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RNA-Seq and RNC-Seq of HeLa cells stably expressing NDV NP protein

ABSTRACT: The oncolytic potential of NDV has gained significant attention in the context of clinical trials. After systematically comparing the oncolytic activities of different NDV subtypes,we found that all subtypes, except for highly pathogenic genotype VII NDVs, display remarkable infectivity on tumor cell lines.A single amino acid mutation (F450L) in the NP protein at position 450 leads to an approximate 1000000-fold increase in the TCID50 value of genotype VII NDVs on tumor cells.Mechanistic inquiries unveiled that the amino acid at position 450 of the NP protein governs preferential translation of NDV mRNA.The preferential translation that regulates viral mRNA translation is often accompanied by modulation of the host translation machinery. Therefore, we established HeLa cell lines stably expressing NP protein from different NDV strains. Ribosome-bound mRNA was enriched to perform Ribosome Nascent-chain Complex sequencing(RNC-seq), while total cellular mRNA was enriched to conducted RNA-Seq . Untreated cells were used as controls. These experiments aimed to investigate the impact of NP on the host translation system.

ORGANISM(S): Homo sapiens

PROVIDER: GSE233617 | GEO | 2023/06/01

REPOSITORIES: GEO

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Mass spectrometry identification of interacting proteins of the NP protein from genotype IV NDV Herts/33 and the NP protein from genotype VII NDV strain I4

Project description:Newcastle Disease Virus (NDV) is one of the most threatening viruses to the poultry industry, and it also exhibits oncolytic properties. In our research, we identified a non-oncolytic strain, genotype VII NDV strain I4, where the lack of oncolytic ability is attributed to its NP protein. To explore the mechanism of NP-mediated oncolysis by NDV, we focused on the differential interacting proteins of the NP proteins from two strains: the highly oncolytic Herts/33 strain and the poorly oncolytic I4 strain. The mass spectrometry results show the interacting protein profiles of the NP proteins from both virus strains. After infecting HeLa cells with each strain, we conducted immunoprecipitation using antibodies against the NP protein. Due to the lower expression of I4-associated proteins during replication, we further performed immunoprecipitation experiments using separately overexpressed NP proteins from I4 and Herts/33.

2023-07-28 | PXD044165 |

Identification of interacting proteins with the NP protein of the non-oncolytic H-NP366-489I strain and the non-oncolytic Herts/33 strain by mass spectrometry

Project description:Newcastle disease virus (NDV) is one of the most significant threats to the poultry industry, and it also exhibits oncolytic properties. In our research, we have identified a non-oncolytic strain, the genotype VII NDV strain I4. We have found that the lack of oncolytic characteristics in this strain is attributed to amino acids 366-489 in the NP protein. To delve into the mechanism of NP-mediated NDV oncolysis, we initiated further investigations using the highly oncolytic Herts/33 strain's NP and a recombinant NP protein with amino acids 366-489 from the I4 strain substituted in (H-NP366-489I). These proteins represent the interacting partners in the proteomics analysis of NP proteins from both viral strains. After infecting HeLa cells with each of these strains, immunoprecipitation was performed using antibodies targeting the NP protein. Since the H-NP366-489I virus produces fewer proteins during replication, we additionally overexpressed the H-NP366-489I and Herts/33 NP proteins separately for immunoprecipitation experiments.

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Expression data of spleens from chickens infected with different Newcastle Disease Viruses

2012-08-15 | GSE40100 | GEO

Oligoadenylate synthetase-like (OASL) aggravated newcastle disease virus (NDV)-induced necroptosis in glioma cells

Project description:Newcastle disease virus (NDV) has emerged as an oncolytic agent in several cancers. Previous study has shown that NDV exerts cytolytic activity in glioma, however, the underlying mechanism has not been fully uncovered. Here the cytolytic activity of NDV in glioma and the associated mechanisms have been demonstrated. Infection with NDV inhibits cell proliferation and promotes cell apoptosis in LN229 cells. Further investigation showed that cytoplasmic organelle damage and cytoplasmic vacuolation were observed in LN229 cells after NDV infection. JC-1 staining assay proved that NDV caused cell apoptosis of LN229 cells by inducing mitochondrial dysfunction. We next speculated that NDV caused LN229 cells death through inducing necroptosis, but not ferroptosis, since the Fe2+ level did not alter after NDV infection. Furthermore, the NDV-caused cell death in LN229 cells was blocked by necroptosis inhibitor Nec1. Besides, RNA-seq analysis identified the different expression genes in NDV-infected LN229 cells. OASL, an antiviral gene, has been found to be directly induced by NDV infection. We also found that knockdown of OASL enhanced NDV infection-induced LN229 cells necroptosis. In summary, two aspects about cytolytic activity of NDV in glioma have been demonstrated. NDV presented cytolytic activity in glioma cells through inducing necroptosis. Additionally, targeting OASL may provide new strategy for enhancing necroptosis of glioma cells after NDV infection.

2023-05-01 | GSE227791 | GEO

Expression data of spleens from chickens infected with different Newcastle Disease Viruses

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2012-08-14 | E-GEOD-40100 | biostudies-arrayexpress

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2014-06-10 | E-GEOD-58321 | biostudies-arrayexpress

Ribosome profiling reveals sequence-independent post-initiation pausing as a signature of translation

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N6-methyladenosine Modulates Messenger RNA Translation Efficiency

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2015-06-09 | E-GEOD-63591 | biostudies-arrayexpress

Ribosome profiling in wild-type and APOBEC1-deficient BMDMs

Project description:APOBEC1 wildtype (WT) and Apobec1-/- (KO) unstimulated bone marrow-derived macrophages (BMDMs) were subject to ribosome profiling (n=6 for each genotype) in order to investigate the effect of APOBEC1 on translation

2018-12-31 | GSE97286 | GEO

The anti-Shine-Dalgarno sequence drives translational pausing and codon choice in bacteria

Project description:Protein synthesis by ribosomes takes place on a linear substrate but at variable speeds. Transient pausing of ribosomes can impact a variety of co-translational processes, including protein targeting and folding. These pauses are influenced by the sequence of the mRNA. Thus redundancy in the genetic code allows the same protein to be translated at different rates. However, our knowledge of both the position and the mechanism of translational pausing in vivo is highly limited. Here we present a genome-wide analysis of translational pausing in bacteria using ribosome profiling-deep sequencing of ribosome-protected mRNA fragments. This approach enables high-resolution measurement of ribosome density profiles along most transcripts at unperturbed, endogenous expression levels. Unexpectedly, we found that codons decoded by rare tRNAs do not lead to slow translation under nutrient-rich conditions. Instead, Shine-Dalgarno-(SD) like features within coding sequences cause pervasive translational pausing. Using an orthogonal ribosome possessing an altered anti-SD sequence, we demonstrated that pausing is due to hybridization between mRNA and the 16S rRNA of the translating ribosome. In protein coding sequences, internal SD sequences are disfavoured, which leads to biased usage, avoiding codons and codon pairs that resemble canonical SD sites. Our results indicate that internal SD-like sequences are a major determinant of translation rates and a global driving force for the coding of bacterial genomes. Identification of translation pause sites in vivo using ribosome profiling

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