitoTEMPO rescues neutrophil numbers and reduces the bacterial burden in Sod2-deficient zebrafish. Our work highlights the importance of mitochondrial ROS regulation by SOD2 in the context of innate immunity and supports the use of mitochondrion-targeted ROS scavengers as potential adjuvant therapies during severe infections.

Complex cytoplasmic nucleotide-sensing mechanisms can recognize foreign DNA based on a lack of methylation and initiate an immune response to clear the infection. Zebrafish embryos with global DNA hypomethylation caused by mutations in the ubiquitin-like with PHD and ring finger domains 1 (uhrf1) or DNA methyltransferase 1 (dnmt1) genes exhibit a robust interferon induction characteristic of the first line of defense against viral infection. We found that this interferon induction occurred in non-immune cells and examined whether intracellular viral sensing pathways in these cells were the trigger. RNA-seq analysis of uhrf1 and dnmt1 mutants revealed widespread induction of Class I retrotransposons and activation of cytoplasmic DNA viral sensors. Attenuating Sting, phosphorylated Tbk1 and, importantly, blocking reverse transcriptase activity suppressed the expression of interferon genes in uhrf1 mutants. Thus, activation of transposons in cells with global DNA hypomethylation mimics a viral infection by activating cytoplasmic DNA sensors. This suggests that antiviral pathways serve as surveillance of cells that have derepressed intragenomic parasites due to DNA hypomethylation.

Comparative functional genomic studies require the proper identification of gene orthologs to properly exploit animal biomedical research models. To identify gene orthologs, comprehensive, conserved gene synteny analyses are necessary to unwind gene histories that are convoluted by two rounds of early vertebrate genome duplication, and in the case of the teleosts, a third round, the teleost genome duplication (TGD). Recently, the genome of the spotted gar, a holostean outgroup to the teleosts that did not undergo this third genome duplication, was sequenced and applied as an orthology bridge to facilitate the identification of teleost orthologs to human genes and to enhance the power of teleosts as biomedical models. In this study, we apply the spotted gar orthology bridge to help describe the gene history of the vertebrate TNFAIP8 family. Members of the TNFAIP8 gene family have been linked to regulation of immune function and homeostasis and the development of multiple cancer types. Through a conserved gene synteny analysis, we identified zebrafish orthologs to human TNFAIP8L1 and TNFAIP8L3 genes and two co-orthologs to human TNFAIP8L2, but failed to identify an ortholog to human TNFAIP8. Through the application of the orthology bridge, we determined that teleost orthologs to human TNFAIP8 genes were likely lost in a genome inversion event after their divergence from their common ancestor with spotted gar. These findings demonstrate the value of this enhanced approach to gene history analysis and support the development of teleost models to study complex questions related to an array of biomedical issues, including immunity and cancer.

The polymeric immunoglobulin (Ig) receptor (pIgR) is an integral transmembrane glycoprotein that plays an important role in the mammalian immune response by transporting soluble polymeric Igs across mucosal epithelial cells. Single pIgR genes, which are expressed in lymphoid organs including mucosal tissues, have been identified in several teleost species. A single pigr gene has been identified on zebrafish chromosome 2 along with a large multigene family consisting of 29 pigr-like (PIGRL) genes. Full-length transcripts from ten different PIGRL genes that encode secreted and putative inhibitory membrane-bound receptors have been characterized. Although PIGRL and pigr transcripts are detected in immune tissues, only PIGRL transcripts can be detected in lymphoid and myeloid cells. In contrast to pIgR which binds Igs, certain PIGRL proteins bind phospholipids. PIGRL transcript levels are increased after infection with Streptococcus iniae, suggesting a role for PIGRL genes during bacterial challenge. Transcript levels of PIGRL genes are decreased after infection with Snakehead rhabdovirus, suggesting that viral infection may suppress PIGRL function.

Antigen processing and presentation genes found within the MHC are among the most highly polymorphic genes of vertebrate genomes, providing populations with diverse immune responses to a wide array of pathogens. Here, we describe transcriptome, exome, and whole-genome sequencing of clonal zebrafish, uncovering the most extensive diversity within the antigen processing and presentation genes of any species yet examined. Our CG2 clonal zebrafish assembly provides genomic context within a remarkably divergent haplotype of the core MHC region on chromosome 19 for six expressed genes not found in the zebrafish reference genome: mhc1uga, proteasome-β 9b (psmb9b), psmb8f, and previously unknown genes psmb13b, tap2d, and tap2e We identify ancient lineages for Psmb13 within a proteasome branch previously thought to be monomorphic and provide evidence of substantial lineage diversity within each of three major trifurcations of catalytic-type proteasome subunits in vertebrates: Psmb5/Psmb8/Psmb11, Psmb6/Psmb9/Psmb12, and Psmb7/Psmb10/Psmb13. Strikingly, nearby tap2 and MHC class I genes also retain ancient sequence lineages, indicating that alternative lineages may have been preserved throughout the entire MHC pathway since early diversification of the adaptive immune system ∼500 Mya. Furthermore, polymorphisms within the three MHC pathway steps (antigen cleavage, transport, and presentation) are each predicted to alter peptide specificity. Lastly, comparative analysis shows that antigen processing gene diversity is far more extensive than previously realized (with ancient coelacanth psmb8 lineages, shark psmb13, and tap2t and psmb10 outside the teleost MHC), implying distinct immune functions and conserved roles in shaping MHC pathway evolution throughout vertebrates.

Teleost genomes encode multiple multigene families of immunoglobulin domain-containing innate immune receptors (IIIRs) with unknown function and no clear mammalian orthologs. However, the genomic organization of IIIR gene clusters and the structure and signaling motifs of the proteins they encode are similar to those of mammalian innate immune receptor families such as the killer cell immunoglobulin-like receptors (KIRs), leukocyte immunoglobulin-like receptors (LILRs), Fc receptors, triggering receptors expressed on myeloid cells (TREMs) and CD300s. Teleost IIIRs include novel immune-type receptors (NITRs); diverse immunoglobulin domain containing proteins (DICPs); polymeric immunoglobulin receptor-like proteins (PIGRLs); novel immunoglobulin-like transcripts (NILTs) and leukocyte immune-type receptors (LITRs). The accumulation of genomic sequence data has revealed that IIIR gene clusters in zebrafish display haplotypic and gene content variation. This intraspecific genetic variation, as well as significant interspecific variation, frequently confounds the identification of definitive orthologous IIIR sequences between teleost species. Nevertheless, by defining which teleost lineages encode (and do not encode) different IIIR families, predictions can be made about the presence (or absence) of specific IIIR families in each teleost lineage. It is anticipated that further investigations into available genomic resources and the sequencing of a variety of multiple teleost genomes will identify additional IIIR families and permit the modeling of the evolutionary origins of IIIRs.

Bony fish encode multiple multi-gene families of membrane receptors that are comprised of immunoglobulin (Ig) domains and are predicted to function in innate immunity. One of these families, the diverse immunoglobulin (Ig) domain-containing protein (DICP) genes, maps to three chromosomal loci in zebrafish. Most DICPs possess one or two Ig ectodomains and include membrane-bound and secreted forms. Membrane-bound DICPs include putative inhibitory and activating receptors. Recombinant DICP Ig domains bind lipids with varying specificity, a characteristic shared with mammalian CD300 and TREM family members. Numerous DICP transcripts amplified from different lines of zebrafish did not match the zebrafish reference genome sequence suggesting polymorphic and haplotypic variation. The expression of DICPs in three different lines of zebrafish has been characterized employing PCR-based strategies. Certain DICPs exhibit restricted expression in adult tissues whereas others are expressed ubiquitously. Transcripts of a subset of DICPs can be detected during embryonic development suggesting roles in embryonic immunity or other developmental processes. Transcripts representing eleven previously uncharacterized DICP sequences were identified. The assignment of two of these sequences to an unplaced genomic scaffold resulted in the identification of an alternative DICP haplotype that is linked to a MHC class I Z lineage haplotype on zebrafish chromosome 3. The linkage of DICP and MHC Class I genes also is observable in the genomes of the related grass carp (Ctenopharyngodon idellus) and common carp (Cyprinus carpio) suggesting that this is a shared character with the last common Cyprinidae ancestor.

Natural killer (NK) cells effect a form of innate immunity that recognizes and eliminates cells that are infected with certain viruses or have undergone malignant transformation. In mammals, this recognition can be mediated through immunoglobulin- (Ig) and/or lectin-type NK receptors (NKRs). NKR genes in mammals range from minimally polymorphic single copy genes to complex multigene families that exhibit high levels of haplotypic complexity and exhibit significant interspecific variation. Certain single copy NKR genes that are present in one mammal are present as expanded multi-gene families in other mammals. These observations highlight NKRs as one of the most rapidly evolving eukaryotic gene families and likely reflect the influence of pathogens, especially viruses, on their evolution. Although well characterized in human and mice, cytotoxic cells that are functionally similar to NK cells have been identified in species ranging from birds to reptiles, amphibians and fish. Although numerous receptors have been identified in non-mammalian vertebrates that share structural relationships with mammalian NKRs, functionally defining these lower vertebrate molecules as NKRs is confounded by methodological and interpretive complexities. Nevertheless, several lines of evidence suggest that NK-type function or its equivalent has sustained a long evolutionary history throughout vertebrate species.

The Sp family of transcription factors is required for the expression of cell cycle- and developmentally regulated genes, and the deregulated expression of a handful of family members is associated with human tumorigenesis. Sp2 is a relatively poorly characterized member of the Sp family that, although widely expressed, exhibits little or no DNA binding or transcriptional activity in human and mouse cell lines. To begin to address the role(s) played by Sp2 in early metazoan development we have cloned and characterized Sp2 from zebrafish (Danio rerio). We report that 1) the intron/exon organization and amino acid sequence of zebrafish Sp2 is closely conserved with its mammalian orthologues, 2) zebrafish Sp2 weakly stimulates an Sp-dependent promoter in vitro and associates with the nuclear matrix in a DNA-independent fashion, 3) zebrafish Sp2 is inherited as a maternal transcript, is transcribed in zebrafish embryos and adult tissues, and is required for completion of gastrulation, and 4) zebrafish lines carrying transgenes regulated by the Sp2 promoter recapitulate patterns of endogenous Sp2 expression.

Detailed knowledge of the external regulation of gene func-tion is a fundamental necessity in order to annotate sequencedgenomes and to understand biological processes in single cellsand multicellular organisms. One of the most widely used ap-proaches for the down-regulation of specific genes is the ap-plication of antisense agents. Antisense agents are oligomersthat have the ability to hybridize sequence specificslly tomRNAs, inhibiting translation and potentially leading to mRNAdegradation through RNAse H recruitment.

Large granular lymphocyte (LGL) leukemia is commonly associated with poor hematopoiesis. The first case of pulmonary artery hypertension (PAH) was observed in a 57-year-old woman with natural killer (NK)–LGL leukemia and transfusion-dependent anemia. Using a genetic approach, we demonstrated that killing of pulmonary endothelial cells by patient NK cells was mediated by dysregulated balance in activating and inhibitory NK-receptor signaling. Elevated pulmonary artery pressure and erythroid differentiation improved after disrupting the NK-receptor signaling pathway with 4 courses of a farnesyltransferase inhibitor, tipifarnib. Coincidental association between PAH and LGL leukemia suggest a causal relationship between the expanded lymphocyte population and these clinical manifestations. This trial is registered at www.ClinicalTrials.gov as NCI 6823.

Transmembrane bound receptors comprised of extracellular immunoglobulin (Ig) or lectin domains play integral roles in a large number of immune functions including inhibitory and activating responses. The function of many of the activating receptors requires a physical interaction with an adaptor protein possessing a cytoplasmic regulatory motif. The partnering of an activating receptor with an adaptor protein relies on complementary charged residues in the two transmembrane domains. The mammalian natural killer (NK) and Fc receptors (FcR) represent two of many receptor families, which possess activating receptors that partner with adaptor proteins for signaling. Zebrafish represent a powerful experimental model for understanding developmental regulation at early stages of embryogenesis and for efficiently generating transgenic animals. In an effort to understand developmental aspects of immune receptor function, we have accessed the partially annotated zebrafish genome to identify six different adaptor molecules: Dap10, Dap12, Cd3zeta, Cd3zeta-like, FcRgamma and FcRgamma-like that are homologous to those effecting immune function in mammals. Their genomic organizations have been characterized, cDNA transcripts have been recovered, phylogenetic relationships have been defined and their cell lineage-specific expression patterns have been established.

Both inhibitory and activating forms of natural killer (NK) cell receptors are found in mammals. The activating receptors play a direct role in the recognition of virally infected or transformed cells and transduce activating signals into the cell by partnering with an adaptor protein, which contains a cytoplasmic activation motif. Activating NK receptors encoded by the mammalian leukocyte receptor complex (e.g., killer cell immunoglobulin-like receptors) and the natural killer complex (e.g., Ly49s) partner with the adaptor protein DAP12, whereas NK receptors encoded in the CD94/NKG2 complex partner with the adaptor protein DAP10. Novel immune-type receptors (NITRs) found in bony fish share several common features with immunoglobulin-type NK receptors. Nitr9 is a putative activating receptor in zebrafish that induces cytotoxicity within the context of human NK cells. One isoform of Nitr9, Nitr9L, is shown here to preferentially partner with a zebrafish ortholog of Dap12. Cross-linking the Nitr9L-Dap12 complex results in activation of the phosphytidylinositol 3-kinase--AKT--extracellular signal-regulated kinase pathway suggesting that the DAP12-based activating pathway is conserved between bony fish and mammals.

The zebrafish has become a powerful tool for dissecting vertebrate gene function during embryogenesis. Numerous molecular systems have been developed to examine gene function in zebrafish, including transgenics for creating lineage-tracer lines of zebrafish that express a fluorescent protein as a marker for specific populations of cells, and antisense strategies, primarily morpholinos, for knocking down gene function. The focus of this review is to summarize the pros and cons of the currently available systems for functional genomics in zebrafish, and to discuss the need for future methodologies.

The identification of C-type lectin (Group V) natural killer (NK) cell receptors in bony fish has remained elusive. Analyses of the Fugu rubripes genome database failed to identify Group V C-type lectin domains (Zelensky and Gready, BMC Genomics 5:51, 2004) suggesting that bony fish, in general, may lack such receptors. Numerous Group II C-type lectin receptors, which are structurally similar to Group V (NK) receptors, have been characterized in bony fish. By searching the zebrafish genome database we have identified a multi-gene family of Group II immune-related, lectin-like receptors (illrs) whose members possess inhibiting and/or activating signaling motifs typical of Group V NK receptors. Illr genes are differentially expressed in the myeloid and lymphoid lineages, suggesting that they may play important roles in the immune functions of multiple hematopoietic cell lineages.

The novel immune-type receptor (NITR) genes encode a unique multigene family of leukocyte regulatory receptors, which possess an extracellular Ig variable (V) domain and may function in innate immunity. Artificial chromosomes that encode zebrafish NITRs have been assembled into a contig spanning ≈350 kb. Resolution of the complete NITR gene cluster has led to the identification of eight previously undescribed families of NITRs and has revealed the presence of C-type lectins within the locus. A maximum haplotype of 36 NITR genes (138 gene sequences in total) can be grouped into 12 distinct families, including inhibitory and activating receptors. An extreme level of interindividual heterozygosity is reflected in allelic polymorphisms, haplotype variation, and family-specific isoform complexity. In addition, the exceptional diversity of NITR sequences among species suggests divergent evolution of this multigene family with a birth-and-death process of member genes. High-confidence modeling of Nitr V-domain structures reveals a significant shift in the spatial orientation of the Ig fold, in the region of highest interfamily variation, compared with Ig V domains. These studies resolve a complete immune gene cluster in zebrafish and indicate that the NITRs represent the most complex family of activating/inhibitory surface receptors thus far described.

SYNOPSIS. The prototypic forms of teleost novel immune-type receptors (NITRs) consist of a variable (V) region, a unique V-like C2 (V/C2) domain, a transmembrane region and a cytoplasmic tail containing immunoreceptor tyrosine-based inhibition motifs (ITIMs). NITRs encode diversified V regions in large multigene families but do not undergo somatic rearrangement. Studies in four different bony fish model systems have identified a number of different organizational forms of NITRs. Specifically,NITR genes encode Nterminal ectodomains of the V-type but otherwise vary in the: total number of extracellular immunoglobulin domains, number and location of joining (J) region-like motifs, presence of transmembrane regions, presence of charged residues within transmembrane regions, presence of cytoplasmic tails, and/or distribution of ITIM(s) within the cytoplasmic tails. V region-containing NITRs constitute a far more complex family than recognized originally and currently include individual members that potentially function through inhibitory as well as activating mechanisms. The genomic organization of the NITR gene cluster as well as the structural diversity and overall architecture of the NITR proteins is reminiscent of genes encoded at the mammalian leukocyte receptor cluster (LRC); however, there presently is no functional evidence to support an orthologous relationship between NITR and LRC gene products. Comparisons of the predicted structures of the NITRs have identified several short regions of sequence identity and a novel cloning strategy has been devised that selects for secretory and transmembrane proteins that encode these short motifs. Using this approach, related genes termed immune-type receptors (ITRs) have been identified in cartilaginous fish. Taken together, these studies indicate that leukocyte regulatory receptors, including those that mediate natural killer function, might have emerged early in vertebrate evolution and that the NITR/ITR genes represent a new and potentially highly significant link between innate and adaptive immune responses.

Novel immune-type receptor ( NITR) genes that encode two extracellular immunoglobulin domains and cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs) have been described previously in three lineages of bony fish. In the current study, four ITIM-containing NITR cDNAs are identified in the rainbow trout ( Oncorhynchus mykiss), and their expression patterns and genomic complexity are characterized. The ITIM-containing NITR2 gene maps 1.3 cM from an ITIM-containing C-type lectin receptor ( TCL-2) on linkage group XXI. A comprehensive, phylogenetic analysis of NITRs from rainbow trout and three other major lineages of bony fish defines conserved families of NITRs and suggests an ancient lineage of distinct groups of genes. Several probable scenarios that explain the origins of variant forms of NITRs are described.

Two decades of research have established the zebrafish (Danio rerio) as a significant model system for studying vertebrate development and gene structure-function relationships. Recent advances in mutation screening, the creation of genomic resources, including the Zebrafish Genome Project and the development of efficient transgenesis procedures, make this model increasingly attractive for immunological study.

A novel gene, BIVM (for basic, immunoglobulin-like variable motif-containing), has been identified using an electronic search based on the conservation of short sequence motifs within the variable region of immunoglobulin (Ig) genes. BIVM maps to human chromosome 13q32-q33 and is predicted to encode a 503-amino-acid protein with a pI of 9.1. The 5' untranslated region of BIVM is encoded in two exons; the coding portion is encoded in nine exons. BIVM is tightly linked (41 bp) and in the opposite transcriptional orientation to MGC5302 (also known as KDEL1 and EP58) in human. The ubiquitous expression of BIVM in normal tissues and the presence of a 5' CpG island suggest that BIVM is a housekeeping gene. Characterization of BIVM in representative species demonstrates significant conservation throughout deuterostomes; no sequence with significant identity to BIVM has been detected in proteostomes. However, an unusual gene has been identified in the protozoan pathogen Giardia lamblia that is similar to the core sequence of BIVM, suggesting the possibility of a horizontal gene transfer.

Major histocompatibility complex (MHC) molecules play a central role in the immune response and in the recognition of non-self. Found in all jawed vertebrate species, including zebrafish and other teleosts, MHC genes are considered the most polymorphic of all genes. In this review we focus on the multi-faceted diversity of zebrafish MHC class I genes, which are classified into three sequence lineages: U, Z, and L. We examine the polygenic, polymorphic, and haplotypic diversity of the zebrafish MHC class I genes, discussing known and postulated functional differences between the different class I lineages. In addition, we provide the first comprehensive nomenclature for the L lineage genes in zebrafish, encompassing at least 15 genes, and characterize their sequence properties. Finally, we discuss how recent findings have shed new light on the remarkably diverse MHC loci of this species.

Five large multigene families encoding innate-type immune receptors that are comprised of immunoglobulin domains have been identified in bony fish, of which four do not possess definable mammalian orthologs. The members of some of the multigene families exhibit unusually extensive patterns of divergence and the individual family members demonstrate marked variation in interspecific comparisons. As a group, the gene families reveal striking differences in domain type and content, mechanisms of intracellular signaling, basic structural features, haplotype and allelic variation and ligand binding. The potential functional roles of these innate immune receptors, their relationships to immune genes in higher vertebrate species and the basis for their adaptive evolution are of broad interest. Ongoing investigations are expected to provide new insight into alternative mechanisms of immunity.

Immune inhibitory receptor genes that encode a variable (V) region, a unique V-like C2 (V/C2) domain, a transmembrane region, and a cytoplasmic tail containing immunoreceptor tyrosine-based inhibition motifs (ITIMs) have been described previously in two lineages of bony fish. In the present study, eleven related genes encoding distinct structural forms have been identified inIctalurus punctatus(channel catfish), a well characterized immunological model system that represents a third independent bony fish lineage. Each of the different genes encodes an N-terminal V region but differs in the number of extracellular Ig domains, number and location of joining (J) region-like motifs, presence of transmembrane regions, presence of charged residues in transmembrane regions, presence of cytoplasmic tails, and/or distribution of ITIM(s) within the cytoplasmic tails. Variation in the numbers of genomic copies of the different gene types, their patterns of expression, and relative levels of expression in mixed leukocyte cultures (MLC) is reported. V region-containing immune-type genes constitute a far more complex family than recognized originally and include individual members that might function in inhibitory or, potentially activatory manners.

Novel immune-type receptor (NITR) genes, which initially were identified in the Southern pufferfish (Spheroides nephelus), encode products which consist of an extracellular variable (V) and V-like C2 (V/C2) domain, a transmembrane region, and a cytoplasmic tail, which typically possesses an immunoreceptor tyrosine-based inhibition motif (ITIM). Multiple NITR genes have been identified in close, contiguous chromosomal linkage. The V regions of NITRs resemble prototypic forms defined for immunoglobulin (Ig) and T-cell antigen receptor (TCR), are present in multiple families and exhibit regionalized variation in sequence, which also occurs in Ig and TCR. Comparisons of exons encoding transmembrane and cytoplasmic regions of multiple NITRs suggest that exon shuffling has factored in the diversification of the NITR gene complex. Zebrafish (Danio rerio) NITRs exhibit many of these characteristics. NITRs that have been identified in additional species of bony fish demonstrate additional variation in the number of extracellular domains as well as in the presence of intramembranous charged residues, cytoplasmic tails and ITIMs. The presence in NITRs of V regions that are related closely to those found in Ig and TCR, as well as regulatory motifs and other structural features that are characteristic of immune inhibitory receptors encoded at the leukocyte receptor cluster, suggests that the NITRs are representative of an integral stage in the evolution of innate and adaptive immune function.

DNMT2 is a human protein that displays strong sequence similarities to DNA (cytosine-5)-methyltransferases (m5C MTases) of both prokaryotes and eukaryotes. DNMT2 contains all 10 sequence motifs that are conserved among m5C MTases, including the consensus S-adenosyl-l-methionine-binding motifs and the active site ProCys dipeptide. DNMT2 has close homologs in plants, insects and Schizosaccharomyces pombe, but no related sequence can be found in the genomes of Saccharomyces cerevisiae or Caenorhabditis elegans. The crystal structure of a deletion mutant of DNMT2 complexed with S-adenosyl-l-homocysteine (AdoHcy) has been determined at 1.8 Å resolution. The structure of the large domain that contains the sequence motifs involved in catalysis is remarkably similar to that of M.HhaI, a confirmed bacterial m5C MTase, and the smaller target recognition domains of DNMT2 and M.HhaI are also closely related in overall structure. The small domain of DNMT2 contains three short helices that are not present in M.HhaI. DNMT2 binds AdoHcy in the same conformation as confirmed m5C MTases and, while DNMT2 shares all sequence and structural features with m5C MTases, it has failed to demonstrate detectable transmethylase activity. We show here that homologs of DNMT2, which are present in some organisms that are not known to methylate their genomes, contain a specific target-recognizing sequence motif including an invariant CysPheThr tripeptide. DNMT2 binds DNA to form a denaturant-resistant complex in vitro. While the biological function of DNMT2 is not yet known, the strong binding to DNA suggests that DNMT2 may mark specific sequences in the genome by binding to DNA through the specific target-recognizing motif.

Summary: The zebrafish has become a well-established animal model for the analysis of development and of several disease phenotypes. Several of the favorable traits that make it a popular model organism would also be beneficial for the study of normal and abnormal vertebrate development in which DNA methylation may play a role. We report the determination of the full-length cDNA sequence corresponding to the zebrafish DNA (cytosine-5-) methyltransferase gene, Dnmt1. It is 4,907 bases long and has an open reading frame predicted to encode a 1,499 amino acid protein that is similar in size and sequence to a number of other methyltransferases identified in other organisms. genesis 30:213–219, 2001. © 2001 Wiley-Liss, Inc.

In addition to being an excellent model system for studying vertebrate development, the zebrafish has become a great tool for gene discovery by mutational analysis. The recent availability of the zebrafish EST database and radiation hybrid mapping panels has dramatically expanded the framework for genomic research in this species. Developing comparative maps of the zebrafish and human genomes is of particular importance for zebrafish mutagenesis studies in which human orthologs are sought for zebrafish genes. However, only partial cDNA sequences are determined routinely for mapped ESTs, leaving the identity of the EST in question. It previously had been reported that zebrafish linkage group 7 shares conserved synteny with human chromosome 11q13. In an effort to further define this relationship, five full-length zebrafish cDNAs, fth1, slc3a2, prkri, cd81, and pc, as well as one putative human gene, DBX were identified and their map positions ascertained. These six genes, along with men1, fgf3 and cycd1 define two regions of conserved synteny between linkage group 7 and 11q13.

Four variant forms of the V1 (T15-H chain) gene are synthesized in mice. Each V1 variant pairs with a distinct L chain to produce a binding site having specificity for phosphocholine (PC). Transgenic mice expressing variant forms of the V1 gene were analyzed to elucidate the factors driving B cell selection into the peripheral repertoire. In all four lines of H chain transgenic mice analyzed, transgene expression caused complete allelic exclusion of endogenous H chains in the bone marrow (BM), whereas most splenic B cells expressed endogenous H chains. The number of sIgM(+) BM B cells and their sIg receptor number was reduced compared to that of normal transgene-negative controls, suggesting that B cells expressing transgene-encoded H chains were being negatively selected in the BM. Mice expressing autoreactive forms of the V1 transgene with lower affinity for PC (M603H and M167H) exhibit positive selection of PC-specific B cells into the spleen, whereas mice expressing the higher affinity T15H variant exhibited elevated PC-specific B cells in the peritoneal cavity but few V(H)1(+) splenic B cells. These data suggest that the higher affinity T15-id(+) B cells preferentially survive in the peritoneal cavity. When these H chain transgenes were crossed into the mu MT knockout mouse in which surface expression of endogenous H chains is blocked, the percent of splenic V(H)1(+) PC-specific B cells increased up to 5-fold and T15-id(+) B cells were detectable in the spleen of T15H mice. This implies that T15-id(+) PC-specific B cells can be selected into the periphery, but they compete poorly with follicular B cells expressing endogenous Ig.

A ribozyme based gene control element enabled the spatio-temporal regulation of gene function in mammalian cell culture with light.

The spermatozoon and oocyte genomes bear sex-specific methylation patterns that are established during gametogenesis and are required for the allele-specific expression of imprinted genes in somatic tissues. The mRNA for Dnmt1, the predominant maintenance and de novo DNA (cytosine-5)-methyl transferase in mammals, is present at high levels in postmitotic murine germ cells but undergoes alternative splicing of sex-specific 5′ exons, which controls the production and localization of enzyme during specific stages of gametogenesis. An oocyte-specific 5′ exon is associated with the production of very large amounts of active Dnmt1 protein, which is truncated at the N terminus and sequestered in the cytoplasm during the later stages of oocyte growth, while a spermatocyte-specific 5′ exon interferes with translation and prevents production of Dnmt1 during the prolonged crossing-over stage of male meiosis. During the course of postnatal oogenesis, Dnmt1 is present at high levels in nuclei only in growing dictyate oocytes, a stage during which gynogenetic developmental potential is lost and biparental developmental potential is gained.

Trace levels of 5-methylcytosine persist in the DNA of mouse embryonic stem cells that are homozygous for null mutations in Dnmt1 , the gene for the one previously recognized metazoan DNA methyltransferase. This residual 5-methylcytosine may be the product of a candidate second DNA methyltransferase, Dnmt2, that has now been identified in human and mouse. Dnmt2 contains all the sequence motifs diagnostic of DNA (cytosine-5)-methyltransferases but appears to lack the large N-terminal regulatory domain common to other eukaryotic methyltransferases. Dnmt2 is more similar to a putative DNA methyltransferase of the fission yeast Schizosaccharomyces pombe than to Dnmt1. Dnmt2 produces multiple mRNA species that are present at low levels in all tissues of human and mouse and is not restricted to those cell types known to be active in de novo methylation. The human DNMT2 gene was mapped to chromosome 10p12-10p14 in a panel of radiation hybrids. Dnmt2 is a candidate for the activity that methylates newly integrated retroviral DNA and maintains trace levels of 5-methylcytosine in the DNA of embryonic stem cells homozygous for null mutations in Dnmt1.

Most of the 5-methylcytosine in mammalian DNA resides in transposons, which are specialized intragenomic parasites that represent at least 35% of the genome. Transposon promoters are inactive when methylated and, over time, C → T transition mutations at methylated sites destroy many transposons. Apart from that subset of genes subject to X inactivation and genomic imprinting, no cellular gene in a non-expressing tissue has been proven to be methylated in a pattern that prevents transcription. It has become increasingly difficult to hold that reversible promoter methylation is commonly involved in developmental gene control; instead, suppression of parasitic sequence elements appears to be the primary function of cytosine and methylation, with crucial secondary roles in allele-specific gene expression as seen in X inactivation and genomic imprinting.

Understanding spatial distribution and dynamics of receptors within unperturbed membranes is essential for elucidating their role in antiviral signaling, but conventional studies of detergent-resistant membrane fractions cannot provide this information. Caveolae are integral to numerous signaling pathways and these membrane domains have been previously implicated in viral entry but not antiviral defense. This study shows, for the first time, the importance of spatio-temporal regulation of signaling receptors and the importance of the regulation of clustering for downstream signaling. A novel mechanism for virus evasion of host cell defenses is demonstrated through disruption of clusters of signaling molecules organized within caveolin-rich domains. Viral infection leads to a downregulation in Caveolin-1b (Cav-1b), disrupting clusters of CRFB1, a zebrafish type I interferon receptor (-R) subunit. Super-resolution microscopy has enabled the first single-molecule imaging of CRFB1 association with cav-1b-containing membrane domains. Strikingly, downregulation of Cav-1b, the major protein component of caveolae, caused CRFB1 clusters to disperse. Dispersal of CRFB1 clusters led to a suppressed antiviral immune response both in vitro and in vivo, through abrogation of downstream signaling. This response strongly suggests that CRFB1 organization within cav-1b-containing membrane domains is critical for IFN-mediated antiviral defense and presents a previously undescribed antiviral evasion strategy to alter IFN signaling and the antiviral immune response.