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1.
Am J Physiol Cell Physiol ; 322(4): C787-C793, 2022 Apr 01.
Article in English | MEDLINE | ID: covidwho-1807579

ABSTRACT

Similar to epigenetic DNA modification, RNA can be methylated and altered for stability and processing. RNA modifications, namely, epitranscriptomes, involve the following three functions: writing, erasing, and reading of marks. Methods for measurement and position detection are useful for the assessment of cellular function and human disease biomarkers. After pyrimidine 5-methylcytosine was reported for the first time a hundred years ago, numerous techniques have been developed for studying nucleotide modifications, including RNAs. Recent studies have focused on high-throughput and direct measurements for investigating the precise function of epitranscriptomes, including the characterization of severe acute respiratory syndrome coronavirus 2. The current study presents an overview of the development of detection techniques for epitranscriptomic marks and briefs about the recent progress in this field.


Subject(s)
COVID-19 , Transcriptome , Epigenesis, Genetic , Humans , RNA/genetics , RNA/metabolism , RNA Processing, Post-Transcriptional , Transcriptome/genetics
2.
ACS Synth Biol ; 11(4): 1389-1396, 2022 04 15.
Article in English | MEDLINE | ID: covidwho-1773921

ABSTRACT

We present a cell-free assay for rapid screening of candidate inhibitors of protein binding, focusing on inhibition of the interaction between the SARS-CoV-2 Spike receptor binding domain (RBD) and human angiotensin-converting enzyme 2 (hACE2). The assay has two components: fluorescent polystyrene particles covalently coated with RBD, termed virion-particles (v-particles), and fluorescently labeled hACE2 (hACE2F) that binds the v-particles. When incubated with an inhibitor, v-particle-hACE2F binding is diminished, resulting in a reduction in the fluorescent signal of bound hACE2F relative to the noninhibitor control, which can be measured via flow cytometry or fluorescence microscopy. We determine the amount of RBD needed for v-particle preparation, v-particle incubation time with hACE2F, hACE2F detection limit, and specificity of v-particle binding to hACE2F. We measure the dose response of the v-particles to known inhibitors. Finally, utilizing an RNA-binding protein tdPP7 incorporated into hACE2F, we demonstrate that RNA-hACE2F granules trap v-particles effectively, providing a basis for potential RNA-hACE2F therapeutics.


Subject(s)
Angiotensin-Converting Enzyme 2 , Antiviral Agents , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Antiviral Agents/pharmacology , COVID-19 , Humans , Protein Binding , RNA/metabolism , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/antagonists & inhibitors
3.
PLoS Genet ; 18(3): e1010130, 2022 03.
Article in English | MEDLINE | ID: covidwho-1770640

ABSTRACT

SARS-CoV-2 is a positive-sense, single-stranded RNA virus responsible for the COVID-19 pandemic. It remains unclear whether and to what extent the virus in human host cells undergoes RNA editing, a major RNA modification mechanism. Here we perform a robust bioinformatic analysis of metatranscriptomic data from multiple bronchoalveolar lavage fluid samples of COVID-19 patients, revealing an appreciable number of A-to-I RNA editing candidate sites in SARS-CoV-2. We confirm the enrichment of A-to-I RNA editing signals at these candidate sites through evaluating four characteristics specific to RNA editing: the inferred RNA editing sites exhibit (i) stronger ADAR1 binding affinity predicted by a deep-learning model built from ADAR1 CLIP-seq data, (ii) decreased editing levels in ADAR1-inhibited human lung cells, (iii) local clustering patterns, and (iv) higher RNA secondary structure propensity. Our results have critical implications in understanding the evolution of SARS-CoV-2 as well as in COVID-19 research, such as phylogenetic analysis and vaccine development.


Subject(s)
COVID-19 , SARS-CoV-2 , Adenosine Deaminase/metabolism , COVID-19/genetics , Humans , Nucleotides/metabolism , Pandemics , Phylogeny , RNA/metabolism , RNA Editing/genetics , SARS-CoV-2/genetics
4.
Biomolecules ; 12(3)2022 03 13.
Article in English | MEDLINE | ID: covidwho-1760348

ABSTRACT

Cellular functions are regulated through the gene expression program by the transcription of new messenger RNAs (mRNAs), alternative RNA splicing, and protein synthesis. To this end, the post-translational modifications (PTMs) of proteins add another layer of complexity, creating a continuously fine-tuned regulatory network. ADP-ribosylation (ADPr) is an ancient reversible modification of cellular macromolecules, regulating a multitude of key functional processes as diverse as DNA damage repair (DDR), transcriptional regulation, intracellular transport, immune and stress responses, and cell survival. Additionally, due to the emerging role of ADP-ribosylation in pathological processes, ADP-ribosyltransferases (ARTs), the enzymes involved in ADPr, are attracting growing interest as new drug targets. In this review, an overview of human ARTs and their related biological functions is provided, mainly focusing on the regulation of ADP-ribosyltransferase Diphtheria toxin-like enzymes (ARTD)-dependent RNA functions. Finally, in order to unravel novel gene functional relationships, we propose the analysis of an inventory of human gene clusters, including ARTDs, which share conserved sequences at 3' untranslated regions (UTRs).


Subject(s)
ADP-Ribosylation , RNA , ADP Ribose Transferases/genetics , Biology , Humans , Protein Processing, Post-Translational , RNA/metabolism
5.
Elife ; 112022 01 21.
Article in English | MEDLINE | ID: covidwho-1716085

ABSTRACT

Methyltransferase like-3 (METTL3) and METTL14 complex transfers a methyl group from S-adenosyl-L-methionine to N6 amino group of adenosine bases in RNA (m6A) and DNA (m6dA). Emerging evidence highlights a role of METTL3-METTL14 in the chromatin context, especially in processes where DNA and RNA are held in close proximity. However, a mechanistic framework about specificity for substrate RNA/DNA and their interrelationship remain unclear. By systematically studying methylation activity and binding affinity to a number of DNA and RNA oligos with different propensities to form inter- or intra-molecular duplexes or single-stranded molecules in vitro, we uncover an inverse relationship for substrate binding and methylation and show that METTL3-METTL14 preferentially catalyzes the formation of m6dA in single-stranded DNA (ssDNA), despite weaker binding affinity to DNA. In contrast, it binds structured RNAs with high affinity, but methylates the target adenosine in RNA (m6A) much less efficiently than it does in ssDNA. We also show that METTL3-METTL14-mediated methylation of DNA is largely restricted by structured RNA elements prevalent in long noncoding and other cellular RNAs.


Subject(s)
DNA Methylation/physiology , Methyltransferases/metabolism , DNA, Single-Stranded/metabolism , Deoxyadenosines/metabolism , Humans , RNA/chemistry , RNA/metabolism
6.
Biochem Biophys Res Commun ; 601: 129-136, 2022 04 23.
Article in English | MEDLINE | ID: covidwho-1699331

ABSTRACT

COVID-19, caused by SARS-CoV-2, has been spreading worldwide for more than two years and has led to immense challenges to human health. Despite the great efforts that have been made, our understanding of SARS-CoV-2 is still limited. The viral helicase, NSP13 is an important enzyme involved in SARS-CoV-2 replication and transcription. Here we highlight the important role of the stalk domain in the enzymatic activity of NSP13. Without the stalk domain, NSP13 loses its dsRNA unwinding ability due to the lack of ATPase activity. The stalk domain of NSP13 also provides a rigid connection between the ZBD and helicase domain. We found that the tight connection between the stalk and helicase is necessary for NSP13-mediated dsRNA unwinding. When a short flexible linker was inserted between the stalk and helicase domains, the helicase activity of NSP13 was impaired, although its ATPase activity remained intact. Further study demonstrated that linker insertion between the stalk and helicase domains attenuated the RNA binding ability and affected the thermal stability of NSP13. In summary, our results suggest the crucial role of the stalk domain in NSP13 enzymatic activity and provide mechanistic insight into dsRNA unwinding by SARS-CoV-2 NSP13.


Subject(s)
COVID-19/prevention & control , Methyltransferases/metabolism , RNA Helicases/metabolism , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Binding Sites/genetics , COVID-19/virology , Enzyme Stability , Humans , Methyltransferases/chemistry , Methyltransferases/genetics , Models, Molecular , Mutation , Protein Conformation , RNA/chemistry , RNA/genetics , RNA/metabolism , RNA Helicases/chemistry , RNA Helicases/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Temperature , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics
7.
Cells ; 11(3)2022 01 30.
Article in English | MEDLINE | ID: covidwho-1667057

ABSTRACT

The global outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still ongoing, as is research on the molecular mechanisms underlying cellular infection by coronaviruses, with the hope of developing therapeutic agents against this pandemic. Other important respiratory viruses such as 2009 pandemic H1N1 and H7N9 avian influenza virus (AIV), influenza A viruses, are also responsible for a possible outbreak due to their respiratory susceptibility. However, the interaction of these viruses with host cells and the regulation of post-transcriptional genes remains unclear. In this study, we detected and analyzed the comparative transcriptome profiling of SARS-CoV-2, panH1N1 (A/California/07/2009), and H7N9 (A/Shanghai/1/2013) infected cells. The results showed that the commonly upregulated genes among the three groups were mainly involved in autophagy, pertussis, and tuberculosis, which indicated that autophagy plays an important role in viral pathogenicity. There are three groups of commonly downregulated genes involved in metabolic pathways. Notably, unlike panH1N1 and H7N9, SARS-CoV-2 infection can inhibit the m-TOR pathway and activate the p53 signaling pathway, which may be responsible for unique autophagy induction and cell apoptosis. Particularly, upregulated expression of IRF1 was found in SARS-CoV-2, panH1N1, and H7N9 infection. Further analysis showed SARS-CoV-2, panH1N1, and H7N9 infection-induced upregulation of lncRNA-34087.27 could serve as a competitive endogenous RNA to stabilize IRF1 mRNA by competitively binding with miR-302b-3p. This study provides new insights into the molecular mechanisms of influenza A virus and SARS-CoV-2 infection.


Subject(s)
COVID-19/immunology , Immunity/immunology , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H7N9 Subtype/immunology , Influenza, Human/immunology , RNA/immunology , Transcriptome/immunology , A549 Cells , Animals , COVID-19/genetics , COVID-19/virology , HEK293 Cells , Host-Pathogen Interactions/immunology , Humans , Immunity/genetics , Influenza A Virus, H1N1 Subtype/physiology , Influenza A Virus, H7N9 Subtype/physiology , Influenza, Human/genetics , Influenza, Human/virology , Interferon Regulatory Factor-1/genetics , Interferon Regulatory Factor-1/immunology , Interferon Regulatory Factor-1/metabolism , MicroRNAs/genetics , MicroRNAs/immunology , MicroRNAs/metabolism , Pandemics/prevention & control , RNA/genetics , RNA/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/immunology , RNA, Long Noncoding/metabolism , RNA, Messenger/genetics , RNA, Messenger/immunology , RNA, Messenger/metabolism , RNA-Seq/methods , SARS-CoV-2/physiology , Signal Transduction/genetics , Signal Transduction/immunology , Transcriptome/genetics
8.
Elife ; 112022 01 20.
Article in English | MEDLINE | ID: covidwho-1634530

ABSTRACT

Despite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single-molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, subgenomic RNAs, and viral proteins. Our absolute quantification of viral RNAs and replication factories revealed that SARS-CoV-2 genomic RNA is long-lived after entry, suggesting that it avoids degradation by cellular nucleases. Moreover, we observed that SARS-CoV-2 replication is highly variable between cells, with only a small cell population displaying high burden of viral RNA. Unexpectedly, the B.1.1.7 variant, first identified in the UK, exhibits significantly slower replication kinetics than the Victoria strain, suggesting a novel mechanism contributing to its higher transmissibility with important clinical implications.


Subject(s)
COVID-19/virology , RNA, Viral/metabolism , SARS-CoV-2/pathogenicity , Animals , Chlorocebus aethiops/genetics , RNA/metabolism , RNA, Viral/genetics , SARS-CoV-2/genetics , Vero Cells , Viral Proteins/metabolism , Virus Replication/physiology
9.
Int J Biol Macromol ; 203: 466-480, 2022 Apr 01.
Article in English | MEDLINE | ID: covidwho-1630871

ABSTRACT

The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory sequences (TRS). Here, we dissect the structural features of the N protein N-terminal domain (N-NTD) and N-NTD plus the SR-rich motif (N-NTD-SR) upon binding to single and double-stranded TRS DNA, as well as their activities for dsTRS melting and TRS-induced liquid-liquid phase separation (LLPS). Our study gives insights on the specificity for N-NTD(-SR) interaction with TRS. We observed an approximation of the triple-thymidine (TTT) motif of the TRS to ß-sheet II, giving rise to an orientation difference of ~25° between dsTRS and non-specific sequence (dsNS). It led to a local unfavorable energetic contribution that might trigger the melting activity. The thermodynamic parameters of binding of ssTRSs and dsTRS suggested that the duplex dissociation of the dsTRS in the binding cleft is entropically favorable. We showed a preference for TRS in the formation of liquid condensates when compared to NS. Moreover, our results on DNA binding may serve as a starting point for the design of inhibitors, including aptamers, against N, a possible therapeutic target essential for the virus infectivity.


Subject(s)
COVID-19/virology , Nucleic Acids/metabolism , Nucleocapsid Proteins/metabolism , Protein Interaction Domains and Motifs , SARS-CoV-2/physiology , Binding Sites , DNA/chemistry , DNA/metabolism , Gene Expression Regulation, Viral , Host-Pathogen Interactions , Humans , Hydrogen Bonding , Models, Molecular , Nucleic Acids/chemistry , Nucleocapsid Proteins/chemistry , Protein Binding , RNA/chemistry , RNA/metabolism , Spectrum Analysis , Structure-Activity Relationship
10.
Sci Rep ; 12(1): 364, 2022 01 10.
Article in English | MEDLINE | ID: covidwho-1617003

ABSTRACT

RNA-binding proteins (RBPs) interact with and determine the fate of many cellular RNAs directing numerous essential roles in cellular physiology. Nuclear Factor 90 (NF90) is an RBP encoded by the interleukin enhancer-binding factor 3 (ILF3) gene that has been found to influence RNA metabolism at several levels, including pre-RNA splicing, mRNA turnover, and translation. To systematically identify the RNAs that interact with NF90, we carried out iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation) analysis in the human embryonic fibroblast cell line HEK-293. Interestingly, many of the identified RNAs encoded proteins involved in the response to viral infection and RNA metabolism. We validated a subset of targets and investigated the impact of NF90 on their expression levels. Two of the top targets, IRF3 and IRF9 mRNAs, encode the proteins IRF3 and IRF9, crucial regulators of the interferon pathway involved in the SARS-CoV-2 immune response. Our results support a role for NF90 in modulating key genes implicated in the immune response and offer insight into the immunological response to the SARS-CoV-2 infection.


Subject(s)
COVID-19/metabolism , Immunoprecipitation/methods , Nuclear Factor 90 Proteins/metabolism , RNA-Binding Proteins/metabolism , RNA/metabolism , SARS-CoV-2/metabolism , COVID-19/virology , Cells, Cultured , HEK293 Cells , Humans , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Interferon-Stimulated Gene Factor 3, gamma Subunit/genetics , Interferon-Stimulated Gene Factor 3, gamma Subunit/metabolism , Nuclear Factor 90 Proteins/genetics , Protein Binding , RNA/genetics , RNA Interference , RNA Stability , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Seq/methods , SARS-CoV-2/genetics , SARS-CoV-2/physiology
11.
Life Sci Alliance ; 5(3)2022 03.
Article in English | MEDLINE | ID: covidwho-1552086

ABSTRACT

Murine neural stem cells (NSCs) were recently shown to release piRNA-containing exosomes/microvesicles (Ex/Mv) for exerting antiviral immunity, but it remains unknown if these Ex/Mv could target SARS-CoV-2 and whether the PIWI-piRNA system is important for these antiviral actions. Here, using in vitro infection models, we show that hypothalamic NSCs (htNSCs) Ex/Mv provided an innate immunity protection against SARS-CoV-2. Importantly, enhanced antiviral actions were achieved by using induced Ex/Mv that were derived from induced htNSCs through twice being exposed to several RNA fragments of SARS-CoV-2 genome, a process that was designed not to involve protein translation of these RNA fragments. The increased antiviral effects of these induced Ex/Mv were associated with increased expression of piRNA species some of which could predictably target SARS-CoV-2 genome. Knockout of piRNA-interacting protein PIWIL2 in htNSCs led to reductions in both innate and induced antiviral effects of Ex/Mv in targeting SARS-CoV-2. Taken together, this study demonstrates a case suggesting Ex/Mv from certain cell types have innate and adaptive immunity against SARS-CoV-2, and the PIWI-piRNA system is important for these antiviral actions.


Subject(s)
Argonaute Proteins/metabolism , COVID-19/immunology , COVID-19/metabolism , Cell-Derived Microparticles/metabolism , Exosomes , RNA, Small Interfering/metabolism , RNA/metabolism , SARS-CoV-2 , A549 Cells , Angiotensin-Converting Enzyme 2/metabolism , Animals , Genome, Viral , Humans , Hypothalamus/metabolism , Immune System , Immunity, Innate , In Vitro Techniques , Mice
12.
J Exp Med ; 219(1)2022 01 03.
Article in English | MEDLINE | ID: covidwho-1510855

ABSTRACT

As SARS-CoV-2 continues to cause morbidity and mortality around the world, there is an urgent need for the development of effective medical countermeasures. Here, we assessed the antiviral capacity of a minimal RIG-I agonist, stem-loop RNA 14 (SLR14), in viral control, disease prevention, post-infection therapy, and cross-variant protection in mouse models of SARS-CoV-2 infection. A single dose of SLR14 prevented viral infection in the lower respiratory tract and development of severe disease in a type I interferon (IFN-I)-dependent manner. SLR14 demonstrated remarkable prophylactic protective capacity against lethal SARS-CoV-2 infection and retained considerable efficacy as a therapeutic agent. In immunodeficient mice carrying chronic SARS-CoV-2 infection, SLR14 elicited near-sterilizing innate immunity in the absence of the adaptive immune system. In the context of infection with variants of concern (VOCs), SLR14 conferred broad protection against emerging VOCs. These findings demonstrate the therapeutic potential of SLR14 as a host-directed, broad-spectrum antiviral for early post-exposure treatment and treatment of chronically infected immunosuppressed patients.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , RNA/metabolism , SARS-CoV-2/drug effects , Animals , COVID-19/metabolism , Disease Models, Animal , Immunity, Innate/drug effects , Interferon Type I/metabolism , Mice , Mice, Inbred BALB C
13.
Biomolecules ; 11(11)2021 10 20.
Article in English | MEDLINE | ID: covidwho-1480577

ABSTRACT

SARS-CoV-2 contains certain molecules that are related to the presence of immunothrombosis. Here, we review the pathogen and damage-associated molecular patterns. We also study the imbalance of different molecules participating in immunothrombosis, such as tissue factor, factors of the contact system, histones, and the role of cells, such as endothelial cells, platelets, and neutrophil extracellular traps. Regarding the pathogenetic mechanism, we discuss clinical trials, case-control studies, comparative and translational studies, and observational studies of regulatory or inhibitory molecules, more specifically, extracellular DNA and RNA, histones, sensors for RNA and DNA, as well as heparin and heparinoids. Overall, it appears that a network of cells and molecules identified in this axis is simultaneously but differentially affecting patients at different stages of COVID-19, and this is characterized by endothelial damage, microthrombosis, and inflammation.


Subject(s)
Alarmins , COVID-19/virology , SARS-CoV-2 , Thrombosis/virology , Angiotensin-Converting Enzyme 2/metabolism , Animals , Blood Coagulation , Blood Platelets/virology , COVID-19/complications , DNA/metabolism , Extracellular Traps , Heparin/metabolism , Histones/metabolism , Humans , Mice , Neuropilin-1/metabolism , RNA/metabolism , Signal Transduction , Thrombin/metabolism , Thromboplastin/metabolism , Thrombosis/complications
14.
Phys Chem Chem Phys ; 23(40): 22957-22971, 2021 Oct 20.
Article in English | MEDLINE | ID: covidwho-1462045

ABSTRACT

The identification of chemical compounds able to bind specific sites of the human/viral proteins involved in the SARS-CoV-2 infection cycle is a prerequisite to design effective antiviral drugs. Here we conduct a molecular dynamics study with the aim to assess the interactions of ivermectin, an antiparasitic drug with broad-spectrum antiviral activity, with the human Angiotensin-Converting Enzyme 2 (ACE2), the viral 3CLpro and PLpro proteases, and the viral SARS Unique Domain (SUD). The drug/target interactions have been characterized in silico by describing the nature of the non-covalent interactions found and by measuring the extent of their time duration along the MD simulation. Results reveal that the ACE2 protein and the ACE2/RBD aggregates form the most persistent interactions with ivermectin, while the binding with the remaining viral proteins is more limited and unspecific.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/metabolism , Coronavirus 3C Proteases/metabolism , Coronavirus Papain-Like Proteases/metabolism , Ivermectin/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Antiviral Agents/chemistry , Binding Sites , Coronavirus 3C Proteases/chemistry , Coronavirus Papain-Like Proteases/chemistry , G-Quadruplexes , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Ivermectin/chemistry , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding , Protein Domains , RNA/genetics , RNA/metabolism , SARS-CoV-2
16.
Biochemistry ; 60(24): 1869-1875, 2021 06 22.
Article in English | MEDLINE | ID: covidwho-1387102

ABSTRACT

Remdesivir is an antiviral drug initially designed against the Ebola virus. The results obtained with it both in biochemical studies in vitro and in cell line assays in vivo were very promising, but it proved to be ineffective in clinical trials. Remdesivir exhibited far better efficacy when repurposed against SARS-CoV-2. The chemistry that accounts for this difference is the subject of this study. Here, we examine the hypothesis that remdesivir monophosphate (RMP)-containing RNA functions as a template at the polymerase site for the second run of RNA synthesis, and as mRNA at the decoding center for protein synthesis. Our hypothesis is supported by the observation that RMP can be incorporated into RNA by the RNA-dependent RNA polymerases (RdRps) of both viruses, although some of the incorporated RMPs are subsequently removed by exoribonucleases. Furthermore, our hypothesis is consistent with the fact that RdRp of SARS-CoV-2 selects RMP for incorporation over AMP by 3-fold in vitro, and that RMP-added RNA can be rapidly extended, even though primer extension is often paused when the added RMP is translocated at the i + 3 position (with i the nascent base pair at an initial insertion site of RMP) or when the concentrations of the subsequent nucleoside triphosphates (NTPs) are below their physiological concentrations. These observations have led to the hypothesis that remdesivir might be a delayed chain terminator. However, that hypothesis is challenged under physiological concentrations of NTPs by the observation that approximately three-quarters of RNA products efficiently overrun the pause.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Coronavirus RNA-Dependent RNA Polymerase/genetics , Ebolavirus/drug effects , SARS-CoV-2/drug effects , Virus Replication/drug effects , Adenosine Monophosphate/genetics , Adenosine Monophosphate/metabolism , Alanine/genetics , Alanine/metabolism , Antiviral Agents/metabolism , Base Pairing , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Enzyme Inhibitors/metabolism , Models, Molecular , Protein Biosynthesis/drug effects , RNA/genetics , RNA/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism
17.
J Biol Chem ; 297(3): 101018, 2021 09.
Article in English | MEDLINE | ID: covidwho-1380706

ABSTRACT

The coronaviral nonstructural protein 9 (Nsp9) is essential for viral replication; it is the primary substrate of Nsp12's pseudokinase domain within the viral replication transcription complex, an association that also recruits other components during different stages of RNA reproduction. In the unmodified state, Nsp9 forms an obligate homodimer via an essential GxxxG protein-interaction motif, but its ssRNA-binding mechanism remains unknown. Using structural biological techniques, here we show that a base-mimicking compound identified from a small molecule fragment screen engages Nsp9 via a tetrameric Pi-Pi stacking interaction that induces the formation of a parallel trimer-of-dimers. This oligomerization mechanism allows an interchange of "latching" N-termini, the charges of which contribute to a series of electropositive channels that suggests a potential interface for viral RNA. The identified pyrrolo-pyrimidine compound may also serve as a potential starting point for the development of compounds seeking to probe Nsp9's role within SARS-CoV-2 replication.


Subject(s)
COVID-19/virology , Pyrimidine Nucleotides/metabolism , RNA-Binding Proteins/metabolism , SARS-CoV-2/metabolism , Viral Proteins/metabolism , Nuclear Magnetic Resonance, Biomolecular/methods , Protein Binding , RNA/metabolism , SARS-CoV-2/physiology , Virus Replication
18.
Nat Commun ; 12(1): 5152, 2021 08 26.
Article in English | MEDLINE | ID: covidwho-1376195

ABSTRACT

The immunological features that distinguish COVID-19-associated acute respiratory distress syndrome (ARDS) from other causes of ARDS are incompletely understood. Here, we report the results of comparative lower respiratory tract transcriptional profiling of tracheal aspirate from 52 critically ill patients with ARDS from COVID-19 or from other etiologies, as well as controls without ARDS. In contrast to a "cytokine storm," we observe reduced proinflammatory gene expression in COVID-19 ARDS when compared to ARDS due to other causes. COVID-19 ARDS is characterized by a dysregulated host response with increased PTEN signaling and elevated expression of genes with non-canonical roles in inflammation and immunity. In silico analysis of gene expression identifies several candidate drugs that may modulate gene expression in COVID-19 ARDS, including dexamethasone and granulocyte colony stimulating factor. Compared to ARDS due to other types of viral pneumonia, COVID-19 is characterized by impaired interferon-stimulated gene (ISG) expression. The relationship between SARS-CoV-2 viral load and expression of ISGs is decoupled in patients with COVID-19 ARDS when compared to patients with mild COVID-19. In summary, assessment of host gene expression in the lower airways of patients reveals distinct immunological features of COVID-19 ARDS.


Subject(s)
COVID-19/genetics , RNA/genetics , Respiratory Distress Syndrome/genetics , Trachea/immunology , Adult , Aged , Aged, 80 and over , COVID-19/immunology , COVID-19/virology , Case-Control Studies , Cohort Studies , Critical Illness , Cytokines/genetics , Cytokines/immunology , Female , Gene Expression Profiling , Humans , Male , Middle Aged , RNA/metabolism , Respiratory Distress Syndrome/immunology , Respiratory Distress Syndrome/virology , SARS-CoV-2/physiology , Sequence Analysis, RNA
19.
Nat Commun ; 12(1): 5113, 2021 08 25.
Article in English | MEDLINE | ID: covidwho-1373413

ABSTRACT

SARS-CoV-2 is a major threat to global health. Here, we investigate the RNA structure and RNA-RNA interactions of wildtype (WT) and a mutant (Δ382) SARS-CoV-2 in cells using Illumina and Nanopore platforms. We identify twelve potentially functional structural elements within the SARS-CoV-2 genome, observe that subgenomic RNAs can form different structures, and that WT and Δ382 virus genomes fold differently. Proximity ligation sequencing identify hundreds of RNA-RNA interactions within the virus genome and between the virus and host RNAs. SARS-CoV-2 genome binds strongly to mitochondrial and small nucleolar RNAs and is extensively 2'-O-methylated. 2'-O-methylation sites are enriched in viral untranslated regions, associated with increased virus pair-wise interactions, and are decreased in host mRNAs upon virus infection, suggesting that the virus sequesters methylation machinery from host RNAs towards its genome. These studies deepen our understanding of the molecular and cellular basis of SARS-CoV-2 pathogenicity and provide a platform for targeted therapy.


Subject(s)
COVID-19/virology , Host Microbial Interactions , RNA, Viral/metabolism , RNA/metabolism , SARS-CoV-2/physiology , COVID-19/genetics , COVID-19/metabolism , COVID-19/physiopathology , DNA Methylation , Genome, Viral , Humans , Nucleic Acid Conformation , RNA/chemistry , RNA/genetics , RNA, Viral/chemistry , RNA, Viral/genetics , SARS-CoV-2/chemistry , SARS-CoV-2/genetics
20.
Trends Biochem Sci ; 46(4): 270-283, 2021 04.
Article in English | MEDLINE | ID: covidwho-958915

ABSTRACT

RNA G-quadruplexes (RG4s) are four-stranded structures known to control gene expression mechanisms, from transcription to protein synthesis, and DNA-related processes. Their potential impact on RNA biology allows these structures to shape cellular processes relevant to disease development, making their targeting for therapeutic purposes an attractive option. We review here the current knowledge on RG4s, focusing on the latest breakthroughs supporting the notion of transient structures that fluctuate dynamically in cellulo, their interplay with RNA modifications, their role in cell compartmentalization, and their deregulation impacting the host immune response. We emphasize RG4-binding proteins as determinants of their transient conformation and effectors of their biological functions.


Subject(s)
G-Quadruplexes , Biology , DNA , Protein Biosynthesis , RNA/metabolism
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