Virus-specific editing identification approach reveals the landscape of A-to-I editing and its impacts on SARS-CoV-2 characteristics and evolution.

Upon SARS-CoV-2 infection, viral intermediates specifically activate the IFN response through MDA5-mediated sensing and accordingly induce ADAR1 p150 expression, which might lead to viral A-to-I RNA editing. Here, we developed an RNA virus-specific editing identification pipeline, surveyed 7622 RNA-seq data from diverse types of samples infected with SARS-CoV-2, and constructed an atlas of A-to-I RNA editing sites in SARS-CoV-2. We found that A-to-I editing was dynamically regulated, varied between tissue and cell types, and was correlated with the intensity of innate immune response. On average, 91 editing events were deposited at viral dsRNA intermediates per sample. Moreover, editing hotspots were observed, including recoding sites in the spike gene that affect viral infectivity and antigenicity. Finally, we provided evidence that RNA editing accelerated SARS-CoV-2 evolution in humans during the epidemic. Our study highlights the ability of SARS-CoV-2 to hijack components of the host antiviral machinery to edit its genome and fuel its evolution, and also provides a framework and resource for studying viral RNA editing.

Adenosine-to-Inosine RNA Editing of Alu Double-Stranded (ds)RNAs Is Markedly Decreased in Multiple Sclerosis and Unedited Alu dsRNAs Are Potent Activators of Proinflammatory Transcriptional Responses.

Sensors that detect dsRNA stimulate IFN responses as a defense against viral infection. IFN responses are also well documented in a variety of human autoimmune diseases, including relapsing-remitting multiple sclerosis (MS), in which increased IFN responses result from increased levels of double-stranded endogenous Alu RNAs. Mechanisms underlying increases in double-stranded Alu RNAs in MS are obscure. We find widespread loss of adenosine-to-inosine editing of Alu RNAs in MS. Unedited Alu RNAs are potent activators of both IFN and NF-κB responses via the dsRNA sensors, RIG-I, and TLR3. Minor editing of highly active Alu elements abrogates the ability to activate both transcriptional responses. Thus, adenosine-to-inosine editing may also represent an important defense against autoimmune diseases such as MS.

ADAR1 Regulates Early T Cell Development via MDA5-Dependent and -Independent Pathways.

ADAR1 is an RNA-editing enzyme that is abundant in the thymus. We have previously reported that ADAR1 is required for establishing central tolerance during the late stage of thymocyte development by preventing MDA5 sensing of endogenous dsRNA as nonself. However, the role of ADAR1 during the early developmental stage remains unknown. In this study, we demonstrate that early thymocyte-specific deletion of ADAR1 in mice caused severe thymic atrophy with excessive apoptosis and impaired transition to a late stage of development accompanied by the loss of TCR expression. Concurrent MDA5 deletion ameliorated apoptosis but did not restore impaired transition and TCR expression. In addition, forced TCR expression was insufficient to restore the transition. However, simultaneous TCR expression and MDA5 deletion efficiently ameliorated the impaired transition of ADAR1-deficient thymocytes to the late stage. These findings indicate that RNA-editing-dependent and -independent functions of ADAR1 synergistically regulate early thymocyte development.

Retroviruses drive the rapid evolution of mammalian APOBEC3 genes.

APOBEC3 (A3) genes are members of the AID/APOBEC gene family that are found exclusively in mammals. A3 genes encode antiviral proteins that restrict the replication of retroviruses by inducing G-to-A mutations in their genomes and have undergone extensive amplification and diversification during mammalian evolution. Endogenous retroviruses (ERVs) are sequences derived from ancient retroviruses that are widespread mammalian genomes. In this study we characterize the A3 repertoire and use the ERV fossil record to explore the long-term history of coevolutionary interaction between A3s and retroviruses. We examine the genomes of 160 mammalian species and identify 1,420 AID/APOBEC-related genes, including representatives of previously uncharacterized lineages. We show that A3 genes have been amplified in mammals and that amplification is positively correlated with the extent of germline colonization by ERVs. Moreover, we demonstrate that the signatures of A3-mediated mutation can be detected in ERVs found throughout mammalian genomes and show that in mammalian species with expanded A3 repertoires, ERVs are significantly enriched for G-to-A mutations. Finally, we show that A3 amplification occurred concurrently with prominent ERV invasions in primates. Our findings establish that conflict with retroviruses is a major driving force for the rapid evolution of mammalian A3 genes.

RNA editing derived epitopes function as cancer antigens to elicit immune responses.

In addition to genomic mutations, RNA editing is another major mechanism creating sequence variations in proteins by introducing nucleotide changes in mRNA sequences. Deregulated RNA editing contributes to different types of human diseases, including cancers. Here we report that peptides generated as a consequence of RNA editing are indeed naturally presented by human leukocyte antigen (HLA) molecules. We provide evidence that effector CD8+ T cells specific for edited peptides derived from cyclin I are present in human tumours and attack tumour cells that are presenting these epitopes. We show that subpopulations of cancer patients have increased peptide levels and that levels of edited RNA correlate with peptide copy numbers. These findings demonstrate that RNA editing extends the classes of HLA presented self-antigens and that these antigens can be recognised by the immune system.

Extensive RNA editing and splicing increase immune self-representation diversity in medullary thymic epithelial cells.

BACKGROUND: In order to become functionally competent but harmless mediators of the immune system, T cells undergo a strict educational program in the thymus, where they learn to discriminate between self and non-self. This educational program is, to a large extent, mediated by medullary thymic epithelial cells that have a unique capacity to express, and subsequently present, a large fraction of body antigens. While the scope of promiscuously expressed genes by medullary thymic epithelial cells is well-established, relatively little is known about the expression of variants that are generated by co-transcriptional and post-transcriptional processes. RESULTS: Our study reveals that in comparison to other cell types, medullary thymic epithelial cells display significantly higher levels of alternative splicing, as well as A-to-I and C-to-U RNA editing, which thereby further expand the diversity of their self-antigen repertoire. Interestingly, Aire, the key mediator of promiscuous gene expression in these cells, plays a limited role in the regulation of these transcriptional processes. CONCLUSIONS: Our results highlight RNA processing as another layer by which the immune system assures a comprehensive self-representation in the thymus which is required for the establishment of self-tolerance and prevention of autoimmunity.

Somatic hypermutation in immunity and cancer: Critical analysis of strand-biased and codon-context mutation signatures.

For 30 years two general mechanisms have competed to explain somatic hypermutation of immunoglobulin (Ig) genes. The first, the DNA-based model, is focused only on DNA substrates. The modern form is the Neuberger "DNA Deamination Model" based on activation-induced cytidine deaminase (AID) and short-patch error-prone DNA repair by DNA Polymerase-η operating around AID C-to-U lesions. The other is an RNA-based mechanism or the "Reverse Transcriptase Model" of SHM which produces strand-biased mutations at A:T and G:C base pairs. This involves error-prone cDNA synthesis via an RNA-dependent DNA polymerase copying the Ig pre-mRNA template and integrating the now error-filled cDNA copy back into the normal chromosomal site. The modern form of this mechanism depends on AID dC-to-dU lesions and long tract error-prone cDNA synthesis of the transcribed strand by DNA Polymerase-η acting as a reverse transcriptase. The evidence for and against each mechanism is critically evaluated. The conclusion is that all the SHM molecular data gathered since 1980 supports directly or indirectly the RNA/RT-based mechanism. All the data and critical analyses are systematically laid out so the reader can evaluate this conclusion for themselves. Recently we have investigated whether similar RNA/RT-based mutator mechanisms explain how de novo mutations arise in somatic tissues (cancer genomes). The data analyses indeed suggest that cancers arise via dysregulated "Ig-like SHM responses" involving rogue DNA and RNA deaminations coupled to genome-wide RT events. Further, Robyn Lindley has recently shown that the strand-biased mutations in cancer genome genes are also in "codon-context." This has been termed Targeted Somatic Mutation (TSM) to highlight that mutations are far more targeted than previously thought in somatic tissues associated with disease. The TSM process implies an "in-frame DNA reader" whereby DNA and RNA deaminases at transcribed regions are guided in their mutagenic action, by the codon reading frame of the DNA.

Skewed primary Igκ repertoire and V-J joining in C57BL/6 mice: implications for recombination accessibility and receptor editing.

Previous estimates of the diversity of the mouse Ab repertoire have been based on fragmentary data as a result of many technical limitations, in particular, the many samples necessary to provide adequate coverage. In this study, we used 5'-coding end amplification of Igκ mRNAs from bone marrow, splenic, and lymph node B cells of C57BL/6 mice combined with amplicon pyrosequencing to assess the functional and nonfunctional Vκ repertoire. To evaluate the potential effects of receptor editing, we also compared V/J associations and usage in bone marrows of mouse mutants under constitutive negative selection or an altered ability to undergo secondary recombination. To focus on preimmune B cells, our cell sorting strategy excluded memory B cells and plasma cells. Analysis of ~90 Mbp, representing >250,000 individual transcripts from 59 mice, revealed that 101 distinct functional Vκ genes are used but at frequencies ranging from ~0.001 to ~10%. Usage of seven Vκ genes made up >40% of the repertoire. A small class of transcripts from apparently nonfunctional Vκ genes was found, as were occasional transcripts from several apparently functional genes that carry aberrant recombination signals. Of 404 potential V-J combinations (101 Vκs × 4 Jκs), 398 (98.5%) were found at least once in our sample. For most Vκ transcripts, all Jκs were used, but V-J association biases were common. Usage patterns were remarkably stable in different selective conditions. Overall, the primary κ repertoire is highly skewed by preferred rearrangements, limiting Ab diversity, but potentially facilitating receptor editing.

BAFF-R expression correlates with positive selection of immature B cells.

The interaction between BAFF and BAFF-R is crucial for the development of mature B cells. Here, we report that the expression of BAFF-R is first detectable on a fraction of mouse CD19(+) CD93(+) IgM(+) CD23(-) and human CD19(+) CD10(+) IgM(+) BM B cells. This BAFF-R(+) BM B-cell population shows higher levels of surface IgM expression and decreased RAG-2 transcripts than BAFF-R(-) immature B cells. When cultured, mouse BAFF-R(-), but not BAFF-R(+) immature B cells spontaneously undergo B-cell receptor editing. However, BAFF-R(+) immature B cells cultured in the presence of an anti-κ light chain antibody are induced to undergo receptor editing. This receptor editing correlates with down-modulation of surface BAFF-R expression and the up-regulation of RAG-2 at the RNA level. B-cell receptor (BCR) cross-linking on splenic T1 B cells results in down-modulation of the BAFF-R, and receptor editing and RAG-2 up-regulation in a minor fraction of B cells. BCR cross-linking on splenic T2/3 B cells results in partly down and partly up-modulation of BAFF-R expression and no evidence for receptor editing. Overall, our data indicate that BAFF-R expression is tightly regulated during B-cell development in mouse and human and its expression is correlated with positive selection.

Direct reduction of antigen receptor expression in polyclonal B cell populations developing in vivo results in light chain receptor editing.

Secondary Ab V region gene segment rearrangement, termed receptor editing, is a major mechanism contributing to B lymphocyte self-tolerance. However, the parameters that determine whether a B cell undergoes editing are a current subject of debate. We tested the role that the level of BCR expression plays in the regulation of receptor editing in a polyclonal population of B cells differentiating in vivo. Expression of a short hairpin RNA for κ L chain RNA in B cells resulted in reduction in levels of this RNA and surface BCRs. Strikingly, fully mature and functional B cells that developed in vivo and efficiently expressed the short hairpin RNA predominantly expressed BCRs containing λ light chains. This shift in L chain repertoire was accompanied by inhibition of development, increased Rag gene expression, and increased λ V gene segment-cleavage events at the immature B cell stage. These data demonstrated that reducing the translation of BCRs that are members of the natural repertoire at the immature B cell stage is sufficient to promote editing.

Is transcription the dominant force during dynamic changes in gene expression?

Dynamic changes in gene expression punctuate lymphocyte development and are a characteristic of lymphocyte activation. A prevailing view has been that these changes are driven by DNA transcription factors, which are the dominant force in gene expression. Accumulating evidence is challenging this DNA centric view and has highlighted the prevalence and dynamic nature of RNA handling mechanisms. Alternative splicing and differential polyadenylation appear to be more widespread than first thought. Changes in mRNA decay rates also affect the abundance of transcripts and this mechanism may contribute significantly to gene expression. Additional RNA handling mechanisms that control the intracellular localization of mRNA and association with translating ribosomes are also important. Thus, gene expression is regulated through the coordination of transcriptional and post-transcriptional mechanisms. Developing a more "RNA centric" view of gene expression will allow a more systematic understanding of how gene expression and cell function are integrated.

A developmental characterization of mesolimbocortical serotonergic gene expression changes following early immune challenge.

An immunogenic challenge during early postnatal development leads to long-term changes in behavioural and physiological measures reflecting enhanced emotionality and anxiety. Altered CNS serotonin (5-HT) signalling during the third postnatal week is thought to modify the developing neurocircuitry governing anxiety-like behaviour. Changes in 5-HT signalling during this time window may underlie increased emotionality reported in early immune challenge rodents. Here we examine both the spatial and temporal profile of 5-HT related gene expression, including 5HT1A, 2A, 2C receptors, the 5-HT transporter (5HTT), and tryptophan hydroxylase 2 (TPH2) during early development (postnatal day [P]14, P17, P21, P28) in mice challenged with lipopolysaccharide (LPS) during the first postnatal week. Expression levels were measured using in situ hybridization in regions associated with mediating emotive behaviours: the dorsal raphe (DR), hippocampus, amygdala, and prefrontal cortex (PFC). Increased TPH2 and 5HTT expression in the ventrolateral region of the DR of LPS-mice accompanied decreased expression of ventral DR 5HT1A and dorsal DR 5HTT. In the forebrain, 5HT1A and 2A receptors were increased, whereas 5HT2C receptors were decreased in the hippocampus. Decreased mRNA expression of 5HT2C was detected in the amygdala and PFC of LPS-treated pups; 5HT1A was increased in the PFC. The majority of these changes were restricted to P14-21. These transient changes in 5-HT expression coincide with the critical time window in which 5-HT disturbance leads to permanent modification of anxiety-related behaviours. This suggests that alterations in CNS 5-HT during development may underlie the enhanced emotionality associated with an early immune challenge.

Diverse functions for DNA and RNA editing in the immune system.

Polynucleotide DNA and RNA editing enzymes alter nucleic acid sequences and can thereby modify encoded informational content. Two major families of polynucleotide editing enzymes, the AID/APOBEC cytidine deaminases (which catalyze the deamination of cytidine to uridine) and the adenosine deaminases acting on RNA (ADARs, which catalyze the deamination of adenosine to inosine), function in a variety of host defense mechanisms. These enzymes act in innate and adaptive immune pathways, with both host and pathogen targets. DNA editing by the cytidine deaminase AID mediates immunoglobulin somatic hypermutation and class switch recombination, providing the antibody response with the flexibility and diversity to defend against an almost limitless array of varied and rapidly adapting pathogenic challenges. Other cytidine deaminases (APOBEC3) restrict retroviral infection by editing viral retrogenomes. Adenosine deaminases (ADARs) shape innate immune responses by modifying host transcripts that encode immune effectors and their regulators. Here we review current knowledge of polynucleotide DNA and RNA editors with a focus on these and other functions they serve in the immune system.

The antiviral factor APOBEC3G improves CTL recognition of cultured HIV-infected T cells.

The cytidine deaminase APOBEC3G (A3G) enzyme exerts an intrinsic anti-human immunodeficiency virus (HIV) defense by introducing lethal G-to-A hypermutations in the viral genome. The HIV-1 viral infectivity factor (Vif) protein triggers degradation of A3G and counteracts this antiviral effect. The impact of A3G on the adaptive cellular immune response has not been characterized. We examined whether A3G-edited defective viruses, which are known to express truncated or misfolded viral proteins, activate HIV-1-specific (HS) CD8+ cytotoxic T lymphocytes (CTLs). To this end, we compared the immunogenicity of cells infected with wild-type or Vif-deleted viruses in the presence or absence of the cytidine deaminase. The inhibitory effect of A3G on HIV replication was associated with a strong activation of cocultivated HS-CTLs. CTL activation was particularly marked with Vif-deleted HIV and with viruses harboring A3G. Enzymatically inactive A3G mutants failed to enhance CTL activation. We also engineered proviruses bearing premature stop codons in their genome as scars of A3G editing. These viruses were not infectious but potently activated HS-CTLs. Therefore, the pool of defective viruses generated by A3G represents an underestimated source of viral antigens. Our results reveal a novel function for A3G, acting not only as an intrinsic antiviral factor but also as an inducer of the adaptive immune system.

Two distinct populations of H chain-edited B cells show differential surrogate L chain dependence.

Developing autoreactive B cells may edit (change) their specificity by secondary H or L chain gene rearrangement. Recently, using mice hemizygous for a site-directed VDJH and VJkappa transgene (tg) encoding an autoreactive Ab, we reported ongoing L chain editing not only in bone marrow cells with a pre-B/immature B cell phenotype but also in immature/transitional splenic B cells. Using the same transgenic model, we report here that editing at the H chain locus appears to occur exclusively in bone marrow cells with a pro-B phenotype. H chain editing is shown to involve VH replacement at the tg allele or VH rearrangement at the wild-type (wt) allele when the tg is inactivated by nonproductive VH replacement. VH replacement/rearrangement at the tg/wt alleles was found to entail diverse usage of VH genes. Whereas the development of edited B cells expressing the wt allele was dependent on the lambda5 component of the surrogate L chain, the development of B cells expressing the tg allele, including those with VH replacement, appeared to be lambda5 independent. We suggest that the unique CDR3 region of the tg-encoded muH chain is responsible for the lambda5 independence of tg-expressing B cells.

Molecular mechanism for generation of antibody memory.

Activation-induced cytidine deaminase (AID) is the essential enzyme inducing the DNA cleavage required for both somatic hypermutation and class switch recombination (CSR) of the immunoglobulin gene. We originally proposed the RNA-editing model for the mechanism of DNA cleavage by AID. We obtained evidence that fulfils three requirements for CSR by this model, namely (i) AID shuttling between nucleus and cytoplasm, (ii) de novo protein synthesis for CSR, and (iii) AID-RNA complex formation. The alternative hypothesis, designated as the DNA-deamination model, assumes that the in vitro DNA deamination activity of AID is representative of its physiological function in vivo. Furthermore, the resulting dU was removed by uracil DNA glycosylase (UNG) to generate a basic site, followed by phosphodiester bond cleavage by AP endonuclease. We critically examined each of these provisional steps. We identified a cluster of mutants (H48A, L49A, R50A and N51A) that had particularly higher CSR activities than expected from their DNA deamination activities. The most striking was the N51A mutant that had no ability to deaminate DNA in vitro but retained approximately 50 per cent of the wild-type level of CSR activity. We also provide further evidence that UNG plays a non-canonical role in CSR, namely in the repair step of the DNA breaks. Taking these results together, we favour the RNA-editing model for the function of AID in CSR.

Influence of B cell antigen receptor expression level on pathways of B cell tolerance induction.

We have described an Ig-transgenic, autoreactive B cell clonotype that undergoes a novel tolerance pathway. Early in development this clonotype expresses average BCR levels, but these levels are progressively down-regulated as development proceeds efficiently to the mature, follicular compartment. This clonotype does not display conventional features of anergy and can be induced to undergo apoptosis and receptor editing in in vitro bone marrow cultures, but these pathways are not taken in vivo. These data suggested that autoantigen-driven down-regulation of BCR levels and, hence, avidity for autoantigen allows this clonotype to bypass conventional tolerance mechanisms. To test this idea, we enforced elevated levels of expression of BCR in this clonotype by making the transgenic Igh locus homozygous. This resulted in retarded clonotype development and L chain receptor editing in vivo. These data support a pivotal role for adaptive, autoantigen-induced adjustment of BCR expression levels in the regulation of primary B cell development and tolerance.