Segment-specific kinetics of mRNA, cRNA and vRNA during influenza infection
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ABSTRACT:
PROVIDER: PRJNA681243 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13160500_1.fastq.gz | Fastqsanger.gz | |||
| SRR13160500_2.fastq.gz | Fastqsanger.gz | |||
| SRR13160501_1.fastq.gz | Fastqsanger.gz | |||
| SRR13160501_2.fastq.gz | Fastqsanger.gz | |||
| SRR13160502_1.fastq.gz | Fastqsanger.gz |
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Project description:Influenza A virus (IAV) is a segmented negative-sense RNA virus and is the cause of major global epidemics and pandemics. The replication of IAV is complex, involving both transcription and replication, during which three distinct RNA species, namely mRNA, cRNA, and vRNA are generated for all eight genome segments. While understanding IAV replication kinetics is important for drug development and improving vaccine production, current methods for studying IAV kinetics has been limited by the ability to detect all three different RNA species in a scalable manner. Here were report the development of a novel pipeline using total stranded RNA-Seq, named Influenza Virus Enumerator of RNA Transcripts (InVERT) which allows for the simultaneous quantification of all three RNA species of IAV. Using InVERT provides a full landscape of the IAV replication kinetics, in which we found that different groups of viral genes followed different traits of kinetics. During a cycle of infection, the RNA-dependent RNA Polymerase (RdRP) produced more vRNA than mRNA while some other genes (NP, NS, HA) consistently make more mRNA than vRNA. vRNA expression levels do not correlate with the cRNA expression, suggesting complex regulations of vRNA synthesis. Furthermore, by studying the kinetics of a virus lacking the capacity to generate new polymerase complexes, we found evidence that further supports the model that cRNA synthesis requires newly synthesized RdRP and that incoming RdRP can only generate mRNA.
2021-02-19 | GSE162281 | GEO
Project description:A century ago, influenza A virus (IAV) infection caused the 1918 flu pandemic and killed an estimated 20-40 million people. Pandemic IAV outbreaks occur when strains from animal reservoirs acquire the ability to infect and spread among humans. The molecular details of this species barrier are incompletely understood. We combined metabolic pulse labeling and quantitative shotgun proteomics to globally monitor protein synthesis upon infection of human cells with a human- and a bird-adapted IAV strain. While production of host proteins was remarkably similar, we observed striking differences in the kinetics of viral protein synthesis over the course of infection. Most importantly, the matrix protein M1 was inefficiently produced by the bird-adapted strain at later stages. We show that impaired production of M1 from bird-adapted strains is caused by increased splicing of the M segment RNA to alternative isoforms. Experiments with reporter constructs and recombinant influenza viruses revealed that strain-specific M segment splicing is controlled by the 3’ splice site and functionally important for permissive infection. Independent in silico and biochemical evidence shows that avian-adapted M segments have evolved different conserved RNA structure features than human-adapted sequences. Thus, our data identifies M segment RNA splicing as a viral determinant of host range.
2019-11-11 | PXD015474 | Pride
Project description:A century ago, influenza A virus (IAV) infection caused the 1918 flu pandemic and killed an estimated 20-40 million people. Pandemic IAV outbreaks occur when strains from animal reservoirs acquire the ability to infect and spread among humans. The molecular details of this species barrier are incompletely understood. We combined metabolic pulse labeling and quantitative shotgun proteomics to globally monitor protein synthesis upon infection of human cells with a human- and a bird-adapted IAV strain. While production of host proteins was remarkably similar, we observed striking differences in the kinetics of viral protein synthesis over the course of infection. Most importantly, the matrix protein M1 was inefficiently produced by the bird-adapted strain at later stages. We show that impaired production of M1 from bird-adapted strains is caused by increased splicing of the M segment RNA to alternative isoforms. Experiments with reporter constructs and recombinant influenza viruses revealed that strain-specific M segment splicing is controlled by the 3’ splice site and functionally important for permissive infection. Independent in silico and biochemical evidence shows that avian-adapted M segments have evolved different conserved RNA structure features than human-adapted sequences. Thus, our data identifies M segment RNA splicing as a viral determinant of host range.
2019-11-11 | PXD015475 | Pride
Project description:During influenza A virus (IAV) infections, viral proteins are targeted by cellular E3 ligases for modification with ubiquitin. Here, we decipher and functionally explore the ubiquitin landscape of the IAV polymerase during infection of human alveolar epithelial cells by applying mass spectrometry analysis of immuno-purified K-ε-GG- (di-glycyl)-remnant-bearing peptides. We identified 59 modified lysines across all three subunits of the viral polymerase of which 17 distinctively affected mRNA transcription, vRNA replication and the generation of recombinant viruses via non-proteolytic mechanisms. Moreover, our results demonstrate that the ubiquitinated residue K578 in the PB1 thumb domain is crucial for the dynamic structural transitions of the viral polymerase that are required for vRNA replication. Mutations K578A and K578R impeded the steps of cRNA stabilization and vRNA transcription, respectively, and affected NP binding as well as polymerase dimerization. Collectively, our results indicate that ubiquitin-mediated disruption of the charge-dependent interaction between PB1-K578 and PB2-E72 is required to coordinate polymerase dimerization and facilitate vRNA replication, which demonstrates that IAV exploit the cellular ubiquitin system to modulate the activity of the viral polymerase for the regulation of viral replication.
2023-03-11 | PXD030816 | Pride
Project description:Influenza A virus (IAV) is a segmented negative-sense RNA virus and is the cause of major epidemics and pandemics. The replication of IAV is complex, involving the production of three distinct RNA species; mRNA, cRNA, and vRNA for all eight genome segments. While understanding IAV replication kinetics is important for drug development and improving vaccine production, current methods for studying IAV kinetics has been limited by the ability to detect all three different RNA species in a scalable manner. Here we report the development of a novel pipeline using total stranded RNA-Seq, which we named Influenza Virus Enumerator of RNA Transcripts (InVERT), that allows for the simultaneous quantification of all three RNA species produced by IAV. Using InVERT, we provide a full landscape of the IAV replication kinetics and found that different groups of viral genes follow different kinetics. The segments coding for RNA-dependent RNA Polymerase (RdRP) produced more vRNA than mRNA while some other segments (NP, NS, HA) consistently made more mRNA than vRNA. vRNA expression levels did not correlate with cRNA expression, suggesting complex regulation of vRNA synthesis. Furthermore, by studying the kinetics of a virus lacking the capacity to generate new polymerase complexes, we found evidence that further supports the model that cRNA synthesis requires newly synthesized RdRP and that incoming RdRP can only generate mRNA. Overall, InVERT is a powerful tool for quantifying IAV RNA species to elucidate key features of IAV replication.ImportanceInfluenza A virus (IAV) is a respiratory pathogen that has caused significant mortality throughout history and remains a global threat to human health. Although much is known about IAV replication, the regulation of IAV replication dynamics is not completely understood. This is due in part to both technical limitations and the complexity of the virus replication, which has a segmented genome and produces three distinct RNA species for each gene segment. We developed a new approach that allows the methodical study of IAV replication kinetics, shedding light on many interesting features of IAV replication biology. This study advances our understanding of the kinetics of IAV replication and will help to facilitate future research in the field.
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Project description:Due to the RNA nature of their genomes, influenza viruses have to utilize many RNA-binding proteins (RBPs) of both viral and host origin, for their replication. To uncover the comprehensive vRNA-host protein interactions, we performed affinity purification coupled with mass spectrometry (AP-MS) analysis of influenza vRNA complexes. The eight vRNA segments of H7N9 were transcribed and individually labeled with biotin in vitro and incubated with IAV virus-infected THP-1 cells, and vRNA complexes were enriched streptavidin magnetic beads and analyzed by mass spectrometry
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Project description:Influenza virus polymerase transcribes or replicates the segmented RNA genome (vRNA) into respectively viral mRNA or full-length copies and initiates RNA synthesis by binding the conserved 3' and 5' vRNA ends (the promoter). In recent structures of promoter-bound polymerase, the cap-binding and endonuclease domains are configured for cap snatching, which generates capped transcription primers. Here, we present a FluB polymerase structure with a bound complementary cRNA 5' end that exhibits a major rearrangement of the subdomains within the C-terminal two-thirds of PB2 (PB2-C). Notably, the PB2 nuclear localization signal (NLS)- containing domain translocates ~90 A ̊ to bind to the endonuclease domain. FluA PB2-C alone and RNA-free FluC polymerase are similarly arranged. Biophysical and cap-dependent endonuclease assays show that in solution the polymerase explores different conformational distributions depending on which RNA is bound. The inherent flexibility of the polymerase allows it to adopt alternative conformations that are likely important during polymerase maturation into active progeny RNPs.</br></br>Extra contact information:</br><a href="mailto:cusack@embl.fr">Stephen Cusack</a>, EMBL Grenoble Outstation, Unit of Virus Host-Cell Interactions, France ( corresponding author and lab head )
2016-01-04 | PXD002234 | Pride
Project description:Segment 3 of influenza A virus contains a second open reading frame accessed via robosomal frameshifting. The frameshift product, PA-Z, comprises the endonuclease domain of viral PA protein with C-terminal demain encoded by the X-ORF and functions to repress cellular gene expression. PA-X also modulates IAV virulence in a mouse infection model. The effect of PA-X deltions were examined using 2 separate mutant influenza viruses (1918-FS/1918-PCT1) and the results compared to wild-type 1918 influenza virus. Balb/c mice were infected and samples harvested at days 3, 5 and 8 days. Four animals/condition were used. Gene expression levels from infected animals were compared to mock animals.
2012-07-11 | E-GEOD-38112 | biostudies-arrayexpress
Project description:Influenza viruses pose a significant burden on global human health. Influenza has a broad cellular tropism in the airway, but how infection of different epithelial cell types impacts replication kinetics and burden in the airways is not fully understood. Using primary human airway cultures, which recapitulate the diverse epithelial cell landscape of the human airways, we investigated the impact of cell type composition on virus tropism and replication kinetics. Cultures were highly diverse across multiple donors and 30 independent differentiation conditions and supported a range of influenza replication. Although many cell types were susceptible to influenza, ciliated and secretory cells were predominantly infected. Despite the strong tropism preference for secretory and ciliated cells, which consistently make up 75% or more of infected cells, only ciliated cells were associated with increased virus production. Surprisingly, infected secretory cells were associated with overall reduced virus output. These data highlight the heterogeneous outcomes of influenza virus infections in primary human airway cultures and the disparate impacts of infected cell identity on burst size, even among preferentially infected cell types.
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Project description:Segment 3 of influenza A virus contains a second open reading frame accessed via robosomal frameshifting. The frameshift product, PA-Z, comprises the endonuclease domain of viral PA protein with C-terminal demain encoded by the X-ORF and functions to repress cellular gene expression. PA-X also modulates IAV virulence in a mouse infection model.
2012-07-12 | GSE38112 | GEO