Adenosine Deaminase ADAR1 Research Articles

PTIR1 acts as an isoform of DDX58 and promotes tumor immune resistance through activation of UCHL5

Cancer evades host immune surveillance by virtue of poor immunogenicity. Here, we report an immune suppressor, designated as PTIR1, that acts as a promotor of tumor immune resistance. PTIR1 is selectively induced in human cancers via alternative splicing of DDX58 (RIG-I), and its induction is closely related to poor outcome in patients with cancer. Through blocking the recruitment of leukocytes, PTIR1 facilitates cancer immune escape and tumor-intrinsic resistance to immunotherapeutic treatments. Unlike RIG-I, PTIR1 is capable of binding to the C terminus of UCHL5 and activates its ubiquitinating function, which in turn inhibits immunoproteasome activity and limits neoantigen processing and presentation, consequently blocking Tcell recognition and attack against cancer. Moreover, we find that the adenosine deaminase ADAR1 induces A-to-I RNA editing on DDX58 transcript, thus triggering PTIR1 production. Collectively, our data uncover the immunosuppressive role of PTIR1 in tumorigenesis and propose that ADAR1-PTIR1-UCHL5 signaling is a potential cancer immunotherapeutic target.

NEIL1 Recoding due to RNA Editing Impacts Lesion-Specific Recognition and Excision.

A-to-I RNA editing is widespread in human cells but is uncommon in the coding regions of proteins outside the nervous system. An unusual target for recoding by the adenosine deaminase ADAR1 is the pre-mRNA of the base excision DNA repair enzyme NEIL1 that results in the conversion of a lysine (K) to arginine (R) within the lesion recognition loop and alters substrate specificity. Differences in base removal by unedited (UE, K242) vs edited (Ed, R242) NEIL1 were evaluated using a series of oxidatively modified DNA bases to provide insight into the chemical and structural features of the lesion base that impact isoform-specific repair. We find that UE NEIL1 exhibits higher activity than Ed NEIL1 toward the removal of oxidized pyrimidines, such as thymine glycol, uracil glycol, 5-hydroxyuracil, and 5-hydroxymethyluracil. Gas-phase calculations indicate that the relative rates in excision track with the more stable lactim tautomer and the proton affinity of N3 of the base lesion. These trends support the contribution of tautomerization and N3 protonation in NEIL1 excision catalysis of these pyrimidine base lesions. Structurally similar but distinct substrate lesions, 5-hydroxycytosine and guanidinohydantoin, are more efficiently removed by the Ed NEIL1 isoform, consistent with the inherent differences in tautomerization, proton affinities, and lability. We also observed biphasic kinetic profiles and lack of complete base removal with specific combinations of the lesion and NEIL1 isoform, suggestive of multiple lesion binding modes. The complexity of NEIL1 isoform activity implies multiple roles for NEIL1 in safeguarding accurate repair and as an epigenetic regulator.

RNA sensing via the RIG‐I‐like receptor LGP2 is essential for the induction of a type I IFN response in ADAR1 deficiency

RNA editing by the adenosine deaminase ADAR1 prevents innate immune responses to endogenous RNAs. In ADAR1‐deficient cells, unedited self RNAs form base‐paired structures that resemble viral RNAs and inadvertently activate the cytosolic RIG‐I‐like receptor (RLR) MDA5, leading to an antiviral type I interferon (IFN) response. Mutations in ADAR1 cause Aicardi‐Goutières Syndrome (AGS), an autoinflammatory syndrome characterized by chronic type I IFN production. Conversely, ADAR1 loss and the consequent type I IFN production restricts tumor growth and potentiates the activity of some chemotherapeutics. Here, we show that another RIG‐I‐like receptor, LGP2, also has an essential role in the induction of a type I IFN response in ADAR1‐deficient human cells. This requires the canonical function of LGP2 as an RNA sensor and facilitator of MDA5‐dependent signaling. Furthermore, we show that the sensitivity of tumor cells to ADAR1 loss requires LGP2 expression. Finally, type I IFN induction in tumor cells depleted of ADAR1 and treated with some chemotherapeutics fully depends on LGP2 expression. These findings highlight a central role for LGP2 in self RNA sensing with important clinical implications.

Adenosine-to-inosine editing of endogenous Z-form RNA by the deaminase ADAR1 prevents spontaneous MAVS-dependent type I interferon responses

SummaryNucleic acids are powerful triggers of innate immunity and can adopt the Z-conformation, an unusual left-handed double helix. Here, we studied the biological function(s) of Z-RNA recognition by the adenosine deaminase ADAR1, mutations in which cause Aicardi-Goutières syndrome. Adar1mZα/mZα mice, bearing two point mutations in the Z-nucleic acid binding (Zα) domain that abolish Z-RNA binding, displayed spontaneous induction of type I interferons (IFNs) in multiple organs, including in the lung, where both stromal and hematopoietic cells showed IFN-stimulated gene (ISG) induction. Lung neutrophils expressed ISGs induced by the transcription factor IRF3, indicating an initiating role for neutrophils in this IFN response. The IFN response in Adar1mZα/mZα mice required the adaptor MAVS, implicating cytosolic RNA sensing. Adenosine-to-inosine changes were enriched in transposable elements and revealed a specific requirement of ADAR1’s Zα domain in editing of a subset of RNAs. Thus, endogenous RNAs in Z-conformation have immunostimulatory potential curtailed by ADAR1, with relevance to autoinflammatory disease in humans.

ADAR1 edits the SenZ and SenZ-ability of RNA

Some RNAs can assume a Z conformation, an unusual, left-handed turn. In this issue of Immunity, three studies report that mutations in the Zα-RNA binding domain of the adenosine deaminase ADAR1 are sufficient to induce autoinflammatory disease in mice, which models human Aicardí-Goutières syndrome, highlighting the important role of Z-RNA editing in limiting innate immune recognition of endogenous RNA.

Recognition of DNA adducts by edited and unedited forms of DNA glycosylase NEIL1

Pre-mRNA encoding human NEIL1 undergoes editing by adenosine deaminase ADAR1 that converts a single adenosine to inosine, and this conversion results in an amino acid change of lysine 242 to arginine. Previous investigations of the catalytic efficiencies of the two forms of the enzyme revealed differential release of thymine glycol (ThyGly) from synthetic oligodeoxynucleotides, with the unedited form, NEIL1 K242 being ≈30-fold more efficient than the edited NEIL1 K242R. In contrast, when these enzymes were reacted with oligodeoxynucleotides containing guanidinohydantoin or spiroiminohydantoin, the edited K242R form was ≈3-fold more efficient than the unedited NEIL1. However, no prior studies have investigated the efficiencies of these two forms of NEIL1 on either high-molecular weight DNA containing multiple oxidatively-induced base damages, or oligodeoxynucleotides containing a bulky alkylated formamidopyrimidine. To understand the extent of changes in substrate recognition, γ-irradiated calf thymus DNA was treated with either edited or unedited NEIL1 and the released DNA base lesions analyzed by gas chromatography-tandem mass spectrometry. Of all the measured DNA lesions, imidazole ring-opened 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) were preferentially released by both NEIL1 enzymes with K242R being ≈1.3 and 1.2-fold more efficient than K242 on excision of FapyAde and FapyGua, respectively. Consistent with the prior literature, large differences (≈7.5 to 12-fold) were measured in the excision of ThyGly from genomic DNA by the unedited versus edited NEIL1. In contrast, the edited NEIL1 was more efficient (≈3 to 5-fold) on release of 5-hydroxycytosine. Excision kinetics on DNA containing a site-specific aflatoxin B1-FapyGua adduct revealed an ≈1.4-fold higher rate by the unedited NEIL1. Molecular modeling provides insight into these differential substrate specificities. The results of this study and in particular, the comparison of substrate specificities of unedited and edited NEIL1 using biologically and clinically important base lesions, are critical for defining its role in preservation of genomic integrity.

ADAR1: "Editor-in-Chief" of Cytoplasmic Innate Immunity.

Specialized receptors that recognize molecular patterns such as double stranded RNA duplexes—indicative of viral replication—are potent triggers of the innate immune system. Although their activation is beneficial during viral infection, RNA transcribed from endogenous mobile genetic elements may also act as ligands potentially causing autoimmunity. Recent advances indicate that the adenosine deaminase ADAR1 through RNA editing is involved in dampening the canonical antiviral RIG-I-like receptor-, PKR-, and OAS-RNAse L pathways to prevent autoimmunity. However, this inhibitory effect must be overcome during viral infections. In this review we discuss ADAR1's critical role in balancing immune activation and self-tolerance.

AKT-dependent phosphorylation of the adenosine deaminases ADAR-1 and -2 inhibits deaminase activity.

Murine thymoma viral oncogene homolog (AKT) kinases target both cytosolic and nuclear substrates for phosphorylation. Whereas the cytosolic substrates are known to be closely associated with the regulation of apoptosis and autophagy or metabolism and protein synthesis, the nuclear substrates are, for the most part, poorly understood. To better define the role of nuclear AKT, potential AKT substrates were isolated from the nuclear lysates of leukemic cell lines using a phosphorylated AKT substrate antibody and identified in tandem mass spectrometry. Among the proteins identified was adenosine deaminase acting on RNA (ADAR)1p110, the predominant nuclear isoform of the adenosine deaminase acting on double-stranded RNA. Coimmunoprecipitation studies and in vitro kinase assays revealed that AKT-1, -2, and -3 interact with both ADAR1p110 and ADAR2 and phosphorylate these RNA editases. Using site-directed mutagenesis of suspected AKT phosphorylation sites, AKT was found to primarily phosphorylate ADAR1p110 and ADAR2 on T738 and T553, respectively, and overexpression of the phosphomimic mutants ADAR1p110 (T738D) and ADAR2 (T553D) resulted in a 50-100% reduction in editase activity. Thus, activation of AKT has a direct and major impact on RNA editing.-Bavelloni, A., Focaccia, E., Piazzi, M., Raffini, M., Cesarini, V., Tomaselli, S., Orsini, A., Ratti, S., Faenza, I., Cocco, L., Gallo, A., Blalock, W. L. AKT-dependent phosphorylation of the adenosine deaminases ADAR-1 and -2 inhibits deaminase activity.

Inosine induces context-dependent recoding and translational stalling.

RNA modifications are present in all classes of RNAs. They control the fate of mRNAs by affecting their processing, translation, or stability. Inosine is a particularly widespread modification in metazoan mRNA arising from deamination of adenosine catalyzed by the RNA-targeting adenosine deaminases ADAR1 or ADAR2. Inosine is commonly thought to be interpreted as guanosine by cellular machines and during translation. Here, we systematically test ribosomal decoding using mass spectrometry. We show that while inosine is primarily interpreted as guanosine it can also be decoded as adenosine, and rarely even as uracil. Decoding of inosine as adenosine and uracil is context-dependent. In addition, mass spectrometry analysis indicates that inosine causes ribosome stalling especially when multiple inosines are present in the codon. Indeed, ribosome profiling data from human tissues confirm inosine-dependent ribosome stalling in vivo. To our knowledge this is the first study where decoding of inosine is tested in a comprehensive and unbiased way. Thus, our study shows novel, unanticipated functions for inosines in mRNAs, further expanding coding potential and affecting translational efficiency.

Editing of Cellular Self-RNAs by Adenosine Deaminase ADAR1 Suppresses Innate Immune Stress Responses

Adenosine deaminases acting on double-stranded RNA (ADARs) catalyze the deamination of adenosine (A) to produce inosine (I) in double-stranded (ds) RNA structures, a process known as A-to-I RNA editing. dsRNA is an important trigger of innate immune responses, including interferon (IFN) production and action. We examined the role of A-to-I RNA editing by two ADARs, ADAR1 and ADAR2, in the sensing of self-RNA in the absence of pathogen infection, leading to activation of IFN-induced, RNA-mediated responses in mouse embryo fibroblasts. IFN treatment of Adar1(-/-) cells lacking both the p110 constitutive and p150 IFN-inducible ADAR1 proteins induced formation of stress granules, whereas neither wild-type (WT) nor Adar2(-/-) cells displayed a comparable stress granule response following IFN treatment. Phosphorylation of protein synthesis initiation factor eIF2α at serine 51 was increased in IFN-treated Adar1(-/-) cells but not in either WT or Adar2(-/-) cells following IFN treatment. Analysis by deep sequencing of mouse exonic loci containing A-to-I-editing sites revealed that the majority of editing in mouse embryo fibroblasts was carried out by ADAR1. IFN treatment increased editing in both WT and Adar2(-/-) cells but not in either Adar1(-/-) or Adar1(-/-) (p150) cells or Stat1(-/-) or Stat2(-/-) cells. Hyper-edited sites found in predicted duplex structures showed strand bias of editing for some RNAs. These results implicate ADAR1 p150 as the major A-to-I editor in mouse embryo fibroblasts, acting as a feedback suppressor of innate immune responses otherwise triggered by self-RNAs possessing regions of double-stranded character.

ID: 32: Roles of A-to-I editing by RNA adenosine deaminase ADAR1 in innate immunity RNA sensing

ADAR1, an interferon inducible adenosine deaminase acting on RNA, catalyzes the C6 deamination of adenosine (A) to produce inosine (I) in RNA with double-stranded (ds) character, leading to genetic recoding and altered RNA structure as I pairs as G instead of A. Adar1 expression occurs from multiple promoters and encodes an IFN-inducible p150 cytoplasmic protein and a constitutively expressed N-terminally truncated p110 nuclear protein. The roles of ADAR1 p150 and p110 in uninfected and virus-infected cells were examined using human cell clones stably deficient in ADAR1, mouse cells genetically deficient in ADAR1, and by complementation strategies. Exemplified by measles virus (MV) infected cells, ADAR1 suppressed activation of PKR and IRF3, suppressed IFN β induction, and protected cells from stress granule formation and cytopathic damage. RNA expression profiles and A-to-I editing were characterized by qPCR and RNA deep sequencing, with differences found between CKO mutant and parental MV infections, and between untreated and IFN-treated uninfected cells. Both non-self (viral) and self (cellular) RNAs with ds character were identified and their editing characterized. Results obtained with mouse cells genetically deficient in either adar1 or the related deaminase adar2, or stat1 or stat2, suggest a central role of ADAR1 in overall A-to-I editing and p150 in the IFN-induced editing and innate immunity RNA sensing. In summary, ADAR1 behaved as an anti-apoptotic host factor that suppressed innate immune activities and was pro-viral. Our findings furthermore implicate a balanced interplay between PKR and ADAR1, two IFN inducible dsRNA binding enzymes, in modulating IFN β protein production and stress responses.

S-22 : RNA adenosine deaminase ADAR1 and RNA-dependent protein kinase PKR: Opposing modulators of antiviral innate immunity

ADAR1, an interferon inducible adenosine deaminase acting on RNA, catalyzes the C6 deamination of adenosine (A) to produce inosine (I) in RNA substrates with double-stranded character, leading to genetic recoding and altered RNA structure. Adar1 expression occurs from multiple promoters and encodes either an IFN-inducible p150 cytoplasmic protein or a constitutively expressed N-terminally truncated p110 nuclear protein. The roles of ADAR1 p150 and p110 in uninfected cells and during virus infection were examined using human cell clones made stably deficient in ADAR1, mouse cells genetically deficient in ADAR1, and by complementation strategies. The effects of ADAR1 deficiency on virus growth, IFNb induction, and stress responses including formation of stress granules (SG) and cell death were opposite to those established for dsRNA-dependent PKR kinase deficiency. Exemplified by measles virus (MV) infection, ADAR1 suppressed activation of PKR and IRF3 and protected cells from SG formation and cytopathic damage. MV-induced SG formation was PKR-dependent and impaired by catalytically active p150 but not p110 ADAR1. C KO mutant MV was an effective inducer of IFNb by IPS-1-dependent signaling that was amplified by PKR. RNA expression profiles were characterized by qPCR and RNA deep sequencing, with differences found between C KO mutant and parental MV, and untreated and IFN-treated cells. Significant amounts of dsRNA accumulate during C KO mutant infection. Results obtained with mouse cells genetically deficient in either adar1 or the related deaminase adar2 , or stat1 or stat2 , suggest a central role of ADAR1 in overall A-to-I editing and p150 in the IFN-induced editing. In summary, ADAR1 behaved as an anti-apoptotic host factor that, in some instances, suppressed innate immune activities and was pro-viral. Our findings furthermore implicate a balanced interplay between PKR and ADAR1 in modulating IFNb protein production and stress responses following virus infection. (Supported by NIAID, NIH).

Induction of stress granules by interferon and down-regulation by the cellular RNA adenosine deaminase ADAR1

Measles virus (MV) deficient in C protein (Cko) expression efficiently induces both stress granules (SG) and interferon (IFNβ), whereas isogenic wild-type (WT) and V mutant (Vko) viruses do not. We therefore examined the effect of IFNβ pretreatment on SG formation, and the roles played by the IFN-inducible double-stranded (ds) RNA-dependent protein kinase (PKR) and dsRNA adenosine deaminase (ADAR1). SG formation in ADAR1-sufficient cells infected with WT or Vko mutant virus was enhanced by IFN treatment and was PKR-dependent. SG formation in Cko virus-infected cells was already high without IFN treatment and was not further enhanced by IFN. IFN treatment alone, in the absence of infection, induced SG formation in ADAR1-deficient but not ADAR1-sufficient cells. Type I IFN-induced enhancement in SG formation occurred by a canonical IFN signaling response dependent upon STAT1 and STAT2. These results further establish ADAR1 as a suppressor of the interferon and SG innate immune responses.

Double-stranded RNA adenosine deaminase ADAR1 enhances both T cell susceptibility to human T-cell leukemia virus type 1 and 2 and viral replication

Type I interferons represent the first line of defense against pathogens. This family of cytokines activates the expression of antiviral proteins, such as the protein kinase R (PKR), an inhibitor of viral mRNA translation, and the double-stranded RNA adenosine deaminase ADAR1. ADAR1 has the ability to convert adenosine (A) into guanosine (G), thereby introducing mutations in the viral genome during its replication. A to G editing was previously reported in cells expressing HTLV-2 or STLV-3 viruses but not investigating in HTLV-1 expressing cells (Ko et al. J. Gen Virol. 2013). Consequently we investigated whether ADAR1 expression was associated or not with an antiviral effect in the course of HTLV-1 and HTLV-2 infections. We first show that ADAR1 expression is increased in ATL patient peripheral blood mononuclear cells, in HTLV-1 and HTLV-2 transformed cell lines as well as in activated primary peripheral blood lymphocytes. Strikingly, in cells transfected with HTLV-1 and HTLV-2 molecular clones, ADAR1 over-expression enhances viral replication and viral egress through PKR functional inhibition, as demonstrated by western-blot analyses, luciferase assays, ELISA and infection experiments. We also demonstrate that this effect is independent of ADAR catalytic activity. In addition, ADAR1 expression enhances the susceptibility of a non-infected T cell line to HTLV-1 and HTLV-2 infection. Altogether, our results demonstrate that an interferon-induced protein exerts a proviral role in the context of HTLV infection by enhancing cells susceptibility to infection and increasing viral replication.

Stress Granule Formation Induced by Measles Virus Is Protein Kinase PKR Dependent and Impaired by RNA Adenosine Deaminase ADAR1

ADAR1, an interferon (IFN)-inducible double-stranded (ds) RNA-specific adenosine deaminase, downregulates host innate responses, including activation of the dsRNA-dependent protein kinase (PKR) and induction of IFN-β mRNA. Conversely, PKR amplifies IFN-β induction by measles virus (MV) and inhibits virus protein synthesis. Formation of stress granules (SGs), cytoplasmic aggregates of stalled translation complexes and RNA-binding proteins, is a host response to virus infection mediated by translation initiation factor eIF2α phosphorylation. We examined the roles of PKR and ADAR1 in SG formation using HeLa cells stably deficient in either PKR (PKR(kd)) or ADAR1 (ADAR1(kd)) compared to control (CON(kd)) cells. Infection with either wild-type (WT) MV or an isogenic mutant lacking C protein expression (C(ko)) comparably induced formation of SG in ADAR1(kd) cells, whereas only the C(ko) mutant was an efficient inducer in control cells. Both ADAR1 and PKR colocalized with SG following infection. MV-induced; SG formation was PKR dependent but impaired by ADAR1. Complementation of ADAR1(kd) cells by expression of either p150 WT isoform or the p150 Zα (Y177A) Z-DNA-binding mutant of ADAR1 restored suppression of host responses, including SG formation and PKR activation. In contrast, neither the p110 WT isoform nor the p150 catalytic (H910A, E912A) mutant of ADAR1 complemented the ADAR1(kd) phenotype. These results further establish ADAR1 as a suppressor of host innate responses, including activation of PKR and the subsequent SG response.

Hyperediting of human T-cell leukemia virus type 2 and simian T-cell leukemia virus type 3 by the dsRNA adenosine deaminase ADAR-1

RNA editing mediated by adenosine deaminases acting on RNA (ADARs) converts adenosine (A) to inosine (I) residues in dsRNA templates. While ADAR-1-mediated editing was essentially described for RNA viruses, the present work addresses the issue for two δ-retroviruses, human T-cell leukemia virus type 2 and simian T-cell leukemia virus type 3 (HTLV-2 and STLV-3). We examined whether ADAR-1 could edit HTLV-2 and STLV-3 virus genomes in cell culture and in vivo. Using a highly sensitive PCR-based method, referred to as 3DI-PCR, we showed that ADAR-1 could hypermutate adenosine residues in HTLV-2. STLV-3 hypermutation was obtained without using 3DI-PCR, suggesting a higher mutation frequency for this virus. Detailed analysis of the dinucleotide editing context showed preferences for 5' ArA and 5' UrA. In conclusion, the present observations demonstrate that ADAR-1 massively edits HTLV-2 and STLV-3 retroviruses in vitro, but probably remains a rare phenomenon in vivo.

Host Response to Polyomavirus Infection Is Modulated by RNA Adenosine Deaminase ADAR1 but Not by ADAR2

Adenosine deaminases acting on RNA (ADARs) catalyze the C-6 deamination of adenosine (A) to produce inosine (I), which behaves as guanine (G), thereby altering base pairing in RNAs with double-stranded character. Two genes, adar1 and adar2, are known to encode enzymatically active ADARs in mammalian cells. Furthermore, two size forms of ADAR1 are expressed by alternative promoter usage, a short (p110) nuclear form that is constitutively made and a long (p150) form that is interferon inducible and present in both the cytoplasm and nucleus. ADAR2 is also a constitutively expressed nuclear protein. Extensive A-to-G substitution has been described in mouse polyomavirus (PyV) RNA isolated late times after infection, suggesting modification by ADAR. To test the role of ADAR in PyV infection, we used genetically null mouse embryo fibroblast cells deficient in either ADAR1 or ADAR2. The single-cycle yields and growth kinetics of PyV were comparable between adar1(-/-) and adar2(-/-) genetic null fibroblast cells. While large T antigen was expressed to higher levels in adar1(-/-) cells than adar2(-/-) cells, less difference was seen in VP1 protein expression levels between the two knockout MEFs. However, virus-induced cell killing was greatly enhanced in PyV-infected adar1(-/-) cells compared to that of adar2(-/-) cells. Complementation with p110 protected cells from PyV-induced cytotoxicity. UV-irradiated PyV did not display any enhanced cytopathic effect in adar1(-/-) cells. Reovirus and vesicular stomatitis virus single-cycle yields were comparable between adar1(-/-) and adar2(-/-) cells, and neither reovirus nor VSV showed enhanced cytotoxicity in adar1(-/-)-infected cells. These results suggest that ADAR1 plays a virus-selective role in the host response to infection.

RNA editing changes the lesion specificity for the DNA repair enzyme NEIL1

Editing of the pre-mRNA for the DNA repair enzyme NEIL1 causes a lysine to arginine change in the lesion recognition loop of the protein. The two forms of NEIL1 are shown here to have distinct enzymatic properties. The edited form removes thymine glycol from duplex DNA 30 times more slowly than the form encoded in the genome, whereas editing enhances repair of the guanidinohydantoin lesion by NEIL1. In addition, we show that the NEIL1 recoding site is a preferred editing site for the RNA editing adenosine deaminase ADAR1. The edited adenosine resides in an A-C mismatch in a hairpin stem formed by pairing of exon 6 to the immediate upstream intron 5 sequence. As expected for an ADAR1 site, editing at this position is increased in human cells treated with interferon α. These results suggest a unique regulatory mechanism for DNA repair and extend our understanding of the impact of RNA editing.

RNA adenosine deaminase ADAR1 deficiency leads to increased activation of protein kinase PKR and reduced vesicular stomatitis virus growth following interferon treatment

Two size forms of ADAR1 adenosine deaminase are known, one constitutively expressed (p110) and the other interferon (IFN)-induced (p150). To test the role of ADAR1 in viral infection, HeLa cells with ADAR1 stably knocked down and 293 cells overexpressing ADAR1 were utilized. Overexpression of p150 ADAR1 had no significant effect on the yield of vesicular stomatitis virus. Likewise, reduction of p110 and p150 ADAR1 proteins to less than approximately 10 to 15% of parental levels (ADAR1-deficient) had no significant effect on VSV growth in the absence of IFN treatment. However, inhibition of virus growth following IFN treatment was approximately 1 log(10) further reduced compared to ADAR1-sufficient cells. The level of phosphorylated protein kinase PKR was increased in ADAR1-deficient cells compared to ADAR1-sufficient cells following IFN treatment, regardless of viral infection. These results suggest that ADAR1 suppresses activation of PKR and inhibition of VSV growth in response to IFN treatment.

Adenosine Deaminase ADAR1 Increases Gene Expression at the Translational Level by Decreasing Protein Kinase PKR-Dependent eIF-2α Phosphorylation

ADAR1 (adenosine deaminase acting on RNA) catalyzes the deamination of adenosine to inosine on RNA substrates with double-stranded character. Here, we show that coexpression of ADAR1 in mammalian cells markedly increases plasmid-based gene expression in transfected cells. The enhanced expression was independent of the nature of the promoter (viral and cellular) used to drive gene expression, of the protein reporter (luciferase and RRP) tested, and of the human cell line examined (293T and HeLa). Exogenous protein levels were increased by ∼20-fold to ∼50-fold when ADAR1 was coexpressed, whereas RNA transcript levels changed by less than 2-fold. The activation of PKR (protein kinase regulated by RNA) protein kinase and the phosphorylation of translation initiation factor eIF-2α seen following plasmid DNA transfection were both greatly reduced in ADAR1-transfected cells. Stable knockdown of the PKR kinase increased reporter gene expression in the absence, but not in the presence, of ADAR1 coexpression. Both size forms of ADAR1—the p150-inducible form and the p110-like constitutive form—enhanced plasmid-based gene expression. Taken together, these results indicate that the ADAR1 deaminase increases exogenous gene expression at the translational level by decreasing PKR-dependent eIF-2α phosphorylation.