key: cord-0868058-qoejvdnb authors: Mourier, Tobias; Sadykov, Mukhtar; Carr, Michael J.; Gonzalez, Gabriel; Hall, William W.; Pain, Arnab title: Host-directed editing of the SARS-CoV-2 genome date: 2020-11-05 journal: Biochem Biophys Res Commun DOI: 10.1016/j.bbrc.2020.10.092 sha: 341423c4e3d761d7f735149522957c073f45d888 doc_id: 868058 cord_uid: qoejvdnb The extensive sequence data generated from SARS-CoV-2 during the 2020 pandemic has facilitated the study of viral genome evolution over a brief period of time. This has highlighted instances of directional mutation pressures exerted on the SARS-CoV-2 genome from host antiviral defense systems. In this brief review we describe three such human defense mechanisms, the apolipoprotein B mRNA editing catalytic polypeptide-like proteins (APOBEC), adenosine deaminase acting on RNA proteins (ADAR), and reactive oxygen species (ROS), and discuss their potential implications on SARS-CoV-2 evolution. most common observed substitutions in SARS-CoV-2 genomes -might be associated with the mutagenic activity of ROS [42] . Adenosine deamination in dsRNAs is attributed to adenosine deaminase acting on RNA (ADAR) enzymes, which are able to convert A-to-I (inosine), but mainly in Alu sequences (an endogenous retroelement) [43] . There are three ADAR genes encoded in the human genome; the first two, ADAR1 and ADAR2, are interferon-inducible and catalytically active for adenosine deamination, while ADAR3, which is expressed mainly in the brain, has no reported ADAR activity [44, 45] . The human ADAR1 gene is expressed in most tissues [46] . It has two isoforms, ADAR1p110, constitutively expressed in the majority of cell types, principally acting in the nucleus, and interferon-stimulated ADAR1p150, which primarily operates in the cytoplasm [47] . ADAR1 is considered a "master regulator" of cytoplasmic innate immunity regulating multiple sensors, such as Mda5, RIG-I, OAS and PKR, which detect intracellular dsRNA (which can arise during the replication-transcription process of (+)ssRNA viruses including SARS-CoV-2) and these sensors are essential in fighting viral infections [45] . Mutations at the ADAR1 locus have been linked to human genetic diseases, including the Aicardi-Goutières syndrome, an inflammatory disorder that phenocopies congenital viral infection [48] , and the pigmentation disorder, dyschromatosis symmetrica hereditaria [49] . [50] . By analyzing the extent of editing at Alu sequences (known to be targets of ADAR1), the authors estimated ADAR1 activity and found evidence for this on both viral and human transcripts. Low levels of editing were observed at early timepoints (4 hours post-infection), where both the activity of ADAR and interferon activation is low. However, after 24 hours post-infection, higher levels of A-to-I editing were recorded, although accounting for <1% of sites. Clearly, nucleotide variation due to sequencing or polymerase errors might also contribute to the observed substitutions [50] . Besides sequence analysis approaches to show ADAR1 activity, direct interaction between viral RNA and proteins has been established. Using RNA antisense purification and mass spectrometry (RAP-MS), Schmidt and colleagues identified RNA-protein interactions in SARS-CoV-2-infected human cells [33] . From this, notably both ADAR and APOBEC were found as frequent interactors with SARS-CoV-2 RNA. In contrast to the above findings, a few studies have found no evidence of ADAR1 activity acting on SARS-CoV-2. DNA nanoball sequencing from Vero cells infected with SARS-CoV-2 suggested no ADAR-mediated editing [51] . Further, the same study performed an independent J o u r n a l P r e -p r o o f analysis of the previous dataset presented by Kim et al. (2020) [51] and did not detect any A-to-G editing [50] . Using predicted secondary structures of target sites, Klimczak et al. (2020) found statistically significant ADAR editing in rubella virus genomes, another (+)ssRNA virus), but not in SARS-CoV-2 genomes [52] . Double-stranded RNAs are the pathogen-associated molecular pattern associated with the strong induction of cellular stress and interferon responses. ADAR1 is one of the major regulators of self-tolerance and innate immune activation that involves recognition of dsRNA and its further processing by downstream antiviral pathways [45, 53] . ADAR1 can exert either antiviral or proviral effects dependent upon the infecting virus [54] . For example, hyperediting of HCV and LCMV viral genomes lead to antiviral effects, while ADAR1 editing of influenza A RNA enhances viral protein expression [54] . It was recently shown that COVID-19 leads to suboptimal interferon responses in comparison to other respiratory viruses [28] , and one might speculate that this may arise if ADAR1 activity contributes to the low type I interferon response. ADAR marks the dsRNA with the A-to-I deamination, allowing the edited RNA duplexes to escape other molecular sensors of dsRNA [45] . There is a threshold for the tolerable number of dsRNAs present within the cytosol, which, when exceeded leads to autoimmunity but favors viral infection [55, 56] . ADAR-mediated J o u r n a l P r e -p r o o f editing of viral RNAs might result in levels of edited RNAs above this threshold. It is not uncommon that some viral infections can lead to autoimmune complications after recovery [57] . The high incidence of different autoimmune conditions has been recorded after the resolution of COVID-19 in adult and, also, pediatric patients are in line with this observation [58] [59] [60] [61] [62] . Besides a potential contribution of ADAR in the evasion of type I interferon responses, the virus also has various proteins that inhibit type I interferon induction and signaling [63] . Previously, knockdown of ADAR1 was shown to lead to viral inhibition, which enhanced interferon stimulation in primary macrophages [64] . ADAR1 inhibitors might thus be another strategy to boost antiviral response in viruses that trigger suboptimal interferon responses as seen during SARS-CoV-2 infection. Upon entry into the host cell, SARS-CoV-2 exists as a single-stranded, positive-sense RNA. This strand is then replicated -during which the virus exists as a double-stranded RNA -and the resulting negative-strand then acts as a template for both replication and transcription (both types of products, thus being positive-sense) [65, 66] . From short-read sequences mapped onto the SARS-CoV-2 reference genome, Graudenzi et al. (2020) identified C-to-U changes [42] . C-to-U change occurring on the negative strand will result in observed G-to-A changes using this approach. The authors observed a ratio of 17:1 between C-to-U and G-to-A changes, consistent with APOBEC predominantly working on the J o u r n a l P r e -p r o o f positive strand RNA. Remarkably, a similar ratio of 17:1 was observed between C-to-A and Gto-U changes, which would be the result of ROS-induced mutagenesis on the positive and negative strands, respectively [42] . This consistency prompted the speculation that the 17:1 ratio reflects the molar ratio between the two viral strands. The percentages presented in Figure 1 are derived from comparing approximately 80,000 assembled consensus genomes to the SARS-CoV-2 reference genome (MN908947.1) and registering all detected changes [21] . This means that a change needs to be present in the majority of viral transcripts in order to be included in the consensus genome, and that it will only be counted once regardless if it is present in one or all of the sample genomes [21] . Therefore, it is not expected that these percentages will reflect the ratios reported by Graudenzi et al. (2020) [42] . It is noteworthy, however, that the percentages in Figure 1 are similar for A-to-G and U-to-C changes, potential hallmarks of ADAR activity (that works on double-stranded RNA), whereas this is not the case for C-to-U and G-to-A, neither for C-to-A and G-to-U, consistent with APOBEC and ROS, respectively, predominantly acting on the positive stranded viral RNA. Global travel, societal interactions and the interconnected modern world provide abundant opportunities for the rapid spreading of viral infections that are becoming severe health and an economic burden for humanity [67] . A better understanding of viral genome changes can help design better diagnostics, therapeutics and prophylactic vaccines [68, 69] . Herein, we have discussed how host innate immune defenses might drive nucleotide substitutions and genomic J o u r n a l P r e -p r o o f evolution in SARS-CoV-2 in a directional manner. These described hypermutation patterns despite the relatively moderate mutation rate is evidence of the adaptation process of a virus of recent zoonotic origin [70] . Di Giorgio et al. [25] have identified a third class of changes comprising A-to-T/T-to-A transversions in SARS-CoV-2 genomes, but the mechanistic basis for this is currently unknown and requires further study. APOBEC, ROS and ADAR are effective sources of nucleotide changes and from Figure 1 , it is evident that these editing agents may potentially account for the vast number of observed changes, certainly if assuming that they may also act on dsRNAs. The SARS-CoV-2 pandemic offers a challenge of global dimensions and successfully controlling the spread of the virus will heavily depend on insight into the biology of the virus. In this respect, host-directed genome editing is likely to play a substantial role and may hypothetically confer susceptibility and potentially a degree of innate resistance for individuals harboring certain haplotypes. As SARS-CoV-2 will inevitably become the most closely monitored virus in terms of real-time sequence data, this concerted effort of the global scientific community is essential to ameliorate the profound burden of disease elicited by this zoonotic pathogen. Matrix showing the distribution of genomic changes in SARS-CoV-2 sequences deposited at GISAID (https://www.gisaid.org/; [71] ) as of October 2nd, 2020. Changes are accumulated across 79,887 samples and mapped onto the reference SARS-CoV-2 genome sequence, and the percentages of changes were recorded as previously described [21] . Changes at individual sites may therefore represent multiple independent events, and the most prominent changes are most likely underestimated [21] . The three types of changes resulting from the activity of ROS, APOBEC, and ADAR (G-to-U, C-to-U, and A-to-G, see main text) are highlighted in red, blue, and green, respectively. The types of changes that would result of the same host factors acting on the complement strand on double-stranded RNA are similarly colored but in a checkered pattern. Coronavirus disease (COVID-2019) situation reports. 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