key: cord-0711644-k3nb2l6d
authors: Vlachogiannis, Nikolaos I.; Verrou, Kleio-Maria; Stellos, Konstantinos; Sfikakis, Petros P.; Paraskevis, Dimitrios
title: The role of A-to-I RNA editing in infections by RNA viruses: Possible implications for SARS-CoV-2 infection
date: 2021-02-25
journal: Clin Immunol
DOI: 10.1016/j.clim.2021.108699
sha: c169074c66788c0a64fb62683c80f348a9cdde80
doc_id: 711644
cord_uid: k3nb2l6d

RNA editing is a fundamental biological process with 2 major forms, namely adenosine-to-inosine (A-to-I, recognized as A-to-G) and cytosine-to-uracil (C-to-U) deamination, mediated by ADAR and APOBEC enzyme families, respectively. A-to-I RNA editing has been shown to directly affect the genome/transcriptome of RNA viruses with significant repercussions for viral protein synthesis, proliferation and infectivity, while it also affects recognition of double-stranded RNAs by cytosolic receptors controlling the host innate immune response. Recent evidence suggests that RNA editing may be present in SARS-CoV-2 genome/transcriptome. The majority of mapped mutations in SARS-CoV-2 genome are A-to-G/U-to-C(opposite strand) and C-to-U/G-to-A(opposite strand) substitutions comprising potential ADAR-/APOBEC-mediated deamination events. A single nucleotide substitution can have dramatic effects on SARS-CoV-2 infectivity as shown by the D614G(A-to-G) substitution in the spike protein. Future studies utilizing serial sampling from patients with COVID-19 are warranted to delineate whether RNA editing affects viral replication and/or the host immune response to SARS-CoV-2.

Currently, more than 170 known RNA base modifications expand the RNA alphabet from 4 to hundreds of individual nucleotides [1] . The most abundant RNA modification in humans is RNA editing, which comes in 2 main forms, namely adenosine-to-inosine (A-to-I) and

cytosine-to-uracil (C-to-U) deamination, mediated by the ADAR and APOBEC family of enzymes, respectively [2] [3] [4] . Inosine (I) is in turn recognized as guanosine (G) by polymerases during RNA-dependent RNA replication (viral replication) and by ribosomes during translation [2, 5, 6] . A-to-I RNA editing is a widespread phenomenon in the human transcriptome, mainly located in the endogenous Alu retroelements, which locally form double-stranded RNA regions, a pre-requisite for the binding and catalytic deamination by ADARs [7, 8] . A-to-I RNA editing has been shown to affect multiple facets of the RNA metabolism [2, 5, 9] , while we and others have previously shown that ADAR1-induced RNA editing is enhanced under chronic inflammatory conditions leading to stabilization of proinflammatory transcripts, thus having a "fuel-on-fire" effect on the perpetuation of the inflammatory response [10, 11] . More importantly, A-to-I RNA editing has been shown to directly affect the genome and transcriptome of RNA viruses with significant repercussions for viral protein synthesis, proliferation and infectivity [6, 12] . Of interest, recent data suggest that RNA editing may also take place in the genome/transcriptome of SARS-CoV-2, the virus responsible for the ongoing COVID-19 pandemic. As of February 2021, COVID-19 accounts for more than 2 million deaths worldwide. Despite the intensive efforts of the scientific and medical community, there is currently no available targeted therapy, while numerous vaccines are in the stage of clinical trials with only a few having reached the clinic. SARS-CoV-2 has an approximately 30 kilobases long, positivesense, single-stranded RNA genome [13, 14] . International efforts have provided early accurate sequencing of the viral genome [14] , while the use of nanopore direct RNA sequencing has enabled the detection of base modifications creating a detailed transcriptomic and epitranscriptomic map of SARS-CoV-2 at single base resolution [15] . Detection of single nucleotide variants (SNVs) in the viral genome has gained significant attention with recent studies showing that substitutional mutations in the spike protein of SARS-CoV-2 may greatly affect its virulence and transmissibility [16, 17] .

In a recent paper examining 33,693 complete SARS-CoV-2 genome sequences, C-to-U (~24%) was the most common base substitution, while A-to-G (~15%) and U-to-C (~14.5%) followed [18] . Base substitutions can be divided into two main categories:

transitions [purine-to-purine (A↔G) and pyrimidine-to-pyrimidine (U↔C)] and transversions (change between a purine and a pyrimidine) [19] . Transitions take place more easily, since they do not require the addition or removal of ribose rings, and bear a lower risk to lead to detrimental amino-acid changes [19] . Therefore, they are generally more common in both viral and human genome throughout evolution [19] . However, the surprisingly high percentage of transitions (~65%) in SARS-CoV-2 genome raises another interesting possibility: the involvement of host RNA editing machinery. In line with this, a recent report showed that 87% of the synonymous substitutions observed between SARS-CoV-2 and the J o u r n a l P r e -p r o o f Journal Pre-proof bat coronavirus RaTG13 could be potentially attributed to deamination of cytosines (65%) and adenosines (22%), respectively, through the host RNA editing machinery [20] .

APOBECs are enzymes that mediate the deamination of cytosine-to-uracil (C-to-U), which, depending on the sequencing strand, can also be viewed as G-to-A. Similarly, the deamination of adenosine-to-inosine (A-to-I) by ADARs, which leads to an adenosine-toguanosine (A-to-G) substitution, can also be detected as U-to-C when in the opposite strand.

While we cannot exclude the possibility that the increased presence of C-to-U and A-to-G mutations in SARS-CoV-2 may have occurred through random mutation events, a series of factors suggest the involvement of host RNA editing machinery:

1) The observed frequency of SNVs does not follow the pattern of RNA-dependent RNA polymerase (RdRP) errors, as revealed by previous mechanistic studies removing the 35' exoribonuclease activity ("proofreading") of coronavirus [21] .

2) C-to-U substitutions observed in SARS-CoV-2 genome/transcriptome follow the [3, [22] [23] [24] . C residues surrounded by A/U both upstream (5') and downstream (3') were ~10-fold more likely to be substituted by U compared to C residues surrounded by either G or C [24] .

3) A-to-G substitutions in SARS-CoV-2 genome show a depletion of G at -1 position [22, 25] , and a slight G enrichment 1-base downstream [25] , which is also observed in human ADAR1/2-induced A-to-I editing events [26] .

supports the potential involvement of the interferon-inducible ADAR1p150 enzyme [25] .

In a first report utilizing nanopore direct RNA sequencing, researchers detected at least 41 RNA modification sites on viral transcripts [15] . Of interest, modified viral RNAs J o u r n a l P r e -p r o o f Journal Pre-proof had shorter poly(A) tails than unmodified RNAs, suggesting that RNA modifications may affect RNA stability and consequently viral protein synthesis [15] . While this initial report excluded the presence of A-to-I RNA editing events, later studies have detected multiple RNA editing sites in the SARS-CoV-2 transcriptome and genome [22, 25] . Di Giorgio et al. [22] . Similarly, C-to-U substitutions following a motif compatible with APOBEC editing were detected in the examined SARS-CoV-2 transcriptome. Of note, 9 (~8.5%) of the observed C-to-U/G-to-A substitutions, but no A-to-G/U-to-C substitutions, led to the creation of a stop codon (nonsense mutations) in the transcriptomic data. Interestingly, none of these 9 nonsense mutations were present in genomic data of SARS-CoV-2 raising the possibility that such editing events may be incompatible with SARS-CoV-2 propagation (Figure 1 ) [22] .

CoV-2 genome consistent with the ADAR1/2-induced RNA editing motif [25] . Moreover, the researchers detected hyper-edited reads [27] (reads with excessive editing that do not easily align to the genome) where A-to-I events accounted for more than 75% of the detected substitutions [25] . Of note, 96% of the observed substitutions in hyper-edited reads occurred in exonic sequences and frequently (64%) led to amino-acid substitutions [25] . More importantly, the authors showed that A-to-G base substitutions were enriched after infection of lung epithelial cells (Calu-3) with SARS-CoV-2 in vitro, coinciding with the spiked increase of type I IFN and ADAR1 expression [25] . Finally, A-to-G and U-to-C substitutions J o u r n a l P r e -p r o o f Journal Pre-proof were observed with equal frequency in both studies [22, 25] , further supporting the involvement of ADARs which act on double-stranded RNA substrates.

A-to-I RNA editing has been previously recognized as a determining factor for the fate of multiple RNA viruses including HIV-1, HCV, HDV, Influenza A and Measles virus ( Table   1) . Host-dependent A-to-I RNA editing of the viral genome or transcriptome can have either pro-viral or anti-viral effects depending on the host-virus interaction (excellently reviewed in [6] ). A-to-I RNA editing in coding regions may affect protein synthesis and consequently proliferation and infectivity of the virus ( Table 1 and Figure 1 ). An excellent example of this comes from the hepatitis delta virus (HDV): HDV encodes two forms of the Hepatitis Delta Antigen (HDAg) protein, namely a shorter form (HDAg-S) that is essential for viral RNA replication and a longer form (HDAg-L) that mediates packaging of the viral genome and HDV particle formation [6] . A-to-I editing of a stop codon (UAG, "amber") is necessary to turn it into tryptophan [UI(=G)G, "W"] thus enabling the production of HDAg-L [28] [29] [30] ( Table 1) . Moreover, ADAR1 may interact with viral proteins, such as Influenza A NS1, through its RNA-binding domains with potential implications for type I IFN pathway activation [31] . Viruses may take advantage of the host RNA editing machinery to avoid recognition by innate immune receptors. More specifically, previous studies have shown that dsRNAs containing multiple IU-pairs suppress the activation of the innate immune receptors MDA5 and RIG-I and subsequently IRF-3, thus inhibiting the induction of the type I IFN pathway [32] (Figure 1) . Similarly, ADAR1 can directly interact with the antiviral PKR protein and prevent its hyperactivation thus promoting viral replication [33] [34] [35] [36] .

J o u r n a l P r e -p r o o f Journal Pre-proof Type I IFN seems to be the determining factor of host response to SARS-CoV-2 [37] . ADAR1, and specifically the cytoplasmic ADAR1p150 isoform, is IFN-inducible [38] , suggesting a potential involvement of ADAR1-induced RNA editing in the host immune response to SARS-CoV-2. However, SARS-CoV-2 seems to avoid extensive A-to-I RNA editing as shown by the low levels (<1%) of A-to-I editing detected in the isolated viral genomes/transcriptomes from patient cells [22, 25] , which is in line with low type I IFNinduced gene expression observed in SARS-CoV-2 infected cells [39] . Whether exogenous IFN administration to COVID-19 patients could affect viral replication partly through induction of multiple RNA editing events (hyper-editing) that can inhibit viral protein synthesis, as has been previously shown for HIV-1 [40] , or mark dsRNA for degradation by specific endonucleases [41] , remains to be proven by future studies.

Finally, the best-studied SARS-CoV-2 mutation to date leading to an amino-acid substitution (D614G) in the spike protein affecting viral binding to ACE2 and consequently cellular entry and virulence is indeed an A-to-G substitution [16] . Whether this was originally an ADAR1-mediated RNA editing event cannot be proven, however it supports the significant repercussions of single nucleotide substitutions in the spike protein for SARS-CoV-2 infectivity [16, 17] through various mechanisms including ACE2 binding affinity [42] , conformational changes leading to an ACE2 binding-competent state [43] or higher availability of spike protein in virions [44] . With CRISPR-Cas13 being intensively 

In vitro Huh-7 cells

Increased A-to-I editing of radiolabeled AMP. Inhibition of HCV replicon (BB7) synthesis.

[46] ADAR1-knockdown 5-to 41-fold increase of HCV replicons.

ADAR2-knockdown Νo effect on HCV replicons.

In vitro Huh-7, HEK293 cells HDAg (A1012) ("Amber/W" site)

HDAg-L production Switch from replication to packaging [28] [29] [30] ADAR1

In vitro Huh-7, HEK293 cells HDAg (A1012) ("Amber/W" site)

Inhibition of HDV-antigenome editing.

Reduced production of HDV virions.

ADAR1p110 is primarily responsible for HDV antigenome editing during replication. [47, 48] ADAR1/ ADAR2 Increased A-to-I editing of the viral envelope RNA in BALF cells of aerosol IFN-γ-treated patients. Inhibition of HIV replication. [40] ADAR1-knockdown Increased viral infectivity.

No effect on viral infectivity.

In vitro HEK293T, A549 cells

Reporter plasmid NS1-ADAR1 interaction increases ADAR1-mediated editing and viral protein expression. [31] Catalytically-inactive ADAR1 overexpression Decreased viral protein expression.

Decreased viral protein expression and viral production.

In Several A-to-G/ U-to-C mutations were observed in SARS-CoV-2 transcriptome (most common single nucleotide variants). Significantly fewer A-to-G/U-to-C substitutions were detected in the viral genome.

No nonsense A-to-G/U-to-C substitutions were detected in SARS-CoV-2 genome or transcriptome, proposing a potential deleterious effect for SARS-CoV-2 replication. [22] Calu-3, Vero cells viral genome/trans criptome Multiple A-to-G/U-to-C substitutions were detected in viral genome (>300 unique Ato-G sites identified).

Increased A-to-G substitutions 12h-24h post-infection of Calu-3 cells with SARS-CoV-2 in vitro coincided with increase of type I interferons and ADAR1.

96% of the observed substitutions in hyperedited transcripts occurred in exonic sequences and frequently (64%) led to amino-acid substitutions [25] *The Table includes 

MODOMICS: a database of RNA modification pathways. 2017 update

A-to-I editing of coding and non-coding RNAs by ADARs

An Overview of the C-to-U RNA Editing Machinery and Its Implication in Human Disease

Dawn of Epitranscriptomic Medicine

Adenosine-to-Inosine RNA Editing in Health and Disease

Adenosine deaminases acting on RNA (ADARs) are both antiviral and proviral

Widespread A-to-I RNA editing of Alu-containing mRNAs in the human transcriptome

Widespread RNA Editing of Embedded Alu Elements in the Human Transcriptome

RNA epigenetics and cardiovascular diseases

Adenosine-to-inosine RNA editing controls cathepsin S expression in atherosclerosis by enabling HuR-mediated posttranscriptional regulation

Increased adenosine-to-inosine RNA editing in rheumatoid arthritis

Functions of the RNA Editing Enzyme ADAR1 and Their Relevance to Human Diseases

Full-genome evolutionary analysis of the novel corona virus (2019-nCoV) rejects the hypothesis of emergence as a result of a recent recombination event

A pneumonia outbreak associated with a new coronavirus of probable bat origin

The Architecture of SARS-CoV-2 Transcriptome

SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus

SARS-CoV-2 D614G variant exhibits efficient replication ex vivo and transmission in vivo

Host Immune Response Driving SARS-CoV-2 Evolution

Evidence for the Selective Basis of Transition-to-Transversion Substitution Bias in Two RNA Viruses

The divergence between SARS-CoV-2 and RaTG13 might be overestimated due to the extensive RNA modification

Coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics

Evidence for hostdependent RNA editing in the transcriptome of SARS-CoV-2

An APOBEC cytidine deaminase mutagenesis pattern is widespread in human cancers

Rampant C→U Hypermutation in the Genomes of SARS-CoV-2 and Other Coronaviruses: Causes and Consequences for Their Short-and Long-Term Evolutionary Trajectories

A-to-I RNA editing in SARS-COV-2: real or artifact?

Double-stranded RNA adenosine deaminases ADAR1 and ADAR2 have overlapping specificities

A genome-wide map of hyper-edited RNA reveals numerous new sites

RNA editing of hepatitis delta virus antigenome by dsRNAadenosine deaminase

Hepatitis Delta Virus RNA Editing Is Highly Specific for the Amber/W Site and Is Suppressed by Hepatitis Delta Antigen

Hepatitis Delta Virus Minimal Substrates Competent for Editing by ADAR1 and ADAR2

The interactomes of influenza virus NS1 and NS2 proteins identify new host factors and provide insights for ADAR1 playing a supportive role in virus replication

Double-stranded RNAs containing multiple IU pairs are sufficient to suppress interferon induction and apoptosis

Enhancement of Replication of RNA Viruses by ADAR1 via RNA Editing and Inhibition of RNA-Activated Protein Kinase

Human ADAR1 Prevents Endogenous RNA from Triggering Translational Shutdown

Protein kinase PKR and RNA adenosine deaminase ADAR1: new roles for old players as modulators of the interferon response

Imagine COVID Group, French COVID Cohort Study Group, CoV-Contact Cohort, Amsterdam UMC Covid-19

Human RNA-specific adenosine deaminase ADAR1 transcripts possess alternative exon 1 structures that initiate from different promoters, one constitutively active and the other interferon inducible

Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19

Adenosine Deaminase Acting on RNA-1 (ADAR1) Inhibits HIV-1 Replication in Human Alveolar Macrophages

Specific cleavage of hyper-edited dsRNAs

SARS-CoV-2 D614G spike mutation increases entry efficiency with enhanced ACE2-binding affinity

Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

SARS-CoV-2 spikeprotein D614G mutation increases virion spike density and infectivity

RNA Editing with CRISPR-Cas13

New Antiviral Pathway That Mediates Hepatitis C Virus Replicon Interferon Sensitivity through ADAR1

Replicating hepatitis delta virus RNA is edited in the nucleus by the small form of ADAR1

Inhibition of Hepatitis Delta Virus RNA Editing by Short Inhibitory RNA-Mediated Knockdown of ADAR1 but Not ADAR2 Expression

Increased RNA Editing and Inhibition of Hepatitis Delta Virus Replication by High-Level Expression of ADAR1 and ADAR2

The large form of ADAR 1 is responsible for enhanced hepatitis delta virus RNA editing in interferonalpha-stimulated host cells

Interferon-α stimulation of liver cells enhances hepatitis delta virus RNA editing in early infection

Double-Stranded RNA Adenosine Deaminases Enhance Expression of Human Immunodeficiency Virus Type 1 Proteins

Editing of HIV-1 RNA by the doublestranded RNA deaminase ADAR1 stimulates viral infection

ADAR2 editing enzyme is a novel human immunodeficiency virus-1 proviral factor

ADAR1 Facilitates HIV-1 Replication in Primary CD4+ T Cells

ADAR1 is a novel multi targeted anti-HIV-1 cellular protein

Measles Virus Defective Interfering RNAs Are Generated Frequently and Early in the Absence of C Protein and Can Be Destabilized by Adenosine Deaminase Acting on RNA-1-Like Hypermutations

RNA editing has two main forms, adenosine-to-inosine (A-to-I, recognized as A-to-G) and cytosine-to-uracil (C-to-U) deamination, mediated by the ADAR and APOBEC family of enzymes

A-to-I RNA editing can directly affect the genome and/or transcriptome of RNA viruses with significant repercussions for viral protein synthesis, proliferation and infectivity

The majority of mapped mutations in SARS-CoV-2 genome are A-to-G/U-to-C (opposite strand) and C-to-U/G-to-A (opposite strand) substitutions comprising potential ADAR-/APOBEC-mediated deamination events

A single nucleotide substitution can have dramatic effects on SARS-CoV-2 infectivity as shown by the D614G (A-to-G) substitution in the spike protein

Future studies utilizing serial sampling from patients with COVID-19 are warranted to delineate whether RNA editing affects viral replication and/or the host immune response to SARS-CoV-2