Mn2+ coordinates Cap-0-RNA to align substrates for efficient 2′-O-methyl transfer by SARS-CoV-2 nsp16

2021 ◽  
Author(s):  
George Minasov ◽  
Monica Rosas-Lemus ◽  
Ludmilla Shuvalova ◽  
Nicole L. Inniss ◽  
Joseph S. Brunzelle ◽  
...  
Keyword(s):  
Divalent Cations ◽  
Critical Role ◽  
Messenger Rnas ◽  
Innate Immune Responses ◽  
Methyl Transfer ◽  
Backbone Conformation ◽  
Binding Groove ◽  
Rna Capping ◽  
Aspartate Residue

AbstractCapping viral messenger RNAs is essential for efficient translation and prevents their detection by host innate immune responses. For SARS-CoV-2, RNA capping includes 2′-O-methylation of the first ribonucleotide by methyltransferase nsp16 in complex with activator nsp10. The reaction requires substrates, a short RNA and SAM, and is catalyzed by divalent cations, with preference for Mn2+. Crystal structures of nsp16-nsp10 with capped RNAs revealed a critical role of metal ions in stabilizing interactions between ribonucleotides and nsp16, resulting in precise alignment of the substrates for methyl transfer. An aspartate residue that is highly conserved among coronaviruses alters the backbone conformation of the capped RNA in the binding groove. This aspartate is absent in mammalian methyltransferases and is a promising site for designing coronavirus-specific inhibitors.

scholarly journals Mn2+ coordinates Cap-0-RNA to align substrates for efficient 2′-O-methyl transfer by SARS-CoV-2 nsp16

2021 ◽  
Vol 14 (689) ◽  
pp. eabh2071
Author(s):  
George Minasov ◽  
Monica Rosas-Lemus ◽  
Ludmilla Shuvalova ◽  
Nicole L. Inniss ◽  
Joseph S. Brunzelle ◽  
...  
Keyword(s):  
Divalent Cations ◽  
Messenger Rnas ◽  
Viral Mrna ◽  
Innate Response ◽  
Methyl Group ◽  
X Ray ◽  
Response System ◽  
Backbone Conformation ◽  
Mrna Capping ◽  
Binding Groove

Capping of viral messenger RNAs is essential for efficient translation, for virus replication, and for preventing detection by the host cell innate response system. The SARS-CoV-2 genome encodes the 2′-O-methyltransferase nsp16, which, when bound to the coactivator nsp10, uses S-adenosylmethionine (SAM) as a donor to transfer a methyl group to the first ribonucleotide of the mRNA in the final step of viral mRNA capping. Here, we provide biochemical and structural evidence that this reaction requires divalent cations, preferably Mn2+, and a coronavirus-specific four-residue insert. We determined the x-ray structures of the SARS-CoV-2 2′-O-methyltransferase (the nsp16-nsp10 heterodimer) in complex with its reaction substrates, products, and divalent metal cations. These structural snapshots revealed that metal ions and the insert stabilize interactions between the capped RNA and nsp16, resulting in the precise alignment of the ribonucleotides in the active site. Comparison of available structures of 2′-O-methyltransferases with capped RNAs from different organisms revealed that the four-residue insert unique to coronavirus nsp16 alters the backbone conformation of the capped RNA in the binding groove, thereby promoting catalysis. This insert is highly conserved across coronaviruses, and its absence in mammalian methyltransferases makes this region a promising site for structure-guided drug design of selective coronavirus inhibitors.

scholarly journals Essential role of IPS-1 in innate immune responses against RNA viruses

2006 ◽  
Vol 203 (7) ◽  
pp. 1795-1803 ◽  
Author(s):  
Himanshu Kumar ◽  
Taro Kawai ◽  
Hiroki Kato ◽  
Shintaro Sato ◽  
Ken Takahashi ◽  
...  
Keyword(s):  
Rna Viruses ◽  
Rna Virus ◽  
Critical Role ◽  
Nuclear Factor Κb ◽  
Type I ◽  
Innate Immune Responses ◽  
Antiviral Responses ◽  
Deficient Mice

IFN-β promoter stimulator (IPS)-1 was recently identified as an adapter for retinoic acid–inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (Mda5), which recognize distinct RNA viruses. Here we show the critical role of IPS-1 in antiviral responses in vivo. IPS-1–deficient mice showed severe defects in both RIG-I– and Mda5-mediated induction of type I interferon and inflammatory cytokines and were susceptible to RNA virus infection. RNA virus–induced interferon regulatory factor-3 and nuclear factor κB activation was also impaired in IPS-1–deficient cells. IPS-1, however, was not essential for the responses to either DNA virus or double-stranded B-DNA. Thus, IPS-1 is the sole adapter in both RIG-I and Mda5 signaling that mediates effective responses against a variety of RNA viruses.

scholarly journals The microbiota plays a critical role in the reactivity of lung immune components to innate ligands

2020 ◽  
Author(s):  
Quentin Marquant ◽  
Daphné Laubreton ◽  
Carole Drajac ◽  
Elliot Mathieu ◽  
Edwige Bouguyon ◽  
...  
Keyword(s):  
Immune Responses ◽  
Critical Role ◽  
Innate Immune ◽  
Immune Reactivity ◽  
Innate Immune Responses ◽  
Plain Language ◽  
Pulmonary Tissue ◽  
Germ Free ◽  
Syncytial Virus

AbstractThe microbiota contributes to shaping efficient and safe immune defenses in the gut. However, little is known about the role of the microbiota in the education of pulmonary innate immune responses. Here, we tested whether the endogenous microbiota can modulate reactivity of pulmonary tissue to pathogen stimuli by comparing the response of specific pathogen-free (SPF) and germ-free (GF) mice. Using SPF and GF mice intranasally exposed to lipopolysaccharide (LPS), a component of Gram-negative bacteria, we observed earlier and greater inflammation in the pulmonary compartment of GF mice than that of SPF mice. Toll-like receptor 4 (TLR4) was more abundantly expressed in the lungs of GF mice than those of SPF mice at steady state, which could predispose the innate immunity of GF mice to strongly react to environmental stimuli. Lung explants were stimulated with different TLR agonists or infected with the human airways pathogen, respiratory syncytial virus (RSV), resulting in greater inflammation under almost all conditions for the GF explants. Finally, alveolar macrophages (AM) from GF mice presented a higher innate immune response upon RSV infection than those of SPF mice. Overall, these data suggest that the presence of microbiota in SPF mice induced a process of innate immune tolerance in the lungs by a mechanism which remains to be elucidated. Our study represents a step forward to establishing the link between the microbiota and the immune reactivity of the lungs.Plain Language summaryMicrobiota represents an important partner of immunologic system at the interface between immune cells and epithelium. It is well known, notably in the gut, that the microbiota contributes in shaping efficient and safe defenses. However, little is known about the role of the microbiota in the education of pulmonary innate immune responses. In this study, we postulate that endogenous microbiota could dampen an excessive reactivity of pulmonary tissue to external stimuli. Thus, we sought to study the innate immune reaction switched on by viral or bacterial ligands in respiratory tract cells coming from mice with or without microbiota (germ-free condition, GF). Altogether, our results show a higher inflammatory reaction in GF condition. This study represents a step forward to better establish the link between the microbiota and the reactivity of the lung tissue. Not only these data demonstrate that the microbiota educates the pulmonary innate immune system, but also contributes the emerging concept of using respiratory commensal bacteria as potential next-generation probiotics to prevent susceptibility to respiratory diseases.

scholarly journals Osteoarthritis and Toll-Like Receptors: When Innate Immunity Meets Chondrocyte Apoptosis

Biology ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 65
Author(s):  
Goncalo Barreto ◽  
Mikko Manninen ◽  
Kari K. Eklund
Keyword(s):  
Inflammatory Responses ◽  
Critical Role ◽  
Innate Immune ◽  
Chondrocyte Apoptosis ◽  
Innate Immune Responses ◽  
Toll Like Receptors ◽  
Chondrocyte Death ◽  
Dominant Feature ◽  
Functional Relevance

Osteoarthritis (OA) has long been viewed as a degenerative disease of cartilage, but accumulating evidence indicates that inflammation has a critical role in its pathogenesis. In particular, chondrocyte-mediated inflammatory responses triggered by the activation of innate immune receptors by alarmins (also known as danger signals) are thought to be involved. Thus, toll-like receptors (TLRs) and their signaling pathways are of particular interest. Recent reports suggest that among the TLR-induced innate immune responses, apoptosis is one of the critical events. Apoptosis is of particular importance, given that chondrocyte death is a dominant feature in OA. This review focuses on the role of TLR signaling in chondrocytes and the role of TLR activation in chondrocyte apoptosis. The functional relevance of TLR and TLR-triggered apoptosis in OA are discussed as well as their relevance as candidates for novel disease-modifying OA drugs (DMOADs).

scholarly journals Critical Role of TLR4 in Human Metapneumovirus Mediated Innate Immune Responses and Disease Pathogenesis

PLoS ONE ◽  
2013 ◽  
Vol 8 (10) ◽  
pp. e78849 ◽  
Author(s):  
Thangam Sudha Velayutham ◽  
Deepthi Kolli ◽  
Teodora Ivanciuc ◽  
Roberto P. Garofalo ◽  
Antonella Casola
Keyword(s):  
Immune Responses ◽  
Critical Role ◽  
Human Metapneumovirus ◽  
Innate Immune ◽  
Innate Immune Responses ◽  
Disease Pathogenesis

scholarly journals Cutting Edge: Critical Role of Intracellular Osteopontin in Antifungal Innate Immune Responses

2010 ◽  
Vol 186 (1) ◽  
pp. 19-23 ◽  
Author(s):  
Makoto Inoue ◽  
Yasuhiro Moriwaki ◽  
Tomohiro Arikawa ◽  
Yu-Hsun Chen ◽  
Young Joo Oh ◽  
...  
Keyword(s):  
Immune Responses ◽  
Critical Role ◽  
Innate Immune ◽  
Cutting Edge ◽  
Innate Immune Responses

scholarly journals Molecular Mechanism of SARS-CoVs Orf6 Targeting the Rae1–Nup98 Complex to Compete With mRNA Nuclear Export

2022 ◽  
Vol 8 ◽  
Author(s):  
Tinghan Li ◽  
Yibo Wen ◽  
Hangtian Guo ◽  
Tingting Yang ◽  
Haitao Yang ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Rna Binding ◽  
Critical Role ◽  
Tight Binding ◽  
Viral Proteins ◽  
Interferon Signaling ◽  
Binding Groove ◽  
Mrna Binding ◽  
Common Target

The accessory protein Orf6 is uniquely expressed in sarbecoviruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which is an ongoing pandemic. SARS-CoV-2 Orf6 antagonizes host interferon signaling by inhibition of mRNA nuclear export through its interactions with the ribonucleic acid export 1 (Rae1)–nucleoporin 98 (Nup98) complex. Here, we confirmed the direct tight binding of Orf6 to the Rae1-Nup98 complex, which competitively inhibits RNA binding. We determined the crystal structures of both SARS-CoV-2 and SARS-CoV-1 Orf6 C-termini in complex with the Rae1–Nup98 heterodimer. In each structure, SARS-CoV Orf6 occupies the same potential mRNA-binding groove of the Rae1–Nup98 complex, comparable to the previously reported structures of other viral proteins complexed with Rae1-Nup98, indicating that the Rae1–Nup98 complex is a common target for different viruses to impair the nuclear export pathway. Structural analysis and biochemical studies highlight the critical role of the highly conserved methionine (M58) of SARS-CoVs Orf6. Altogether our data unravel a mechanistic understanding of SARS-CoVs Orf6 targeting the mRNA-binding site of the Rae1–Nup98 complex to compete with the nuclear export of host mRNA, which further emphasizes that Orf6 is a critical virulence factor of SARS-CoVs.

Barium accumulation in rat pancreatic B cells

1976 ◽  
Vol 22 (2) ◽  
pp. 455-465
Author(s):  
S.L. Howell ◽  
M. Tyhurst
Keyword(s):  
B Cells ◽  
Cyclic Amp ◽  
Divalent Cations ◽  
Critical Role ◽  
Frozen Sections ◽  
X Ray ◽  
Pancreatic B Cells ◽  
Acetate Accumulation ◽  
Rat Islets Of Langerhans

Barium has been used as an electron-opaque substitute for calcium in a study of the distribution of divalent cations between organelles in homogenates or intact rat islets of Langerhans. These were incubated in the presence of barium acetate. Accumulation of electron-opaque deposits was stimulated during incubation of islets in the presence of high glucose concentrations and was diminished in conditions in which intracellular cyclic AMP levels were raised. Mitochondria were found to be the principal sites of accumulation of electron-opaque deposits. Addition of dinitrophenol to homogenates or intact islets abolished mitochondrial barium accumulation. X-ray microanalysis of the deposits in frozen sections showed them to consist predominantly of barium and phosphate. These experiments serve to emphasize further the critical role of mitochondria in the regulation of divalent cation accumulation in B cells, and to confirm that a direct effect on intracellular distribution of divalent cations may represent one important mechanism of action of cyclic AMP in regulating insulin secretion.

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