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1.
Front Immunol ; 13: 818023, 2022.
Article in English | MEDLINE | ID: covidwho-1674343

ABSTRACT

Alu retrotransposons belong to the class of short interspersed nuclear elements (SINEs). Alu RNA is abundant in cells and its repetitive structure forms double-stranded RNAs (dsRNA) that activate dsRNA sensors and trigger innate immune responses with significant pathological consequences. Mechanisms to prevent innate immune activation include deamination of adenosines to inosines in dsRNAs, referred to as A-to-I editing, degradation of Alu RNAs by endoribonucleases, and sequestration of Alu RNAs by RNA binding proteins. We have previously demonstrated that widespread loss of Alu RNA A-to-I editing is associated with diverse human diseases including viral (COVID-19, influenza) and autoimmune diseases (multiple sclerosis). Here we demonstrate loss of A-to-I editing in leukocytes is also associated with inflammatory bowel diseases. Our structure-function analysis demonstrates that ability to activate innate immune responses resides in the left arm of Alu RNA, requires a 5'-PPP, RIG-I is the major Alu dsRNA sensor, and A-to-I editing disrupts both structure and function. Further, edited Alu RNAs inhibit activity of unedited Alu RNAs. Altering Alu RNA nucleotide sequence increases biological activity. Two classes of Alu RNAs exist, one class stimulates both IRF and NF-kB transcriptional activity and a second class only stimulates IRF transcriptional activity. Thus, Alu RNAs play important roles in human disease but may also have therapeutic potential.


Subject(s)
Alu Elements/genetics , Alu Elements/immunology , Inflammatory Bowel Diseases/genetics , Inflammatory Bowel Diseases/immunology , Adenosine , COVID-19 , Humans , Inosine , RNA, Double-Stranded/genetics , RNA, Double-Stranded/immunology , SARS-CoV-2
2.
Genes (Basel) ; 13(1)2021 12 23.
Article in English | MEDLINE | ID: covidwho-1580896

ABSTRACT

ADAR1-mediated deamination of adenosines in long double-stranded RNAs plays an important role in modulating the innate immune response. However, recent investigations based on metatranscriptomic samples of COVID-19 patients and SARS-COV-2-infected Vero cells have recovered contrasting findings. Using RNAseq data from time course experiments of infected human cell lines and transcriptome data from Vero cells and clinical samples, we prove that A-to-G changes observed in SARS-COV-2 genomes represent genuine RNA editing events, likely mediated by ADAR1. While the A-to-I editing rate is generally low, changes are distributed along the entire viral genome, are overrepresented in exonic regions, and are (in the majority of cases) nonsynonymous. The impact of RNA editing on virus-host interactions could be relevant to identify potential targets for therapeutic interventions.


Subject(s)
Adenosine Deaminase/genetics , COVID-19/genetics , Genome, Viral , Host-Pathogen Interactions/genetics , RNA Editing , RNA, Viral/genetics , RNA-Binding Proteins/genetics , SARS-CoV-2/genetics , Adenosine/metabolism , Adenosine Deaminase/immunology , Animals , COVID-19/metabolism , COVID-19/virology , Cell Line, Tumor , Chlorocebus aethiops , DEAD Box Protein 58/genetics , DEAD Box Protein 58/immunology , Deamination , Epithelial Cells/immunology , Epithelial Cells/virology , Host-Pathogen Interactions/immunology , Humans , Immunity, Innate , Inosine/metabolism , Interferon-Induced Helicase, IFIH1/genetics , Interferon-Induced Helicase, IFIH1/immunology , Interferon-beta/genetics , Interferon-beta/immunology , RNA, Double-Stranded/genetics , RNA, Double-Stranded/immunology , RNA, Viral/immunology , RNA-Binding Proteins/immunology , Receptors, Immunologic/genetics , Receptors, Immunologic/immunology , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Transcriptome , Vero Cells
3.
Nat Commun ; 12(1): 6668, 2021 11 18.
Article in English | MEDLINE | ID: covidwho-1526076

ABSTRACT

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.


Subject(s)
Adenosine Triphosphate/metabolism , Inflammation/metabolism , Interferon-Induced Helicase, IFIH1/metabolism , Mutation, Missense , RNA, Double-Stranded/metabolism , RNA, Viral/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/ultrastructure , Cryoelectron Microscopy , HEK293 Cells , Humans , Immunity, Innate/genetics , Inflammation/genetics , Interferon-Induced Helicase, IFIH1/chemistry , Interferon-Induced Helicase, IFIH1/genetics , Models, Molecular , Nucleic Acid Conformation , Protein Binding , Protein Conformation , RNA, Double-Stranded/chemistry , RNA, Double-Stranded/genetics , RNA, Viral/genetics
4.
J Virol ; 95(12)2021 05 24.
Article in English | MEDLINE | ID: covidwho-1501541

ABSTRACT

Long disregarded as junk DNA or genomic dark matter, endogenous retroviruses (ERVs) have turned out to represent important components of the antiviral immune response. These remnants of once-infectious retroviruses not only regulate cellular immune activation, but may even directly target invading viral pathogens. In this Gem, we summarize mechanisms by which retroviral fossils protect us from viral infections. One focus will be on recent advances in the role of ERVs as regulators of antiviral gene expression.


Subject(s)
Endogenous Retroviruses/physiology , Retroelements , Virus Diseases/immunology , Animals , Endogenous Retroviruses/genetics , Enhancer Elements, Genetic , Gene Expression Regulation , Humans , Immunity, Cellular , Promoter Regions, Genetic , RNA, Double-Stranded/genetics , RNA, Double-Stranded/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Receptors, Pattern Recognition/metabolism , Receptors, Virus/antagonists & inhibitors , Receptors, Virus/metabolism , Viral Proteins/metabolism , Virion/metabolism , Virus Diseases/genetics , Virus Diseases/virology
5.
Science ; 374(6567): eabj3624, 2021 Oct 29.
Article in English | MEDLINE | ID: covidwho-1440797

ABSTRACT

Inherited genetic factors can influence the severity of COVID-19, but the molecular explanation underpinning a genetic association is often unclear. Intracellular antiviral defenses can inhibit the replication of viruses and reduce disease severity. To better understand the antiviral defenses relevant to COVID-19, we used interferon-stimulated gene (ISG) expression screening to reveal that 2'-5'-oligoadenylate synthetase 1 (OAS1), through ribonuclease L, potently inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We show that a common splice-acceptor single-nucleotide polymorphism (Rs10774671) governs whether patients express prenylated OAS1 isoforms that are membrane-associated and sense-specific regions of SARS-CoV-2 RNAs or if they only express cytosolic, nonprenylated OAS1 that does not efficiently detect SARS-CoV-2. In hospitalized patients, expression of prenylated OAS1 was associated with protection from severe COVID-19, suggesting that this antiviral defense is a major component of a protective antiviral response.


Subject(s)
2',5'-Oligoadenylate Synthetase/genetics , 2',5'-Oligoadenylate Synthetase/metabolism , COVID-19/genetics , COVID-19/physiopathology , RNA, Double-Stranded/metabolism , RNA, Viral/metabolism , SARS-CoV-2/physiology , 5' Untranslated Regions , A549 Cells , Animals , COVID-19/enzymology , COVID-19/immunology , Chiroptera/genetics , Chiroptera/virology , Coronaviridae/enzymology , Coronaviridae/genetics , Coronaviridae/physiology , Endoribonucleases/metabolism , Humans , Interferons/immunology , Isoenzymes/genetics , Isoenzymes/metabolism , Phosphoric Diester Hydrolases/genetics , Phosphoric Diester Hydrolases/metabolism , Polymorphism, Single Nucleotide , Protein Prenylation , RNA, Double-Stranded/chemistry , RNA, Double-Stranded/genetics , RNA, Viral/chemistry , RNA, Viral/genetics , Retroelements , SARS-CoV-2/genetics , Severity of Illness Index , Virus Replication
6.
Nucleic Acids Res ; 49(18): 10604-10617, 2021 10 11.
Article in English | MEDLINE | ID: covidwho-1406489

ABSTRACT

RNA hydrolysis presents problems in manufacturing, long-term storage, world-wide delivery and in vivo stability of messenger RNA (mRNA)-based vaccines and therapeutics. A largely unexplored strategy to reduce mRNA hydrolysis is to redesign RNAs to form double-stranded regions, which are protected from in-line cleavage and enzymatic degradation, while coding for the same proteins. The amount of stabilization that this strategy can deliver and the most effective algorithmic approach to achieve stabilization remain poorly understood. Here, we present simple calculations for estimating RNA stability against hydrolysis, and a model that links the average unpaired probability of an mRNA, or AUP, to its overall hydrolysis rate. To characterize the stabilization achievable through structure design, we compare AUP optimization by conventional mRNA design methods to results from more computationally sophisticated algorithms and crowdsourcing through the OpenVaccine challenge on the Eterna platform. We find that rational design on Eterna and the more sophisticated algorithms lead to constructs with low AUP, which we term 'superfolder' mRNAs. These designs exhibit a wide diversity of sequence and structure features that may be desirable for translation, biophysical size, and immunogenicity. Furthermore, their folding is robust to temperature, computer modeling method, choice of flanking untranslated regions, and changes in target protein sequence, as illustrated by rapid redesign of superfolder mRNAs for B.1.351, P.1 and B.1.1.7 variants of the prefusion-stabilized SARS-CoV-2 spike protein. Increases in in vitro mRNA half-life by at least two-fold appear immediately achievable.


Subject(s)
Algorithms , RNA, Double-Stranded/chemistry , RNA, Messenger/chemistry , RNA, Viral/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Base Pairing , Base Sequence , COVID-19/prevention & control , Humans , Hydrolysis , RNA Stability , RNA, Double-Stranded/genetics , RNA, Double-Stranded/immunology , RNA, Messenger/genetics , RNA, Messenger/immunology , RNA, Viral/genetics , RNA, Viral/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Thermodynamics
7.
Mol Ther ; 29(7): 2219-2226, 2021 07 07.
Article in English | MEDLINE | ID: covidwho-1228174

ABSTRACT

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans. Despite several emerging vaccines, there remains no verifiable therapeutic targeted specifically to the virus. Here we present a highly effective small interfering RNA (siRNA) therapeutic against SARS-CoV-2 infection using a novel lipid nanoparticle (LNP) delivery system. Multiple siRNAs targeting highly conserved regions of the SARS-CoV-2 virus were screened, and three candidate siRNAs emerged that effectively inhibit the virus by greater than 90% either alone or in combination with one another. We simultaneously developed and screened two novel LNP formulations for the delivery of these candidate siRNA therapeutics to the lungs, an organ that incurs immense damage during SARS-CoV-2 infection. Encapsulation of siRNAs in these LNPs followed by in vivo injection demonstrated robust repression of virus in the lungs and a pronounced survival advantage to the treated mice. Our LNP-siRNA approaches are scalable and can be administered upon the first sign of SARS-CoV-2 infection in humans. We suggest that an siRNA-LNP therapeutic approach could prove highly useful in treating COVID-19 disease as an adjunctive therapy to current vaccine strategies.


Subject(s)
COVID-19/drug therapy , Drug Delivery Systems/methods , Lipids/chemistry , Nanoparticles/chemistry , RNA, Double-Stranded/administration & dosage , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , SARS-CoV-2/genetics , Administration, Intravenous , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19/metabolism , COVID-19/virology , Female , Gene Silencing , HEK293 Cells , Humans , Lung/metabolism , Male , Mice , Mice, Transgenic , RNA, Double-Stranded/genetics , RNA, Viral/genetics , Transcriptome/drug effects , Treatment Outcome
8.
Vascul Pharmacol ; 140: 106861, 2021 10.
Article in English | MEDLINE | ID: covidwho-1180098

ABSTRACT

The virus responsible for the coronavirus disease of 2019 (COVID-19) is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Evidences suggest that COVID-19 could trigger cardiovascular complications in apparently healthy patients. Coronaviruses are enveloped positive-strand RNA viruses acting as a pathogen-associated molecular pattern (PAMP)/ danger-associated molecular patterns (DAMP). Interestingly, Toll-like receptor (TLR) 3 recognize both PAMPs DAMPs and is activated by viral double-stranded RNA (dsRNA) leading to activation of TIR receptor domain-containing adaptor inducing IFN-ß (TRIF) dependent pathway. New evidence has shown a link between virus dsRNA and increased BP. Hence, we hypothesize that COVID-19 infection may be over activating the TLR3 through dsRNA, evoking further damage to the patients, leading to vascular inflammation and increased blood pressure, favoring the development of several cardiovascular complications, including hypertension.


Subject(s)
COVID-19/genetics , COVID-19/pathology , Hypertension/genetics , RNA, Double-Stranded/genetics , Toll-Like Receptor 3/genetics , Animals , Humans , Hypertension/pathology , Hypertension/virology , Mice , SARS-CoV-2/pathogenicity , Signal Transduction/genetics
9.
J Immunol ; 206(8): 1691-1696, 2021 04 15.
Article in English | MEDLINE | ID: covidwho-1158408

ABSTRACT

Severe COVID-19 disease is associated with elevated inflammatory responses. One form of Aicardi-Goutières syndrome caused by inactivating mutations in ADAR results in reduced adenosine-to-inosine (A-to-I) editing of endogenous dsRNAs, induction of IFNs, IFN-stimulated genes, other inflammatory mediators, morbidity, and mortality. Alu elements, ∼10% of the human genome, are the most common A-to-I-editing sites. Using leukocyte whole-genome RNA-sequencing data, we found reduced A-to-I editing of Alu dsRNAs in patients with severe COVID-19 disease. Dendritic cells infected with COVID-19 also exhibit reduced A-to-I editing of Alu dsRNAs. Unedited Alu dsRNAs, but not edited Alu dsRNAs, are potent inducers of IRF and NF-κB transcriptional responses, IL6, IL8, and IFN-stimulated genes. Thus, decreased A-to-I editing that may lead to accumulation of unedited Alu dsRNAs and increased inflammatory responses is associated with severe COVID-19 disease.


Subject(s)
Adenosine/genetics , Alu Elements/genetics , COVID-19/genetics , Inosine/genetics , RNA Editing/genetics , RNA, Double-Stranded/genetics , SARS-CoV-2 , Severity of Illness Index , Adenosine/metabolism , COVID-19/pathology , Dendritic Cells/metabolism , Dendritic Cells/virology , Genome, Human , Humans , Inosine/metabolism , Interferon Regulatory Factors/metabolism , NF-kappa B/metabolism , RNA-Seq , Signal Transduction/genetics
10.
J Virol ; 94(15)2020 07 16.
Article in English | MEDLINE | ID: covidwho-831394

ABSTRACT

Currently, an effective therapeutic treatment for porcine reproductive and respiratory syndrome virus (PRRSV) remains elusive. PRRSV helicase nsp10 is an important component of the replication transcription complex that plays a crucial role in viral replication, making nsp10 an important target for drug development. Here, we report the first crystal structure of full-length nsp10 from the arterivirus PRRSV, which has multiple domains: an N-terminal zinc-binding domain (ZBD), a 1B domain, and helicase core domains 1A and 2A. Importantly, our structural analyses indicate that the conformation of the 1B domain from arterivirus nsp10 undergoes a dynamic transition. The polynucleotide substrate channel formed by domains 1A and 1B adopts an open state, which may create enough space to accommodate and bind double-stranded RNA (dsRNA) during unwinding. Moreover, we report a unique C-terminal domain structure that participates in stabilizing the overall helicase structure. Our biochemical experiments also showed that deletion of the 1B domain and C-terminal domain significantly reduced the helicase activity of nsp10, indicating that the four domains must cooperate to contribute to helicase function. In addition, our results indicate that nidoviruses contain a conserved helicase core domain and key amino acid sites affecting helicase function, which share a common mechanism of helicase translocation and unwinding activity. These findings will help to further our understanding of the mechanism of helicase function and provide new targets for the development of antiviral drugs.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is a major respiratory disease agent in pigs that causes enormous economic losses to the global swine industry. PRRSV helicase nsp10 is a multifunctional protein with translocation and unwinding activities and plays a vital role in viral RNA synthesis. Here, we report the first structure of full-length nsp10 from the arterivirus PRRSV at 3.0-Å resolution. Our results show that the 1B domain of PRRSV nsp10 adopts a novel open state and has a unique C-terminal domain structure, which plays a crucial role in nsp10 helicase activity. Furthermore, mutagenesis and structural analysis revealed conservation of the helicase catalytic domain across the order Nidovirales (families Arteriviridae and Coronaviridae). Importantly, our results will provide a structural basis for further understanding the function of helicases in the order Nidovirales.


Subject(s)
Porcine respiratory and reproductive syndrome virus/enzymology , RNA Helicases/chemistry , RNA, Double-Stranded/chemistry , RNA, Viral/chemistry , Viral Proteins/chemistry , Porcine respiratory and reproductive syndrome virus/genetics , Protein Domains , RNA Helicases/genetics , RNA, Double-Stranded/genetics , RNA, Viral/genetics , Viral Proteins/genetics
11.
Biomacromolecules ; 21(6): 2440-2454, 2020 06 08.
Article in English | MEDLINE | ID: covidwho-23562

ABSTRACT

Reactive poly(pentafluorophenyl acrylate) (PPFPA)-grafted surfaces offer a versatile platform to immobilize biomolecules. Here, we utilize PPFPA-grafted surface and double-stranded RNA (dsRNA) recognizing J2 antibody to construct a universal virus detection platform with enhanced sensitivity. PPFPA on silicon substrates is prepared, and surface hydrophilicity is modulated by partial substitution of the pentafluorophenyl units with poly(ethylene glycol). Following dsRNA antibody immobilization, the prepared surfaces can distinguish long dsRNAs from single-stranded RNAs of the same length and short dsRNAs. As long dsRNAs are common byproducts of viral transcription/replication, these surfaces can detect the presence of different kinds of viruses without prior knowledge of their genomic sequences. To increase dsRNA detection sensitivity, a two-step method is devised where the captured dsRNAs are visualized with multiple fluorophore-tagged J2 antibodies. We show that the developed platform can differentiate foreign long dsRNAs from cellular dsRNAs and other biomolecules present in the cell lysate. Moreover, when tested against cells infected with hepatitis A or C viruses, both viruses are successfully detected using a single platform. Our study shows that the developed PPFPA platform immobilized with J2 antibody can serve as a primary diagnostic tool to determine the infection status for a wide range of viruses.


Subject(s)
Polymers , RNA, Double-Stranded , RNA, Double-Stranded/genetics
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