Academic literature on the topic 'Apobec1, deaminases, Covid, RNA editing'

The COVID-19 outbreak has become a global health risk, and understanding the response of the host to the SARS-CoV-2 virus will help to combat the disease. RNA editing by host deaminases is an innate restriction process to counter virus infection, but it is not yet known whether this process operates against coronaviruses. Here, we analyze RNA sequences from bronchoalveolar lavage fluids obtained from coronavirus-infected patients. We identify nucleotide changes that may be signatures of RNA editing: adenosine-to-inosine changes from ADAR deaminases and cytosine-to-uracil changes from APOBEC deaminases. Mutational analysis of genomes from different strains of Coronaviridae from human hosts reveals mutational patterns consistent with those observed in the transcriptomic data. However, the reduced ADAR signature in these data raises the possibility that ADARs might be more effective than APOBECs in restricting viral propagation. Our results thus suggest that both APOBECs and ADARs are involved in coronavirus genome editing, a process that may shape the fate of both virus and patient.

Abstract During the course of the COVID-19 pandemic, large-scale genome sequencing of SARS-CoV-2 has been useful in tracking its spread and in identifying variants of concern (VOC). Viral and host factors could contribute to variability within a host that can be captured in next-generation sequencing reads as intra-host single nucleotide variations (iSNVs). Analysing 1347 samples collected till June 2020, we recorded 16 410 iSNV sites throughout the SARS-CoV-2 genome. We found ∼42% of the iSNV sites to be reported as SNVs by 30 September 2020 in consensus sequences submitted to GISAID, which increased to ∼80% by 30th June 2021. Following this, analysis of another set of 1774 samples sequenced in India between November 2020 and May 2021 revealed that majority of the Delta (B.1.617.2) and Kappa (B.1.617.1) lineage-defining variations appeared as iSNVs before getting fixed in the population. Besides, mutations in RdRp as well as RNA-editing by APOBEC and ADAR deaminases seem to contribute to the differential prevalence of iSNVs in hosts. We also observe hyper-variability at functionally critical residues in Spike protein that could alter the antigenicity and may contribute to immune escape. Thus, tracking and functional annotation of iSNVs in ongoing genome surveillance programs could be important for early identification of potential variants of concern and actionable interventions.

Cancer genomes accumulate nucleotide sequence variations that number in the tens of thousands per genome. A prominent fraction of these mutations is thought to arise as a consequence of the off-target activity of DNA/RNA editing cytosine deaminases. These enzymes, collectively called activation induced deaminase (AID)/APOBECs, deaminate cytosines located within defined DNA sequence contexts. The resulting changes of the original C:G pair in these contexts (mutational signatures) provide indirect evidence for the participation of specific cytosine deaminases in a given cancer type. The conventional method used for the analysis of mutable motifs is the consensus approach. Here, for the first time, we have adopted the frequently used weight matrix (sequence profile) approach for the analysis of mutagenesis and provide evidence for this method being a more precise descriptor of mutations than the sequence consensus approach. We confirm that while mutational footprints of APOBEC1, APOBEC3A, APOBEC3B, and APOBEC3G are prominent in many cancers, mutable motifs characteristic of the action of the humoral immune response somatic hypermutation enzyme, AID, are the most widespread feature of somatic mutation spectra attributable to deaminases in cancer genomes. Overall, the weight matrix approach reveals that somatic mutations are significantly associated with at least one AID/APOBEC mutable motif in all studied cancers.

Abstract Background:The APOBEC3 (A3) family of cytidine deaminases in primates is comprised of seven homologous enzymes that are structurally related to the RNA editing enzyme APOBEC1. APOBEC3G (A3G) is a restriction factor for HIV-1 and endogenous retroviruses, and is highly expressed in T lymphocytes. Encapsidation of A3G into HIV-1 particles is essential for its antiviral activity which leads to hypermutation of its cDNA in target cells, and requires RNA binding by A3G to form a ribonucleoprotein complex with viral proteins. A3G can also reduce HIV-1 production in producer cells independently of its DNA deaminating activity. A3G has homologous N-and C-terminal catalytic domains (NTD and CTD) but only the CTD is active for deamination of ssDNAs. The zinc-coordinating catalytic residues as well as non-catalytic residues in A3G-NTD are known to bind RNA and this interaction is required for A3G's binding to the HIV-1 nucleocapsid for recruitment into nascent virions as well as for A3G dimerization. A3G binds to DNA and RNA substrates with similar affinity. Thus far, studies have demonstrated DNA deamination by A3G whereas deamination has not been observed in HIV-1 RNA or synthetic RNA oligonucleotides, thereby, ruling out the RNA editing function of A3G. We recently described that the structurally related enzyme A3A induces widespread site-specific C-to-U RNA editing of cellular transcripts in pro-inflammatory macrophages and in monocytes exposed to hypoxia and/or interferons. We hypothesized that A3G may also have RNA editing function, which may play a role in HIV-1 restriction. Methods:To determine if A3G is capable of RNA editing, we transiently overexpressed the protein in 293T cells, a model routinely used by various labs to study A3G function and its mode of HIV-1 restriction, and then performed transcriptome-wide RNA sequencing (RNA-Seq), Sanger sequencing and site-directed mutagenesis. Results: RNA sequencing analysis showed site-specific RNA editing in hundreds of genes' transcripts, including approximately 200 genes that acquire protein recoding. The transcripts edited by A3G are largely distinct from those edited by A3A. We find that several host genes including NMT1, CHMP4B, MAPK1, ACIN1, MED1, NFAT5, RBM14 which areinvolved in HIV-1 infection acquire pathogenic recoding RNA mutations by A3G-mediated RNA editing. By performing Sanger sequencing of PCR-amplified cDNA, we validated site-specific, non-synonymous C-to-U RNA editing for 21 of 21 (100%) tested sites in 20 genes that we had selected for experimental confirmation. As expected no genomic mutations were seen in the DNA sequences corresponding to the RNA-edited sites in 11 tested genes. The discovery of A3G's RNA editing function prompted us to study the role of the N-terminal domain in RNA editing. We made mutations in the zinc-coordinating and non-catalytic residues in both N-terminal and C-terminal domains of A3G. We demonstrate that mutating zinc-coordinating residues in either N- and C-terminal domains of A3G in 293T cells greatly reduce or abolish editing in its target transcripts. Conclusions: We demonstrate a novel RNA editing function for the A3G cytidine deaminase. Our study shows that the RNAs of genes involved in HIV-1 replication, assembly, transcription and infectivity are targets of A3G-mediated RNA editing. This result raises the possibility that the editing of host transcripts may be a novel mechanism by which HIV-1 infection is inhibited by A3G. Our findings suggest a previously unrecognized role for the N-terminal domain of A3G in RNA editing. A3G is the second of the seven members of the APOBEC3 family of cytidine deaminases and the first two-domain cytidine deaminase for which a previously unrecognized RNA editing activity has been discovered. It suggests that other APOBEC3 proteins may also possess hitherto unknown RNA editing activity that may underpin some of their biological roles. Our findings have the potential to significantly expand on the role of C-to-U RNA editing in epitranscriptomic regulation in T lymphocytes, define specific gene targets of A3G-mediated RNA editing and open new avenues of inquiry on the functions of APOBEC3 genes in HIV-1 restriction. Disclosures No relevant conflicts of interest to declare.

Abstract A mutational signature consistent with APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide) activity has been identified in somatic mutations found in large-scale surveys of ultra-deep sequencing data from many human cancers including chronic lymphocytic leukemia (CLL). APOBEC is a cytidine deaminase family made up of eleven genes, including AID (activation-induced cytidine deaminase) and APOBEC3B, both of which have been implicated in somatic mutation in various cancers, including CLL. These observations have led to the hypothesis that APOBEC cytidine deaminases may be driving somatic mutations leading to the development of more aggressive cancers. Therefore, we examined APOBEC gene family member RNA expression levels in CLL to test for correlations with expression levels and patient outcome. We further examined if CLL cells generated de novo APOBEC family member mutational patterns in the immunoglobulin variable region gene (IGHV) after implantation in a mouse xenograft model of CLL. CLL peripheral blood mononuclear cells (PBMCs) and associated clinical data were collected from patients after informed consent as approved by the Institutional Review Board at the North Shore-Long Island Jewish Health System and in accordance with the Helsinki Declaration. CLL samples were chosen based on availability with no pre-established inclusion/exclusion criteria. CLL RNA expression levels were examined by microarray or quantitative real-time PCR (qPCR). For microarray studies, CLL B cells were purified prior to RNA isolation and acquisition of microarray expression data using Illumina Human WG6 and HT12 bead chips, followed by quantile normalization using GenomeStudio software (Illumina). For qPCR, RNA expression from CLL PBMCs was measured relative to glyceraldehyde 3-phosphate dehydrogenase gene expression by Taqman assay with Roche UPL probes and LightCycler 480. To examine de novo mutations in CLL, the IGHV region was ultra-deep sequenced (Roche 454 FLX system) from human CLL cells recovered from the NOD/Shi-scid,γcnull (NSG) xenograft mouse model of CLL as approved by the Institutional Animal Care and Use Committee at the North Shore-Long Island Jewish Health System. CLL patient (N = 65) RNA expression by microarray showed very low levels of APOBEC1, 2, 3A, 3B, 3D, 4, and AID, modest levels of APOBEC3C and 3H, and high levels of APOBEC3F and 3G. Higher AID expression levels significantly correlated (P <0.05) with shorter time to first treatment (TFT), which was anticipated based on previous reports. Interestingly APOBEC3B and APOBEC3F expression differences showed possible trends correlating with worse patient outcome. Therefore, we tested select APOBEC gene family members by qPCR. For qPCR, we utilized the CLL patient cohort (N= 83) previously found to indicate that AID expression was a risk factor for worse patient outcome in a multivariate analysis (Patten et al. 2012 Blood 120:4802). RNA expression by qPCR followed the same pattern as the microarray data: AID and APOBEC3B had very low levels, APOBEC3H had modest levels, and APOBEC3F and 3G had high levels. Similar to AID, patients could be grouped based on the presence or absence of detectable APOBEC3B, with its presence showing a significant correlation (P <0.05) with worse TFT and overall survival. Higher levels of APOBEC3F and 3H showed a trend towards a correlation with shorter TFT, while differences in APOBEC3G expression had no significant correlation with patient outcome. Thus, not only did we confirm the correlation of AID expression with worse patient outcome, but we also found APOBEC3B and potentially APOBEC3F and 3H correlate with worse patient outcome. To test if CLL cells can acquire de novo mutations indicative of APOBEC gene family member activity, human CLL cells were transferred into NSG mice. After CLL cells proliferated for 4-14 weeks in this xenograft model, the IGHV region was amplified, ultra-deep sequenced, and analyzed for specific mutational characteristics of various APOBEC gene family members. The results of these ongoing analyses will be presented. In conclusion, the expression levels of the APOBEC gene family members AID, APOBEC3B, and potentially APOBEC3F and 3H, correlate with worse patient outcome. These data are consistent with the hypothesis that APOBEC gene family member activity may promote new mutations at sites outside the IG gene loci leading to the evolution of aggressive CLL. Disclosures Barrientos: Pharmacyclics, Celgene, and Genentech: Membership on an entity's Board of Directors or advisory committees; Gilead, Pharmacyclics, and AbbVie: Research Funding.

AbstractDuring COVID-19 pandemic, mutations of SARS-CoV-2 produce new strains that can be more infectious or evade vaccines. Viral RNA mutations can arise from misincorporation by RNA-polymerases and modification by host factors. Analysis of SARS-CoV-2 sequence from patients showed a strong bias toward C-to-U mutation, suggesting a potential mutational role by host APOBEC cytosine deaminases that possess broad anti-viral activity. We report the first experimental evidence demonstrating that APOBEC3A, APOBEC1, and APOBEC3G can edit on specific sites of SARS-CoV-2 RNA to produce C-to-U mutations. However, SARS-CoV-2 replication and viral progeny production in Caco-2 cells are not inhibited by the expression of these APOBECs. Instead, expression of wild-type APOBEC3 greatly promotes viral replication/propagation, suggesting that SARS-CoV-2 utilizes the APOBEC-mediated mutations for fitness and evolution. Unlike the random mutations, this study suggests the predictability of all possible viral genome mutations by these APOBECs based on the UC/AC motifs and the viral genomic RNA structure.

The AID (activation-induced cytidine deaminase)/APOBEC (apolipoprotein B mRNA editing enzyme catalytic subunit) family with its multifaceted mode of action emerges as potent intrinsic host antiviral system that acts against a variety of DNA and RNA viruses including coronaviruses. All family members are cytosine-to-uracil deaminases that either have a profound role in driving a strong and specific humoral immune response (AID) or restricting the virus itself by a plethora of mechanisms (APOBECs). In this article, we highlight some of the key aspects apparently linking the AID/APOBECs and SARS-CoV-2. Among those is our discovery that APOBEC4 shows high expression in cell types and anatomical parts targeted by SARS-CoV-2. Additional focus is given by us to the lymphoid structures and AID as the master regulator of germinal center reactions, which result in antibody production by plasma and memory B cells. We propose the dissection of the AID/APOBECs gene signature towards decisive determinants of the patient-specific and/or the patient group-specific antiviral response. Finally, the patient-specific mapping of the AID/APOBEC polymorphisms should be considered in the light of COVID-19.

Abstract Background APOBEC1 (A1) enzymes are cytidine deaminases involved in RNA editing. In addition to this activity, a few A1 enzymes have been shown to be active on single stranded DNA. As two human ssDNA cytidine deaminases APOBEC3A (A3A), APOBEC3B (A3B) and related enzymes across the spectrum of placental mammals have been shown to introduce somatic mutations into nuclear DNA of cancer genomes, we explored the mutagenic threat of A1 cytidine deaminases to chromosomal DNA. Results Molecular cloning and expression of various A1 enzymes reveal that the cow, pig, dog, rabbit and mouse A1 have an intracellular ssDNA substrate specificity. However, among all the enzymes studied, mouse A1 appears to be singular, being able to introduce somatic mutations into nuclear DNA with a clear 5’TpC editing context, and to deaminate 5-methylcytidine substituted DNA which are characteristic features of the cancer related mammalian A3A and A3B enzymes. However, mouse A1 activity fails to elicit formation of double stranded DNA breaks, suggesting that mouse A1 possess an attenuated nuclear DNA mutator phenotype reminiscent of human A3B. Conclusions At an experimental level mouse APOBEC1 is remarkable among 12 mammalian A1 enzymes in that it represents a source of somatic mutations in mouse genome, potentially fueling oncogenesis. While the order Rodentia is bereft of A3A and A3B like enzymes it seems that APOBEC1 may well substitute for it, albeit remaining much less active. This modifies the paradigm that APOBEC3 and AID enzymes are the sole endogenous mutator enzymes giving rise to off-target editing of mammalian genomes.

Abstract APOBEC1 (APO1), a member of AID/APOBEC nucleic acid cytosine deaminase family, can edit apolipoprotein B mRNA to regulate cholesterol metabolism. This APO1 RNA editing activity requires a cellular cofactor to achieve tight regulation. However, no cofactors are required for deamination on DNA by APO1 and other AID/APOBEC members, and aberrant deamination on genomic DNA by AID/APOBEC deaminases has been linked to cancer. Here, we present the crystal structure of APO1, which reveals a typical APOBEC deaminase core structure, plus a unique well-folded C-terminal domain that is highly hydrophobic. This APO1 C-terminal hydrophobic domain (A1HD) interacts to form a stable dimer mainly through hydrophobic interactions within the dimer interface to create a four-stranded β-sheet positively charged surface. Structure-guided mutagenesis within this and other regions of APO1 clarified the importance of the A1HD in directing RNA and cofactor interactions, providing insights into the structural basis of selectivity on DNA or RNA substrates.

Abstract Background Site-specific C>T DNA base editing has been achieved by recruiting cytidine deaminases to the target C using catalytically impaired Cas proteins; the target C is typically located within 5-nt editing window specified by the guide RNAs. The prototypical cytidine base editor BE3, comprising rat APOBEC1 (rA1) fused to nCas9, can indiscriminately deaminate multiple C’s within the editing window and also create substantial off-target edits on the transcriptome. A powerful countermeasure for the DNA off-target editing is to replace rA1 with APOBEC proteins which selectively edit C’s in the context of specific motifs, as illustrated in eA3A-BE3 which targets TC. However, analogous editors selective for other motifs have not been described. In particular, it has been challenging to target a particular C in C-rich sequences. Here, we sought to confront this challenge and also to overcome the RNA off-target effects seen in BE3. Results By replacing rA1 with an optimized human A3G (oA3G), we developed oA3G-BE3, which selectively targets CC and CCC and is also free of global off-target effects on the transcriptome. Furthermore, we created oA3G-BE4max, an upgraded version of oA3G-BE3 with robust on-target editing. Finally, we showed that oA3G-BE4max has negligible Cas9-independent off-target effects at the genome. Conclusions oA3G-BE4max can edit C(C)C with high efficiency and selectivity, which complements eA3A-editors to broaden the collective editing scope of motif selective editors, thus filling a void in the base editing tool box.

The AID/APOBECs are cytosine deaminases involved in diverse physiological contexts through their ability to edit DNA and RNA. This ability comes with a price: Activation-Induce cytidine Deaminase (AID), the main player in the antibody diversification processes, is responsible for some of the common genetic alterations in mature B-cells tumours; the APOBEC3 subgroup, important actors in virus defence, have been linked to different mutational processes in a number of cancers. Also APOBEC1, an RNA/DNA editing enzyme, also able to restrict lentivirus and mobile elements, can act as a mutator in human cells, and its aberrant expression could be linked to the onset of alterations both at the genomic and transcriptomic level. APOBEC1 RNA editing is a post transcriptional process, its only well-characterized target is the Apolipoprotein B transcript (ApoB) in the small intestine where editing of C6666 induces formation of a stop codon and translation of a truncated protein. Quite different is the situation in Rodents where, thanks to the availability of APOBEC1 knockout mice, hundreds of APOBEC1-dependent editing events, have been discovered beyond ApoB. To date, APOBEC1 deficiencies are not known and in humans and only a few transcripts have been added to the list of targets. Despite the targets known in mice and in humans, we are far from understanding the overall physiological meaning of APOBEC1-induced C to U editing. If we exclude the efforts that have been made to identify and characterise C to U editing in rodents, only few computational approaches have been employed to identify and characterize human APOBEC1 targets. This is the reason why I have used available human RNA-seq data to develop a computational strategy for the identification of APOBEC1 dependent RNA editing events. I used The Cancer Genome Atlas (TCGA) and The Genotype-Tissue Expression (GTEX) to obtain datasets of samples in which APOBEC1 is expressed at different levels. Using these datasets, I divided samples in high and low expression levels of APOBEC1, and through known tools and ad hoc scripts I built different pipelines to identify positions in the transcript that are differentially edited. The pipeline includes several filters: removal of mapping artefacts, germline and somatic single nucleotide variants, removal of homopolymeric regions and so on. Among the several strategies I used, the most promising are those applied to the GTEX small intestine data, where a strict analysis has shown the presence of at least 12 sites, including 3 known targets on the ApoB mRNA. Surprisingly we found evidence of ApoB editing at canonical sites beyond the small intestine, even in absence of measurable APOBEC1 expression. Considering the possible presence of APOBEC1 outside the gastric tissue, to improve our capacity to identify C to U editing in human tissues, I decided to create a database of C>U edited sites using RNA-seq from APOBEC1 -transfected Hek293T cell lines. This database, despite not representing physiologically edited sites, it informs on all positions biologically editable. Crossing these positions with those obtained from the GTEX dataset results in the identification of hundreds of common edited sites. Finally, I tested the hypothesis that APOBEC1 editing affects the transcript stability in Hek293T cells. Preliminary data suggest that APOBEC1 expression could shift the equilibrium between processed RNA and non-processed RNA towards the latter one. The second part of the thesis centers on the study of RNA-off targets induced by Base editors (BEs). In order to improve the safety of this powerful genome editing tool, another PhD student in the lab, Francesco Donati, selected several APOBEC1 mutant that are not able to edit the RNA while maintaining their mutagenicity on DNA. He investigated both the tumorigenicity of these mutants in mice and their use in genome editing. He obtained exonic and transcriptomic data from murine liver tumors and from cells overexpressing the mutant base editors, respectively. I performed the bioinformatic analyses to explore the mutational signature induced by APOBEC1 in mice, and to assess the off-targets effects on RNA and DNA of these base editors. I demonstrated that -contrarily to wild-type APOBEC1- these mutants provide the ability to perform genome editing in absence of detectable off-targets.
The 2019-nCoV outbreak has become a global health risk. Editing by host deaminases is an innate restriction process to counter viruses, and it is not yet known whether it operates against coronaviruses. Here we analyze RNA sequences from bronchoalveolar lavage fluids derived from two Wuhan patients. We identify nucleotide changes that may be signatures of RNA editing: Adenosine-to-Inosine changes from ADAR deaminases and Cytosine-to-Uracil changes from APOBEC ones. A mutational analysis of genomes from different strains of human-hosted Coronaviridae reveals patterns similar to the RNA editing pattern observed in the 2019-nCoV transcriptomes. Our results suggest that both APOBECs and ADARs are involved in Coronavirus genome editing, a process that may shape the fate of both virus and patient.

The AID/APOBECs are cytosine deaminases involved in diverse physiological contexts through their ability to edit DNA and RNA. This ability comes with a price: Activation-Induce cytidine Deaminase (AID), the main player in the antibody diversification processes, is responsible for some of the common genetic alterations in mature B-cells tumours; the APOBEC3 subgroup, important actors in virus defence, have been linked to different mutational processes in a number of cancers. Also APOBEC1, an RNA/DNA editing enzyme, also able to restrict lentivirus and mobile elements, can act as a mutator in human cells, and its aberrant expression could be linked to the onset of alterations both at the genomic and transcriptomic level. APOBEC1 RNA editing is a post transcriptional process, its only well-characterized target is the Apolipoprotein B transcript (ApoB) in the small intestine where editing of C6666 induces formation of a stop codon and translation of a truncated protein. Quite different is the situation in Rodents where, thanks to the availability of APOBEC1 knockout mice, hundreds of APOBEC1-dependent editing events, have been discovered beyond ApoB. To date, APOBEC1 deficiencies are not known and in humans and only a few transcripts have been added to the list of targets. Despite the targets known in mice and in humans, we are far from understanding the overall physiological meaning of APOBEC1-induced C to U editing. If we exclude the efforts that have been made to identify and characterise C to U editing in rodents, only few computational approaches have been employed to identify and characterize human APOBEC1 targets. This is the reason why I have used available human RNA-seq data to develop a computational strategy for the identification of APOBEC1 dependent RNA editing events. I used The Cancer Genome Atlas (TCGA) and The Genotype-Tissue Expression (GTEX) to obtain datasets of samples in which APOBEC1 is expressed at different levels. Using these datasets, I divided samples in high and low expression levels of APOBEC1, and through known tools and ad hoc scripts I built different pipelines to identify positions in the transcript that are differentially edited. The pipeline includes several filters: removal of mapping artefacts, germline and somatic single nucleotide variants, removal of homopolymeric regions and so on. Among the several strategies I used, the most promising are those applied to the GTEX small intestine data, where a strict analysis has shown the presence of at least 12 sites, including 3 known targets on the ApoB mRNA. Surprisingly we found evidence of ApoB editing at canonical sites beyond the small intestine, even in absence of measurable APOBEC1 expression. Considering the possible presence of APOBEC1 outside the gastric tissue, to improve our capacity to identify C to U editing in human tissues, I decided to create a database of C>U edited sites using RNA-seq from APOBEC1 -transfected Hek293T cell lines. This database, despite not representing physiologically edited sites, it informs on all positions biologically editable. Crossing these positions with those obtained from the GTEX dataset results in the identification of hundreds of common edited sites. Finally, I tested the hypothesis that APOBEC1 editing affects the transcript stability in Hek293T cells. Preliminary data suggest that APOBEC1 expression could shift the equilibrium between processed RNA and non-processed RNA towards the latter one. The second part of the thesis centers on the study of RNA-off targets induced by Base editors (BEs). In order to improve the safety of this powerful genome editing tool, another PhD student in the lab, Francesco Donati, selected several APOBEC1 mutant that are not able to edit the RNA while maintaining their mutagenicity on DNA. He investigated both the tumorigenicity of these mutants in mice and their use in genome editing. He obtained exonic and transcriptomic data from murine liver tumors and from cells overexpressing the mutant base editors, respectively. I performed the bioinformatic analyses to explore the mutational signature induced by APOBEC1 in mice, and to assess the off-targets effects on RNA and DNA of these base editors. I demonstrated that -contrarily to wild-type APOBEC1- these mutants provide the ability to perform genome editing in absence of detectable off-targets.
The 2019-nCoV outbreak has become a global health risk. Editing by host deaminases is an innate restriction process to counter viruses, and it is not yet known whether it operates against coronaviruses. Here we analyze RNA sequences from bronchoalveolar lavage fluids derived from two Wuhan patients. We identify nucleotide changes that may be signatures of RNA editing: Adenosine-to-Inosine changes from ADAR deaminases and Cytosine-to-Uracil changes from APOBEC ones. A mutational analysis of genomes from different strains of human-hosted Coronaviridae reveals patterns similar to the RNA editing pattern observed in the 2019-nCoV transcriptomes. Our results suggest that both APOBECs and ADARs are involved in Coronavirus genome editing, a process that may shape the fate of both virus and patient.

The thesis is focus on RNA editing mediated by two AID/APOBEC family members. The aim of my work was the investigation of possible novel factors that regulate hAPOBEC1 expression or cofactors which help the deaminase to exert its activity. First, I characterised cellular models for their proliferation and clonogenic activities as well as cell cycle distribution evaluating a combinatorial effect of hAPOBEC1 and RBM47 which lead to a decrease in cell growth. I investigated the role of RNA editing beyond the lipid transport by high-throughput sequencing which provided me information regarding new deamination events, RNA stability, and also a differential gene expression in presence or absence of the editosome components. By Differential expression analysis, I got a list of genes that are differentially expressed in clones with hAPOBEC1 and RBM47 which need to be analysed for their biological meaning. From the mRNA-seq I got a consistent list of putative edited sites even though some of them were validated with no success. Moreover, I applied a genetic library screen to activate a high number of genes in cells expressing RBM47 to evaluate an eventual up-regulation of APOBEC1 and find factors which trigger its expression. The cells in which editing happened have been selected thanks to a specific fluorescent reporter containing ApoB target. The results have still to be analysed. The second aim of my project was to study APOBEC3A regulation, by chemical and genetic screenings, through the development of a specific sensitive reporter system to detect APOBEC3A-mediated RNA editing. In this work I presented the design of an artificial fluorescent reporter containing a target of APOBEC3A like SDHB or DDOST properly built to produce a stop codon in the middle of the target and optimised for the levels of editing. I checked its specificity for APOBEC3A and not for other APOBEC proteins like APOBEC1 and APOBEC3B. This let me also detected a novel putative editing site mediated by APOBEC3A by Sanger sequencing. Moreover, I designed another fluorescent reporter system able to evaluate APOBEC3A RNA editing by fluorescent microscopy. I created stable cell lines expressing all the lentiviral reporter plasmids to further investigate induction of endogenous APOBEC3A and its regulation. In a future perspective the dual fluorescent reporter could be a useful tool to identify novel RNA editing targets upon the application of an activation library screen.