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SND1 binds SARS-CoV-2 negative-sense RNA and promotes viral RNA synthesis through NSP9

ABSTRACT: Regulation of viral RNA biogenesis is fundamental to productive SARS-CoV-2 infection. To characterize host RNA-binding proteins involved in this process, we biochemically identified proteins bound to genomic and subgenomic SARS-CoV-2 RNAs. We find that the host protein SND1 specifically binds to the 5'-end of negative-sense viral RNA and is required for SARS-CoV-2 RNA synthesis. SND1-depleted cells form smaller replication organelles and display diminished virus growth kinetics. We discover that NSP9, a viral RNA-binding protein and direct SND1 interaction partner, is covalently linked to the 5'-ends of positive and negative-sense RNAs produced during infection. These linkages occur at replication-transcription initiation sites, consistent with NSP9 priming viral RNA synthesis. Mechanistically, SND1 remodels NSP9 occupancy and alters the covalent linkage of NSP9 to initiating nucleotides in viral RNA. Our findings implicate NSP9 in the initiation of SARS-CoV-2 RNA synthesis and unravel an unsuspected role of a cellular protein in orchestrating viral RNA production.

ORGANISM(S): Homo sapiens

PROVIDER: GSE217429 | GEO | 2023/10/03

REPOSITORIES: GEO

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SND1 binds SARS-CoV-2 negative-sense RNA and promotes viral RNA synthesis through NSP9

Project description:Regulation of viral RNA biogenesis is fundamental to productive SARS-CoV-2 infection. To characterize host RNA-binding proteins (RBPs) involved in this process, we biochemically identified proteins bound to genomic and subgenomic SARS-CoV-2 RNAs. We find that the host protein SND1 binds the 5ʹ end of negative-sense viral RNA and is required for SARS-CoV-2 RNA synthesis. SND1-depleted cells form smaller replication organelles and display diminished virus growth kinetics. We discover that NSP9, a viral RBP and direct SND1 interaction partner, is covalently linked to the 5ʹ ends of positive and negative-sense RNAs produced during infection. These linkages occur at replication-transcription initiation sites, consistent with NSP9 priming viral RNA synthesis. Mechanistically, SND1 remodels NSP9 occupancy and alters the covalent linkage of NSP9 to initiating nucleotides in viral RNA. Our findings implicate NSP9 in the initiation of SARS-CoV-2 RNA synthesis and unravel an unsuspected role of a cellular protein in orchestrating viral RNA production.

2023-10-05 | PXD037528 | Pride

SND1 binds SARS-CoV-2 negative-sense RNA and promotes viral RNA synthesis through NSP9

Project description:Schmidt N, Ganskih S, Wei Y, Gabel A, Zielinski S, Keshishian H, Lareau CA, , Zimmermann L, Makroczyova J, Pearce C, Krey K, Henig T, Stegmaier S, Moyon L, Horlacher M, Werner S, Aydin J, Olguin-Nava M, Potabattula R, Kibe A, Dölken L, Smyth RP, Caliskan N, Marsico A, Krempl C, Bodem J, Pichlmair A, Carr SA, Chlanda P, Erhard F, Munschauer M Regulation of viral RNA biogenesis is fundamental to productive SARS-CoV-2 infection. To characterize host RNA-binding proteins involved in this process, we biochemically identified proteins bound to genomic and subgenomic SARS-CoV-2 RNAs. We find that the host protein SND1 specifically binds to the 5?-end of negative-sense viral RNA and is required for SARS-CoV-2 RNA synthesis. SND1-depleted cells form smaller replication organelles and display diminished virus growth kinetics. We discover that NSP9, a viral RNA-binding protein and direct SND1 interaction partner, is covalently linked to the 5'-ends of positive and negative-sense RNAs produced during infection. These linkages occur at replication-transcription initiation sites, consistent with NSP9 priming viral RNA synthesis. Mechanistically, SND1 remodels NSP9 occupancy and alters the covalent linkage of NSP9 to initiating nucleotides in viral RNA. Our findings implicate NSP9 in the initiation of SARS-CoV-2 RNA synthesis and unravel an unsuspected role of a cellular protein in orchestrating viral RNA production.

2022-11-09 | MSV000090682 | MassIVE

Nanobodies agains Sars Cov 2 Nsp9 protein protects cells from virus infection

Project description:Nanobodies are emerging as ideal instruments for drug design and several have recently been created to block SARS-Cov-2 entry in the host cell by targeting surface-exposed Spike protein. However, due to the high frequency of mutations that affect Spike, these nanobodies may not efficiently target Spike during viral entry. Here we have established a pipeline that instead targets highly conserved viral proteins that are made only after viral entry into the host cell when the SARS-Cov-2 RNA-based genome is translated. As proof of principle, we designed nanobodies against the SARS-CoV-2 non-structural protein Nsp9, required for replication of the viral genome. To find out if this strategy efficiently blocked viral replication, one of these anti-Nsp9 nanobodies, 2NSP23, previously characterized using immunoassays and NMR spectroscopy for epitope mapping, was encapsulated into lipid nanoparticles (LNP) as mRNA. We show that this nanobody, hereby referred to as LNP-mRNA-2NSP23, is internalized and translated in HEK293 cells. We next infected HEK293-ACE2 cells subjected to LNP-mRNA-2NSP23 with multiple SARS-CoV-2 variants. Analysis of total RNA isolated form infected cells treated or untreated with LNP-mRNA-2NSP23 using qPCR and RNA deep sequencing shows that the LNP-mRNA-2NSP23 nanobody protects HEK293-ACE2 cells and suppresses replication of several SARS-CoV-2 variants. These observations indicate that following translation, the nanobody 2NSP23 inhibits viral replication by targeting Nsp9 in living cells. We propose that LNP-mRNA-2NSP23 may be translated into an innovative technology to generate novel antiviral drugs highly efficient across coronaviruses.

2023-10-10 | GSE244714 | GEO

m6A-RNA mapping, SND1-RNA binding profile mapping and SND1-depletion in KSHV-infected B-lymphocytes

Project description:We have mapped m6A sites and endogenous SND1 binding sites in the viral and cellular transcriptome using TREx BCBL1-Rta cells. In addition, we have depleted SND1 in TREx BCBL1-Rta cells and BCBL1 cells and analyzed their RNA expression profile both during latency and lytic replication.

2019-10-15 | GSE119026 | GEO

Analysis on Replication and Transcription of SARS-CoV-2 and inhition effect of Remdesivir

Project description:SARS-CoV-2, a betacoronavirus with a positive-sense, single-stranded RNA genome, caused the COVID-19 pandemic with unprecedent health and socio-economic crisis. Although its general sense mRNA architecture was reported, that of its negative strand was unexplored. We combined poly(A)-mRNA and ribosomal RNA-depleted sequencing to delineate several fine features of both RNA strands. Together with the transcriptome data under the perturbation of anti-viral drug Remdesivir, this project opens new sights into SARS-CoV-2 replication mechanism and may facilitate the anti-viral drug design.

2021-06-27 | GSE160668 | GEO

Discovery and functional interrogation of the virus and host RNA interactome of SARS-CoV-2 proteins [eCLIP]

Project description:The coronavirus disease 2019 (COVID-19) pandemic was caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Understanding the molecular functions of SARS-CoV-2 proteins is thus imperative to developing effective antiviral treatments. Here, we use enhanced crosslinking and immunoprecipitation to investigate SARS-CoV-2 protein interactions with viral and host RNAs. SARS-CoV-2 proteins, NSP8 and NSP12, are found to specifically bind to untranslated regions of the RNA viral genome, with NSP12 additionally binding to all transcription regulatory sequences. This provides evidence for their central roles in replication and transcription. Moreover, we discovered a potential site of NSP12 mediated genome recombination, which could explain the genetic diversity found in coronaviruses. SARS-CoV-2 proteins exogenously expressed in human lung epithelial cells bind to 4,281 unique host RNAs. Nine SARS-CoV-2 proteins upregulate target gene expression, including NSP12 which upregulates mitochondrial electron transport and N-linked glycosylation proteins. Furthermore, siRNA knockdown of NSP12-targeted proteins in human lung organoid cells demonstrates substantial antiviral effects. Conversely, NSP9 inhibits host gene expression via blocking mRNA export and dampens antiviral inflammation response such as interleukin 1α (IL1α) production. Our extensive viral protein-RNA interactome provides a catalog of potential therapeutic targets and offers insight into the etiology of COVID-19 as a safeguard against future pandemics.

2022-03-09 | GSE173498 | GEO

Discovery and functional interrogation of the virus and host RNA interactome of SARS-CoV-2 proteins [RNA-Seq]

2022-03-09 | GSE173507 | GEO

Discovery and functional interrogation of the virus and host RNA interactome of SARS-CoV-2 proteins [Ribo-Seq]

2022-03-31 | GSE198899 | GEO

Profilling of viral and host transcriptome in SARS-CoV-2 infected A549-ACE2 cells with VIC or B.1.1.7 strains.

Project description: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, sub-genomic 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.

2021-12-31 | GSE184932 | GEO

The PDZ-binding motif of SARS-CoV envelope protein is a determinant of viral pathogenesis

Project description:A recombinant SARS-CoV lacking the envelope (E) protein is attenuated in vivo. Here we report that E protein PDZ-binding motif (PBM), a domain involved in protein-protein interactions, is a major virulence determinant in vivo. Elimination of SARS-CoV E protein PBM by using reverse genetics led to attenuated viruses (SARS-CoV-mutPBM) and to a reduction in the deleterious exacerbate immune response triggered during infection with the parental virus (SARS-CoV-wt). Cellular protein syntenin bound E protein PBM during SARS-CoV infection. Syntenin activates p38 MAPK leading to overexpression of inflammatory cytokines, and we have shown that active p38 MAPK was reduced in lungs of mice infected with SARS-CoVs lacking E protein PBM (SARS-CoV-mutPBM) as compared with the parental virus (SARS-CoV-wt), leading to a decreased expression of inflammatory cytokines and to viral attenuation. Therefore, E protein PBM is a virulence factor that activates pathogenic immune response most likely by using syntenin as a mediator of p38 MAPK induced inflammation. Three biological replicates were independently hybridized (one channel per slide) for each sample type (SARS-CoV-wt, SARS-CoV-mutPBM, Mock). Slides were Sure Print G3 Agilent 8x60K Mouse (G4852A-028005)

2014-10-31 | E-GEOD-52920 | biostudies-arrayexpress

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