Cytosolic Double-stranded RNA Research Articles

Abstract 2838: ZNFX1 is a master regulator and tumor suppressor in epigenetically induced pathogen mimicry, mitochondrial dysfunction and STING-dependent signaling in cancer

Abstract Immunomodulatory agents are an important recent advance in cancer therapy, but utility is often limited by tumor immune evasion mechanisms. Strategic therapeutic activation of intracellular antiviral immune responses offers an opportunity to reverse immune evasion mechanisms and improve treatment outcomes. Anti-cancer agents such as, DNA methyltransferase inhibitors (DNMTis) induce re-expression of endogenous retroviruses (ERVs), leading to cytosolic double-stranded RNA (dsRNA) accumulation that activates interferon/inflammasome signaling. Moreover, poly (ADP ribose) polymerase inhibitors (PARPi) increase cytosolic dsDNA, activating stimulator of interferon (IFN) genes (STING). We reported that DNMTis in combination with PARPis induce STING-dependent signaling in a process termed pathogen mimicry response (PMR). Mitochondria (mt) are an important gateway for antiviral inflammasome signaling, but are not fully understood in cancer. Our studies of the little-known gene, NFX1-type zinc finger-containing 1 (ZNFX1), show that it acts as a master nucleic acid (dsRNA/DNA) sensor for mt gateway function. Bioinformatics analysis in primary ovarian tumors from TCGA and clinical trial RNAseq datasets, shows that increased ZNFX1 expression tracks with tumor stage and grade but inversely correlates with a mt dysfunction signature. In studies of high-grade and endometrial serous carcinoma (OC) cell lines (N=3), transfection of dsRNA/DNA mimics or DNMTi azacytidine (AZA) and PARPi (talazoparib [TAL]) treatments induce increased ZNFX1 expression and binding to mt antiviral protein (MAVs) localized on the mt outer membrane. Functional studies of mt dysfunction in OC cells, show that dsRNA/DNA as well as above viral mimicry drugs increase mt reactive oxygen species (ROS) as measured by mitosox flow cytometry. AZA and TAL treatments also increase fragmented mtDNA and oxidative mtDNA base damage, as measured by long range PCR and 8-oxoguanine (8-oxoG) ELISA assays. These drug treatments also increase release of mtDNA into the cytosol, resulting in STING-dependent inflammasome signaling and cytokine release. Notably, ZNFX1 knockout (KO) attenuates these dynamics, including in bioinformatics of RNA seq data, thus defining this gene as essential for IFN/inflammasome signaling induced by mtDNA damage. Further pathway analysis of ZNFX1 KO vs wild-type (WT) RNAseq data suggest increased proliferation and epithelial to mesenchymal transition (EMT) that was validated by in vitro and in vivo assays. Not only is ZNFX1 a master regular of the above mt dynamics, but it is a tumor suppressor in OC. Abrogating ZNFX1 expression in OC cells triggers increased cell proliferation, invasive capacity and, in-vivo, increased tumor growth. This work then suggests novel approaches to improve immune therapy responses in OC through manipulating ZNFX1. Citation Format: Lora Stojanovic, Rachel Abbotts, Kaushlendra Tripathi, Collin M. Coon, Sheng Liu, Jun Wan, Michael Topper, Stephen Baylin, Kenneth Nephew, Feyruz Rassool. ZNFX1 is a master regulator and tumor suppressor in epigenetically induced pathogen mimicry, mitochondrial dysfunction and STING-dependent signaling in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2838.

Abstract PR-005: ZNFX1 is a master regulator for epigenetic reprograming of mitochondrial inflammasome signaling and pathogen mimicry in cancer cells

Abstract Immunomodulatory agents represent an important recent advance in cancer therapy, but utility is often limited by tumor immune evasion mechanisms. Strategic therapeutic activation of intracellular antiviral immune responses offers an opportunity to reverse immune evasion mechanisms and improve treatment outcomes. Anti-cancer agents such as, DNA methyltransferase inhibitors (DNMTis) induce re-expression of endogenous retroviral elements (ERVs), leading to cytosolic double-stranded RNA (dsRNA) accumulation that activates interferon/inflammasome signaling. Moreover, poly (ADP ribose) polymerase inhibitors (PARPi) increase cytosolic dsDNA leading to activation of stimulator of interferon genes (STING). We previously reported in triple negative breast cancer (TNBC) and ovarian cancer (OC) that DNMTis in combination with PARPi induce STING-dependent interferon/inflammasome signaling in a process termed pathogen mimicry response (PMR). Mitochondria (mt) are an important gateway for antiviral inflammasome signaling, but upstream activating events in cancer are not understood. We now show NFX1-type zinc finger–containing 1 (ZNFX1), a little-studied innate immune gene, acts as a master nucleic acid (dsRNA/DNA) sensor for this mt gateway function. Importantly, in primary ovarian tumors from TCGA and clinical trial RNAseq datasets, increased ZNFX1 expression tracks with tumor stage and grade but inversely correlates with a mt dysfunction signature. In studies of high grade serous (TYK-nu, OVCAR4) as well as endometrial ovarian (A2780) cancer cells, transfection of dsRNA/DNA mimics or DNMTi azacytidine (AZA) and PARPi (talazoparib) treatments induce increased ZNFX1 expression and binding to mt antiviral protein (MAVs) localized on the mt outer membrane, using immunofluorescence-based proximity ligation assays. In studies of mt dysfunction, we further show that dsRNA/DNA as well as above-described anti-cancer drugs increase mt reactive oxygen species (ROS), using flow cytometry of ROS dye mitoSOX. DNMTi and PARPi treatments also increase fragmented mtDNA and oxidative mtDNA base damage, as measured long range PCR and 8-oxoguanine (8-oxoG) ELISA assays, respectively. Importantly, these drug treatment increase release of mtDNA into the cytosol, resulting in STING-dependent inflammasome signaling and cytokine release. ZNFX1 knockout robustly attenuates these dynamics, thus defining this immune gene as essential for interferon/inflammasome signaling induced by mtDNA damage. Our data indicates a master role for the little-studied dsRNA/DNA sensor ZNFX1 in initiating a pathogen mimicry response to DNMTi and PARPi combination treatment, and further highlights the critical role of the mt in cellular antiviral signaling mechanisms. Importantly, this work suggests a novel avenue for manipulation of inflammasome signaling to improve cancer therapy responses and disease outcomes. Citation Format: Lora Stojanovic, Rachel Abbotts, Kaushlendra Tripathi, Collin M. Coon, Sheng Liu, Jun Wan, Michael J. Topper, Kenneth P. Nephew, Stephen B. Baylin, Feyruz V. Rassool. ZNFX1 is a master regulator for epigenetic reprograming of mitochondrial inflammasome signaling and pathogen mimicry in cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr PR-005.

Contrasting functions of ATP hydrolysis by MDA5 and LGP2 in viral RNA sensing

Cytosolic long dsRNA, among the most potent proinflammatory signals, is recognized by melanoma differentiation-associated protein 5 (MDA5). MDA5 binds dsRNA cooperatively forming helical filaments. ATP hydrolysis by MDA5 fulfills a proofreading function by promoting dissociation of shorter endogenous dsRNs from MDA5 while allowing longer viral dsRNAs to remain bound leading to activation of interferon-β responses. Here, we show that adjacent MDA5 subunits in MDA5-dsRNA filaments hydrolyze ATP cooperatively, inducing cooperative filament disassembly. Consecutive rounds of ATP hydrolysis amplify the filament footprint, displacing tightly bound proteins from dsRNA. Our electron microscopy and biochemical assays show that LGP2 binds to dsRNA at internal binding sites through noncooperative ATP hydrolysis. Unlike MDA5, LGP2 has low nucleic acid selectivity and can hydrolyze GTP and CTP as well as ATP. Binding of LGP2 to dsRNA promotes nucleation of MDA5 filament assembly resulting in shorter filaments. Molecular modeling identifies an internally bound MDA5-LGP2-RNA complex, with the LGP2 C-terminal tail forming the key contacts with MDA5. These contacts are specifically required for NTP-dependent internal RNA binding. We conclude that NTPase-dependent binding of LGP2 to internal dsRNA sites complements NTPase-independent binding to dsRNA ends, via distinct binding modes, to increase the number and signaling output of MDA5-dsRNA complexes.

Lysine catabolism reprograms tumour immunity through histone crotonylation.

Cancer cells rewire metabolism to favour the generation of specialized metabolites that support tumour growth and reshape the tumour microenvironment1,2. Lysine functions as a biosynthetic molecule, energy source and antioxidant3-5, but little is known about its pathological role in cancer. Here we show that glioblastoma stem cells (GSCs) reprogram lysine catabolism through the upregulation of lysine transporter SLC7A2 and crotonyl-coenzyme A (crotonyl-CoA)-producing enzyme glutaryl-CoA dehydrogenase (GCDH) with downregulation of the crotonyl-CoA hydratase enoyl-CoA hydratase short chain 1 (ECHS1), leading to accumulation of intracellular crotonyl-CoA and histone H4 lysine crotonylation. A reduction in histone lysine crotonylation by either genetic manipulation or lysine restriction impaired tumour growth. In the nucleus, GCDH interacts with the crotonyltransferase CBP to promote histone lysine crotonylation. Loss of histone lysine crotonylation promotes immunogenic cytosolic double-stranded RNA (dsRNA) and dsDNA generation through enhanced H3K27ac, which stimulates the RNA sensor MDA5 and DNA sensor cyclic GMP-AMP synthase (cGAS) to boost type I interferon signalling, leading to compromised GSC tumorigenic potential and elevated CD8+ T cell infiltration. A lysine-restricted diet synergized with MYC inhibition or anti-PD-1 therapy to slow tumour growth. Collectively, GSCs co-opt lysine uptake and degradation to shunt the production of crotonyl-CoA, remodelling the chromatin landscape to evade interferon-induced intrinsic effects on GSC maintenance and extrinsic effects on immune response.

Virus-Like Cytosolic and Cell-Free Oxidatively Damaged Nucleic Acids Likely Drive Inflammation, Synapse Degeneration, and Neuron Death in Alzheimer's Disease.

Oxidative stress, inflammation, and amyloid-β are Alzheimer's disease (AD) hallmarks that cause each other and other AD hallmarks. Most amyloid-β-lowering, antioxidant, anti-inflammatory, and antimicrobial AD clinical trials failed; none stopped or reversed AD. Although signs suggest an infectious etiology, no pathogen accumulated consistently in AD patients. Neuropathology, neuronal cell culture, rodent, genome-wide association, epidemiological, biomarker, and clinical studies, plus analysis using Hill causality criteria and revised Koch's postulates, indicate that the virus-like oxidative damage-associated molecular-pattern (DAMP) cytosolic and cell-free nucleic acids accumulated in AD patients' brains likely drive neuroinflammation, synaptotoxicity, and neurotoxicity. Cytosolic oxidatively-damaged mitochondrial DNA accumulated outside mitochondria dose-dependently in preclinical AD and AD patients' hippocampal neurons, and in AD patients' neocortical neurons but not cerebellar neurons or glia. In oxidatively-stressed neural cells and rodents' brains, cytosolic oxidatively-damaged mitochondrial DNA accumulated and increased antiviral and inflammatory proteins, including cleaved caspase-1, interleukin-1β, and interferon-β. Cytosolic double-stranded RNA and DNA are DAMPs that induce antiviral interferons and/or inflammatory proteins by oligomerizing with various innate-immune pattern-recognition receptors, e.g., cyclic GMP-AMP synthase and the nucleotide-binding-oligomerization-domain-like-receptor-pyrin-domain-containing-3 inflammasome. In oxidatively-stressed neural cells, cytosolic oxidatively-damaged mitochondrial DNA caused synaptotoxicity and neurotoxicity. Depleting mitochondrial DNA prevented these effects. Additionally, cell-free nucleic acids accumulated in AD patients' blood, extracellular vesicles, and senile plaques. Injecting cell-free nucleic acids bound to albumin oligomers into wild-type mice's hippocampi triggered antiviral interferon-β secretion; interferon-β injection caused synapse degeneration. Deoxyribonuclease-I treatment appeared to improve a severe-AD patient's Mini-Mental Status Exam by 15 points. Preclinical and clinical studies of deoxyribonuclease-I and a ribonuclease for AD should be prioritized.

Inborn errors of OAS-RNase L in SARS-CoV-2-related multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) is a rare and severe condition that follows benign COVID-19. We report autosomal recessive deficiencies of OAS1, OAS2, or RNASEL in five unrelated children with MIS-C. The cytosolic double-stranded RNA (dsRNA)-sensing OAS1 and OAS2 generate 2'-5'-linked oligoadenylates (2-5A) that activate the single-stranded RNA-degrading ribonuclease L (RNase L). Monocytic cell lines and primary myeloid cells with OAS1, OAS2, or RNase L deficiencies produce excessive amounts of inflammatory cytokines upon dsRNA or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) stimulation. Exogenous 2-5A suppresses cytokine production in OAS1-deficient but not RNase L-deficient cells. Cytokine production in RNase L-deficient cells is impaired by MDA5 or RIG-I deficiency and abolished by mitochondrial antiviral-signaling protein (MAVS) deficiency. Recessive OAS-RNase L deficiencies in these patients unleash the production of SARS-CoV-2-triggered, MAVS-mediated inflammatory cytokines by mononuclear phagocytes, thereby underlying MIS-C.

Adenosine deaminase acting on RNA-1 is essential for early B lymphopoiesis.

Adenosine deaminase acting on RNA-1 (ADAR1) is a ubiquitously expressed RNA deaminase catalyzing adenosine-to-inosine editing to prevent hyperactivated cytosolic double-stranded RNA (dsRNA) response mediated by MDA5. Here, we demonstrate that ADAR1 is essential for early B lymphopoiesis from late pro-B and large pre-B cell stages onward. ADAR1 exerts its requisite role via both MDA5-dependent and -independent pathways. Interestingly, the MDA5-dependent mechanisms regulate early pro-B to large pre-B cell transition by promoting early B cell survival. In contrast, the MDA5-independent mechanisms control large pre-B to small pre-B cell transition by regulating pre-B cell receptor (pre-BCR) expression. Moreover, we show that protein kinase R (PKR) and oligoadenylate synthetase/ribonuclease (OAS/RNase) L pathways are dispensable for ADAR1's role in early B lymphopoiesis. Importantly, we demonstrate that p150 isoform of ADAR1 exclusively accounts for ADAR1's function in early B lymphopoiesis, and its conventional dsRNA-binding, but not the Z-DNA/RNA-binding or the RNA-editing, activity is required for ADAR1's function in B cell development. Thus, our findings suggest that ADAR1 regulates early B lymphopoiesis through various mechanisms.

Abstract 6301: DNA methyltransferase inhibitors increase ERV reactivation and STING-dependent interferon/inflammasome signaling in TP53 mutant AML

Abstract DNA methyltransferase inhibitors (DNMTis), which transcriptionally activate hypermethylated genes in cancers and leukemias, also activate endogenous retroviruses (ERVs), leading to increased cytosolic double-stranded (ds) RNA and interferon (IFN) signaling, in a process termed viral mimicry. The tumor suppressor TP53 has been reported to cooperate with DNA methylation and IFN signaling to maintain transcriptional silencing of various repeat sequences, including ERVs. We now report that DNMTis, used to treat acute myeloid leukemia (AML) patients unfit for chemotherapy, robustly induce expression of ERVs and STING-dependent IFN/inflammasome signaling in AML with mutations in TP53(approximately 10%), a subset with poor prognosis. First, our studies of ERV transcripts (N=13) levels in TP53 mutant (KASUMI, KG1-1A, U937) and wild-type (WT; MOLM-14, OCI/AML2, and OCI/AML3) AML cell lines and primary cells (N=6 mutant, N=6 WT) show that, while TP53 mutant cells have low baseline ERV expression, DNMTi treatment significantly increases ERV expression, compared with WT TP53 cells (p<0.05). Moreover, inhibiting TP53 in WT TP53 AML cell lines with the specific TP53 inhibitor pifithrin significantly upregulates ERVs, validating the role of TP53 in suppressing ERVs in AML cells. Second, while TCGA data analysis showed that baseline STING transcript are low in TP53 mutant vs WT AML, phospho-STING (activated) is actually increased in TP53 mutant AML cells and DNMTi treatment further activates STING. Increased expression of downstream IFN/inflammasome genes in TP53 mutant AML treated with DNMTi, compared with results in WT cells, validates these results. Moreover, DNMTi combination with poly (ADP-ribose) polymerase inhibitors (PARPis), which we previously reported to activate STING in breast and ovarian cancer though increasing cytosolic dsDNA and dsRNA, further augments STING activation and IFN/inflammasome signaling (p<0.05). Third, the IFN/inflammasome signaling response to DNMTI/PARPI signaling is directly linked with decreased expression of DNA ds break repair (DSBR) genes and activity, leading to homologous recombination defects (HRD). Fourth, the mechanism for both IFN/inflammasome signaling and HRD induction is dependent on STING, since both CRISPR/CAS KO of STING or treatment with the STING inhibitor H-151 can rescue these effects in TP53 mutant and WT AML cells. Our findings increase understanding of the therapy potential for combining DNMTi+PARPi treatment in AML and suggest use of this paradigm to treat mutant TP53 AML. These results also suggest that activating STING may be a key strategy for increasing IFN/inflammasome signaling in treatment of TP53 mutant AML and that immune responses may be further augmented with immune therapy in AML and other cancers. Citation Format: Aksinija A. Kogan, Michael J. Topper, Lora Stojanovic, Lena J. McLaughlin, Tammy J. Kingsbury, Maria R. Baer, Michael Kessler, Stephen B. Baylin, Feyruz V. Rassool. DNA methyltransferase inhibitors increase ERV reactivation and STING-dependent interferon/inflammasome signaling in TP53 mutant AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6301.

Role of helical structure and dynamics in oligoadenylate synthetase 1 (OAS1) mismatch tolerance and activation by short dsRNAs

The 2'-5'-oligoadenylate synthetases (OAS) are innate immune sensors of cytosolic double-stranded RNA (dsRNA) that play a critical role in limiting viral infection. How these proteins are able to avoid aberrant activation by cellular RNAs is not fully understood, but adenosine-to-inosine (A-to-I) editing has been proposed to limit accumulation of endogenous RNAs that might otherwise cause stimulation of the OAS/RNase L pathway. Here, we aim to uncover whether and how such sequence modifications can restrict the ability of short, defined dsRNAs to activate the single-domain form of OAS, OAS1. Unexpectedly, we find that all tested inosine-containing dsRNAs have an increased capacity to activate OAS1, whether in a destabilizing (I•U) or standard Watson-Crick-like base pairing (I-C) context. Additional variants with strongly destabilizing A•C mismatches or stabilizing G-C pairs also exhibit increased capacity to activate OAS1, eliminating helical stability as a factor in the relative ability of the dsRNAs to activate OAS1. Using thermal difference spectra and molecular dynamics simulations, we identify both increased helical dynamics and specific local changes in helical structure as important factors in the capacity of short dsRNAs to activate OAS1. These helical features may facilitate more ready adoption of the distorted OAS1-bound conformation or stabilize important structures to predispose the dsRNA for optimal binding and activation of OAS1. These studies thus reveal the molecular basis for the greater capacity of some short dsRNAs to activate OAS1 in a sequence-independent manner.

MDA5 disease variant M854K prevents ATP-dependent structural discrimination of viral and cellular RNA

Our innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. Mutations that reduce RNA selectivity can cause autoinflammatory disease. Here, we show how the disease-associated MDA5 variant M854K perturbs MDA5-dsRNA recognition. M854K MDA5 constitutively activates interferon signaling in the absence of exogenous RNA. M854K MDA5 lacks ATPase activity and binds more stably to synthetic Alu:Alu dsRNA. CryoEM structures of MDA5-dsRNA filaments at different stages of ATP hydrolysis show that the K854 sidechain forms polar bonds that constrain the conformation of MDA5 subdomains, disrupting key steps in the ATPase cycle- RNA footprint expansion and helical twist modulation. The M854K mutation inhibits ATP-dependent RNA proofreading via an allosteric mechanism, allowing MDA5 to form signaling complexes on endogenous RNAs. This work provides insights on how MDA5 recognizes dsRNA in health and disease.

DNA Damage-Induced Inflammatory Microenvironment and Adult Stem Cell Response.

Adult stem cells ensure tissue homeostasis and regeneration after injury. Due to their longevity and functional requirements, throughout their life stem cells are subject to a significant amount of DNA damage. Genotoxic stress has recently been shown to trigger a cascade of cell- and non-cell autonomous inflammatory signaling pathways, leading to the release of pro-inflammatory factors and an increase in the amount of infiltrating immune cells. In this review, we discuss recent evidence of how DNA damage by affecting the microenvironment of stem cells present in adult tissues and neoplasms can affect their maintenance and long-term function. We first focus on the importance of self-DNA sensing in immunity activation, inflammation and secretion of pro-inflammatory factors mediated by activation of the cGAS-STING pathway, the ZBP1 pathogen sensor, the AIM2 and NLRP3 inflammasomes. Alongside cytosolic DNA, the emerging roles of cytosolic double-stranded RNA and mitochondrial DNA are discussed. The DNA damage response can also initiate mechanisms to limit division of damaged stem/progenitor cells by inducing a permanent state of cell cycle arrest, known as senescence. Persistent DNA damage triggers senescent cells to secrete senescence-associated secretory phenotype (SASP) factors, which can act as strong immune modulators. Altogether these DNA damage-mediated immunomodulatory responses have been shown to affect the homeostasis of tissue-specific stem cells leading to degenerative conditions. Conversely, the release of specific cytokines can also positively impact tissue-specific stem cell plasticity and regeneration in addition to enhancing the activity of cancer stem cells thereby driving tumor progression. Further mechanistic understanding of the DNA damage-induced immunomodulatory response on the stem cell microenvironment might shed light on age-related diseases and cancer, and potentially inform novel treatment strategies.

ADAR and hnRNPC deficiency synergize in activating endogenous dsRNA-induced type I IFN responses.

Cytosolic double-stranded RNA (dsRNA) initiates type I IFN responses. Endogenous retroelements, notably Alu elements, constitute a source of dsRNA. Adenosine-to-inosine (A-to-I) editing by ADAR induces mismatches in dsRNA and prevents recognition by MDA5 and autoinflammation. To identify additional endogenous dsRNA checkpoints, we conducted a candidate screen in THP-1 monocytes and found that hnRNPC and ADAR deficiency resulted in synergistic induction of MDA5-dependent IFN responses. RNA-seq analysis demonstrated dysregulation of Alu-containing introns in hnRNPC-deficient cells via utilization of unmasked cryptic splice sites, including introns containing ADAR-dependent A-to-I editing clusters. These putative MDA5 ligands showed reduced editing in the absence of ADAR, providing a plausible mechanism for the combined effects of hnRNPC and ADAR. This study contributes to our understanding of the control of repetitive element-induced autoinflammation and suggests that patients with hnRNPC-mutated tumors might maximally benefit from ADAR inhibition-based immunotherapy.

Species-Specific Deamidation of RIG-I Reveals Collaborative Action between Viral and Cellular Deamidases in HSV-1 Lytic Replication.

Retinoic acid-inducible gene I (RIG-I) is a sensor that recognizes cytosolic double-stranded RNA derived from microbes to induce host immune response. Viruses, such as herpesviruses, deploy diverse mechanisms to derail RIG-I-dependent innate immune defense. In this study, we discovered that mouse RIG-I is intrinsically resistant to deamidation and evasion by herpes simplex virus 1 (HSV-1). Comparative studies involving human and mouse RIG-I indicate that N495 of human RIG-I dictates species-specific deamidation by HSV-1 UL37. Remarkably, deamidation of the other site, N549, hinges on that of N495, and it is catalyzed by cellular phosphoribosylpyrophosphate amidotransferase (PPAT). Specifically, deamidation of N495 enables RIG-I to interact with PPAT, leading to subsequent deamidation of N549. Collaboration between UL37 and PPAT is required for HSV-1 to evade RIG-I-mediated antiviral immune response. This work identifies an immune regulatory role of PPAT in innate host defense and establishes a sequential deamidation event catalyzed by distinct deamidases in immune evasion.IMPORTANCE Herpesviruses are ubiquitous pathogens in human and establish lifelong persistence despite host immunity. The ability to evade host immune response is pivotal for viral persistence and pathogenesis. In this study, we investigated the evasion, mediated by deamidation, of species-specific RIG-I by herpes simplex virus 1 (HSV-1). Our findings uncovered a collaborative and sequential action between viral deamidase UL37 and a cellular glutamine amidotransferase, phosphoribosylpyrophosphate amidotransferase (PPAT), to inactivate RIG-I and mute antiviral gene expression. PPAT catalyzes the rate-limiting step of the de novo purine synthesis pathway. This work describes a new function of cellular metabolic enzymes in host defense and viral immune evasion.

Human OAS1 activation is highly dependent on both RNA sequence and context of activating RNA motifs.

2′-5′-Oligoadenylate synthetases (OAS) are innate immune sensors of cytosolic double-stranded RNA (dsRNA) and play a critical role in limiting viral infection. dsRNA binding induces allosteric structural changes in OAS1 that reorganize its catalytic center to promote synthesis of 2′-5′-oligoadenylate and thus activation of endoribonuclease L. Specific RNA sequences and structural motifs can also enhance activation of OAS1 through currently undefined mechanisms. To better understand these drivers of OAS activation, we tested the impact of defined sequence changes within a short dsRNA that strongly activates OAS1. Both in vitro and in human A549 cells, appending a 3′-end single-stranded pyrimidine (3′-ssPy) can strongly enhance OAS1 activation or have no effect depending on its location, suggesting that other dsRNA features are necessary for correct presentation of the motif to OAS1. Consistent with this idea, we also find that the dsRNA binding position is dictated by an established consensus sequence (WWN9WG). Unexpectedly, however, not all sequences fitting this consensus activate OAS1 equivalently, with strong dependence on the identity of both partially conserved (W) and non-conserved (N9) residues. A picture thus emerges in which both specific RNA features and the context in which they are presented dictate the ability of short dsRNAs to activate OAS1.

ADAR1-Dependent RNA Editing Promotes MET and iPSC Reprogramming by Alleviating ER Stress.

RNA editing of adenosine to inosine (A to I) is catalyzed by ADAR1 and dramatically alters the cellular transcriptome, although its functional roles in somatic cell reprogramming are largely unexplored. Here, we show that loss of ADAR1-mediated A-to-I editing disrupts mesenchymal-to-epithelial transition (MET) during induced pluripotent stem cell (iPSC) reprogramming and impedes acquisition of induced pluripotency. Using chemical and genetic approaches, we show that absence of ADAR1-dependent RNA editinginducesaberrant innate immune responses through the double-stranded RNA (dsRNA) sensor MDA5, unleashing endoplasmic reticulum (ER) stress and hindering epithelial fate acquisition. We found that A-to-I editing impedes MDA5 sensing and sequestration of dsRNAs encoding membrane proteins, which promote ER homeostasis by activating the PERK-dependent unfolded protein response pathway to consequently facilitate MET. This study therefore establishes a critical role for ADAR1 and its A-to-I editing activity duringcell fate transitions and delineates a key regulatory layer underlying MET to control efficient reprogramming.

Generation of three isogenic induced Pluripotent Stem Cell lines (iPSCs) from fibroblasts of a patient with Aicardi Goutières Syndrome carrying a c.2471G>A dominant mutation in IFIH1 gene.

Aicardi-Goutières syndrome (AGS) is an early-onset monogenic encephalopathy characterized by intracranial calcification, leukodystrophy and cerebrospinal fluid lymphocytosis. To date, seven genes have been related to AGS. Among these, IFIH1 encodes for MDA5, a cytosolic double-stranded RNA receptor, and is responsible for AGS type 7. We generated three isogenic iPSC clones, using a Sendai virus-based vector, starting from fibroblasts of a patient carrying a dominant mutation in IFIH1. All lines were characterized for genomic integrity, genetic uniqueness, pluripotency, and differentiation capability. Our clones might offer a good model to investigate AGS7 pathophysiological mechanism and to discover new biomarkers for this condition treatment.

RNA regulation of the antiviral protein 2'-5'-oligoadenylate synthetase.

The innate immune system is a broad collection of critical intra- and extra-cellular processes that limit the infectivity of diverse pathogens. The 2'-5'-oligoadenylate synthetase (OAS) family of enzymes are important sensors of cytosolic double-stranded RNA (dsRNA) that play a critical role in limiting viral infection by activating the latent ribonuclease (RNase L) to halt viral replication and establish an antiviral state. Attesting to the importance of the OAS/RNase L pathway, diverse viruses have developed numerous distinct strategies to evade the effects of OAS activation. How OAS proteins are regulated by viral or cellular RNAs is not fully understood but several recent studies have provided important new insights into the molecular mechanisms of OAS activation by dsRNA. Other studies have revealed unanticipated features of RNA sequence and structure that strongly enhance activation of at least one OAS family member. While these discoveries represent important advances, they also underscore the fact that much remains to be learned about RNA-mediated regulation of the OAS/RNase L pathway. In particular, defining the full complement of RNA molecular signatures that activate OAS is essential to our understanding of how these proteins maximize their protective role against pathogens while still accurately discriminating host molecules to avoid inadvertent activation by cellular RNAs. A more complete knowledge of OAS regulation may also serve as a foundation for the development of novel antiviral therapeutic strategies and lead the way to a deeper understanding of currently unappreciated cellular functions of the OAS/RNase L pathway in the absence of infection. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications Translation > Translation Regulation.

Poly I:C Delivery into J774.1 and RAW264.7 Macrophages Induces Rapid Cell Death.

Cytosolic double-stranded RNA (dsRNA) is an important 'molecular signature' for the detection of intracellular viral infections. Although intracellular dsRNA is a known potent inducer of apoptosis, the optimal time and dose for the onset of dsRNA-mediated apoptosis have not been studied in detail. To perform an accurate temporal assessment of the cell death responses in dsRNA-dependent cytotoxicity. Poly I:C (PIC), a synthetic dsRNA molecule was delivered intracellularly into J774.1 and RAW264.7 murine macrophages via electroporation. Cell viability was measured using the MTT assay and apoptosis was determined by sub-G0/G1 DNA content using flow cytometry. Loss of cell viability was seen as early as 3h post-electroporation of macrophages. A significant increase in the sub-G0/G1 DNA content consistent with apoptosis was observed in PIC-electroporated macrophages as early as 3h post electroporation. Intracellular PIC delivery induces rapid macrophage cell death.

Musculoskeletal Disease in MDA5-Related Type I Interferonopathy: A Mendelian Mimic of Jaccoud's Arthropathy.

To define the molecular basis of a multisystem phenotype with progressive musculoskeletal disease of the hands and feet, including camptodactyly, subluxation, and tendon rupture, reminiscent of Jaccoud's arthropathy. We identified 2 families segregating an autosomal-dominant phenotype encompassing musculoskeletal disease and variable additional features, including psoriasis, dental abnormalities, cardiac valve involvement, glaucoma, and basal ganglia calcification. We measured the expression of interferon (IFN)-stimulated genes in the peripheral blood and skin, and undertook targeted Sanger sequencing of the IFIH1 gene encoding the cytosolic double-stranded RNA (dsRNA) sensor melanoma differentiation-associated protein 5 (MDA-5). We also assessed the functional consequences of IFIH1 gene variants using an in vitro IFNβ reporter assay in HEK 293T cells. We recorded an up-regulation of type I IFN-induced gene transcripts in all 5 patients tested and identified a heterozygous gain-of-function mutation in IFIH1 in each family, resulting in different substitutions of the threonine residue at position 331 of MDA-5. Both of these variants were associated with increased IFNβ expression in the absence of exogenous dsRNA ligand, consistent with constitutive activation of MDA-5. These cases highlight the significant musculoskeletal involvement that can be associated with mutations in MDA-5, and emphasize the value of testing for up-regulation of IFN signaling as a marker of the underlying molecular lesion. Our data indicate that both Singleton-Merten syndrome and neuroinflammation described in the context of MDA-5 gain-of-function constitute part of the same type I interferonopathy disease spectrum, and provide possible novel insight into the pathology of Jaccoud's arthropathy.

Growth inhibition by self-DNA: a phenomenon and its multiple explanations.

Growth inhibition by self-DNA: a phenomenon and its multiple explanations.