Tricks and threats of RNA viruses - towards understanding the fate of viral RNA
TL;DR: Human innate cellular defence pathways have evolved to sense and eliminate pathogens, of which, viruses are considered one of the most dangerous as mentioned in this paper, and their relatively simple structure makes the identif...
Abstract: Human innate cellular defence pathways have evolved to sense and eliminate pathogens, of which, viruses are considered one of the most dangerous. Their relatively simple structure makes the identif...
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TL;DR: In this paper , a detection methodology for SARS-CoV-2 RNA (wildtype and variants of concern) in wastewater was developed based on the detection of different target genes (E and ORF1ab) by polyethylene glycol (PEG) precipitation and digital droplet PCR.
24 citations
TL;DR: This review focuses on the recent development in the synthesis of various dinucleotide cap analogs such as dinucleucleotide containing a triazole moiety, phosphorothiolate cap, biotinylated cap, cap analog containing N1 modification, cap Analog containing N2 modification, din nucleotide containing fluorescence probe and TAT, bacterial caps, and trinucleotidecap analogs.
Abstract: The recent FDA approval of the mRNA vaccine for severe acute respiratory syndrome coronavirus (SARS‐CoV‐2) emphasizes the importance of mRNA as a powerful tool for therapeutic applications. The chemically modified mRNA cap analogs contain a unique cap structure, m7G[5′]ppp[5′]N (where N=G, A, C or U), present at the 5′‐end of many eukaryotic cellular and viral RNAs and several non‐coding RNAs. The chemical modifications on cap analog influence orientation's nature, translational efficiency, nuclear stability, and binding affinity. The recent invention of a trinucleotide cap analog provides groundbreaking research in the area of mRNA analogs. Notably, trinucleotide cap analogs outweigh dinucleotide cap analogs in terms of capping efficiency and translational properties. This review focuses on the recent development in the synthesis of various dinucleotide cap analogs such as dinucleotide containing a triazole moiety, phosphorothiolate cap, biotinylated cap, cap analog containing N1 modification, cap analog containing N2 modification, dinucleotide containing fluorescence probe and TAT, bacterial caps, and trinucleotide cap analogs. In addition, the biological applications of these novel cap analogs are delineated.
11 citations
TL;DR: 2’-O-methylation of mRNA cap structure regulates protein biosynthesis in a cell-dependent manner and was found to have a prominent impact on the composition of the protein interactome associated with the RNA bearing mentioned modifications.
Abstract: In higher eukaryotes, m7G-adjacent nucleotides undergo extensive modifications. Ribose of the first or first and second transcribed nucleotides can be subjected to 2’-O-methylation to form cap1 or cap2, respectively. Additionally, when the first transcribed nucleotide is adenosine, it can not only undergo 2’-O-methylation but can also be methylated at position N6 forming N6,2’-O-dimethyladenosine (m6Am). Recent studies have shed some light on the functions of cap1, showing that cap1 in mammalian cells plays a crucial role in distinguishing between ‘self’ and ‘non-self’ RNA during viral infection. Here, we attempted to understand the impact of other cap methylations on RNA-related processes. Therefore, we synthesized tetranucleotide cap analogs and used them for efficient co-transcriptional RNA capping during in vitro transcription. Using this tool, we found that 2’-O-methylation of the second transcribed nucleotide within the mRNA 5’ cap influences protein production levels in a cell-specific manner. The presence of this modification can strongly hamper protein biosynthesis or do not influence protein production levels. Interestingly, 2’-O-methylation of the second transcribed nucleotide as well as the presence of N6,2’-O-dimethyladenosine as the first transcribed nucleotide serve as determinants that define transcripts as ‘self’ and contribute to transcript escape from the host innate immune response. Additionally, cap methylation status does not influence transcript affinity towards translation initiation factor 4E or in vitro susceptibility to decapping by DCP2; however what we observe is resistance of RNA capped with cap2 to DXO-mediated decapping and degradation. Significance Statement Methylation of mRNA cap structure regulates protein biosynthesis in a cell-dependent manner. Among the three known m7G cap modifications, the 2’-O-methylation is dominant. 2’-O-methylation of the first transcribed nucleotide can boost protein production, whereas the same modification of the second transcribed nucleotide can strongly decrease translation. Interestingly, we show that in the JAWS II cell line, 2’-O-methylation of mRNA cap had a prominent impact on the composition of the protein interactome associated with the RNA bearing mentioned modifications. Further analysis revealed that 2’-O-methylation of the second transcribed nucleotide and N6-methylation of adenosine as the first transcribed nucleotide serve as determinants defining transcripts as ‘self’ and contribute to transcript escape from the host innate immune response.
8 citations
TL;DR: In this article, a detection methodology for SARS-CoV-2 RNA (wild-type and variants of concern) in wastewater was developed based on the detection of different target genes (E and ORF1ab) by PEG precipitation and digital droplet PCR.
Abstract: In recent months, wastewater-based epidemiology (WBE) has been shown to be an important tool for early detection of SARS-CoV-2 circulation in the population. In this study, a detection methodology for SARS-CoV-2 RNA (wild-type and variants of concern) in wastewater was developed based on the detection of different target genes (E and ORF1ab) by PEG precipitation and digital droplet PCR. This methodology was used to determine the SARS-CoV-2 concentration and the proportion of N501Y mutation in raw sewage of the wastewater treatment plant of the city of Karlsruhe in southwestern Germany over a period of 1 year (June 2020 to July 2021). Comparison of SARS-CoV-2 concentrations with reported COVID-19 cases in the catchment area showed a significant correlation. Viral RNA titre trends appeared more than 12 days earlier than clinical data, demonstrating the potential of wastewater-based epidemiology as an early warning system. Parallel PCR analysis using seven primer and probe systems revealed similar gene copy numbers with E, ORF, RdRP2 and NSP9 assays. RdPP1 and NSP3 generally resulted in lower copy numbers, and in particular for N1 there was low correlation with the other assays due to outliers. The occurrence of the N501Y mutation in the wastewater of Karlsruhe was consistent with the occurrence of the alpha-variant (B.1.1.7) in the corresponding individual clinical tests. In batch experiments SARS-CoV-2 RNA was stable for several days under anaerobic conditions, but the copy numbers decreased rapidly in the presence of dissolved oxygen. Overall, this study shows that wastewater-based epidemiology is a sensitive and robust approach to detect trends in the spread of SARS-CoV-2 at an early stage, contributing to successful pandemic management.
7 citations
TL;DR: Contextual analysis indicates that the bacteriophage versions of ADPr-targeting domains are more likely to counter soluble ADPr derivatives, while the eukaryotic RNA viral versions might prefer macromolecular ADPr adducts, and comparative genomics is used to predict host systems involved in countering viral ADP ribosylation of host molecules.
Abstract: NAD+ and ADP-ribose (ADPr)-containing molecules are at the interface of virus–host conflicts across life encompassing RNA processing, restriction, lysogeny/dormancy and functional hijacking. We objectively defined the central components of the NAD+–ADPr networks involved in these conflicts and systematically surveyed 21,191 completely sequenced viral proteomes representative of all publicly available branches of the viral world to reconstruct a comprehensive picture of the viral NAD+–ADPr systems. These systems have been widely and repeatedly exploited by positive-strand RNA and DNA viruses, especially those with larger genomes and more intricate life-history strategies. We present evidence that ADP-ribosyltransferases (ARTs), ADPr-targeting Macro, NADAR and Nudix proteins are frequently packaged into virions, particularly in phages with contractile tails (Myoviruses), and deployed during infection to modify host macromolecules and counter NAD+-derived signals involved in viral restriction. Genes encoding NAD+–ADPr-utilizing domains were repeatedly exchanged between distantly related viruses, hosts and endo-parasites/symbionts, suggesting selection for them across the virus world. Contextual analysis indicates that the bacteriophage versions of ADPr-targeting domains are more likely to counter soluble ADPr derivatives, while the eukaryotic RNA viral versions might prefer macromolecular ADPr adducts. Finally, we also use comparative genomics to predict host systems involved in countering viral ADP ribosylation of host molecules.
2 citations
References
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TL;DR: New insights into innate immunity are changing the way the way the authors think about pathogenesis and the treatment of infectious diseases, allergy, and autoimmunity.
10,685 citations
TL;DR: It is shown that cellular response to CpG DNA is mediated by a Toll-like receptor, TLR9, and vertebrate immune systems appear to have evolved a specific Toll- like receptor that distinguishes bacterial DNA from self-DNA.
Abstract: DNA from bacteria has stimulatory effects on mammalian immune cells, which depend on the presence of unmethylated CpG dinucleotides in the bacterial DNA. In contrast, mammalian DNA has a low frequency of CpG dinucleotides, and these are mostly methylated; therefore, mammalian DNA does not have immuno-stimulatory activity. CpG DNA induces a strong T-helper-1-like inflammatory response. Accumulating evidence has revealed the therapeutic potential of CpG DNA as adjuvants for vaccination strategies for cancer, allergy and infectious diseases. Despite its promising clinical use, the molecular mechanism by which CpG DNA activates immune cells remains unclear. Here we show that cellular response to CpG DNA is mediated by a Toll-like receptor, TLR9. TLR9-deficient (TLR9-/-) mice did not show any response to CpG DNA, including proliferation of splenocytes, inflammatory cytokine production from macrophages and maturation of dendritic cells. TLR9-/- mice showed resistance to the lethal effect of CpG DNA without any elevation of serum pro-inflammatory cytokine levels. The in vivo CpG-DNA-mediated T-helper type-1 response was also abolished in TLR9-/- mice. Thus, vertebrate immune systems appear to have evolved a specific Toll-like receptor that distinguishes bacterial DNA from self-DNA.
6,188 citations
TL;DR: It is shown that mammalian TLR3 recognizes dsRNA, and that activation of the receptor induces the activation of NF-κB and the production of type I interferons (IFNs).
Abstract: Toll-like receptors (TLRs) are a family of innate immune-recognition receptors that recognize molecular patterns associated with microbial pathogens, and induce antimicrobial immune responses. Double-stranded RNA (dsRNA) is a molecular pattern associated with viral infection, because it is produced by most viruses at some point during their replication. Here we show that mammalian TLR3 recognizes dsRNA, and that activation of the receptor induces the activation of NF-kappaB and the production of type I interferons (IFNs). TLR3-deficient (TLR3-/-) mice showed reduced responses to polyinosine-polycytidylic acid (poly(I:C)), resistance to the lethal effect of poly(I:C) when sensitized with d-galactosamine (d-GalN), and reduced production of inflammatory cytokines. MyD88 is an adaptor protein that is shared by all the known TLRs. When activated by poly(I:C), TLR3 induces cytokine production through a signalling pathway dependent on MyD88. Moreover, poly(I:C) can induce activation of NF-kappaB and mitogen-activated protein (MAP) kinases independently of MyD88, and cause dendritic cells to mature.
6,066 citations
TL;DR: It is shown that guanosine (G)- and uridine (U)-rich ssRNA oligonucleotides derived from human immunodeficiency virus–1 (HIV-1) stimulate dendritic cells and macrophages to secrete interferon-α and proinflammatory, as well as regulatory, cytokines, and these data suggest that ssRNA represents a physiological ligand for TLR7 and TLR8.
Abstract: Double-stranded ribonucleic acid (dsRNA) serves as a danger signal associated with viral infection and leads to stimulation of innate immune cells. In contrast, the immunostimulatory potential of single-stranded RNA (ssRNA) is poorly understood and innate immune receptors for ssRNA are unknown. We report that guanosine (G)- and uridine (U)-rich ssRNA oligonucleotides derived from human immunodeficiency virus-1 (HIV-1) stimulate dendritic cells (DC) and macrophages to secrete interferon-alpha and proinflammatory, as well as regulatory, cytokines. By using Toll-like receptor (TLR)-deficient mice and genetic complementation, we show that murine TLR7 and human TLR8 mediate species-specific recognition of GU-rich ssRNA. These data suggest that ssRNA represents a physiological ligand for TLR7 and TLR8.
3,847 citations
TL;DR: It is found that RIG-I is essential for the production of interferons in response to RNA viruses including paramyxoviruses, influenza virus and Japanese encephalitis virus, whereas MDA5 is critical for picornavirus detection.
Abstract: The innate immune system senses viral infection by recognizing a variety of viral components (including double-stranded (ds)RNA) and triggers antiviral responses. The cytoplasmic helicase proteins RIG-I (retinoic-acid-inducible protein I, also known as Ddx58) and MDA5 (melanoma-differentiation-associated gene 5, also known as Ifih1 or Helicard) have been implicated in viral dsRNA recognition. In vitro studies suggest that both RIG-I and MDA5 detect RNA viruses and polyinosine-polycytidylic acid (poly(I:C)), a synthetic dsRNA analogue. Although a critical role for RIG-I in the recognition of several RNA viruses has been clarified, the functional role of MDA5 and the relationship between these dsRNA detectors in vivo are yet to be determined. Here we use mice deficient in MDA5 (MDA5-/-) to show that MDA5 and RIG-I recognize different types of dsRNAs: MDA5 recognizes poly(I:C), and RIG-I detects in vitro transcribed dsRNAs. RNA viruses are also differentially recognized by RIG-I and MDA5. We find that RIG-I is essential for the production of interferons in response to RNA viruses including paramyxoviruses, influenza virus and Japanese encephalitis virus, whereas MDA5 is critical for picornavirus detection. Furthermore, RIG-I-/- and MDA5-/- mice are highly susceptible to infection with these respective RNA viruses compared to control mice. Together, our data show that RIG-I and MDA5 distinguish different RNA viruses and are critical for host antiviral responses.
3,508 citations