Combinitorial Approaches to Viral Attenuation
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA474818 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7268720.fastq.gz | Fastqsanger.gz | |||
| SRR7268730.fastq.gz | Fastqsanger.gz | |||
| SRR7268740.fastq.gz | Fastqsanger.gz | |||
| SRR7268750.fastq.gz | Fastqsanger.gz | |||
| SRR7268760.fastq.gz | Fastqsanger.gz |
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Project description:Attenuated viruses have numerous applications, in particular in the context of live viral vaccines. However, purposefully designing attenuated viruses remains challenging, in particular if the attenuation is meant to be resistant to rapid evolutionary recovery. Here we develop and analyze a new attenuation method, promoter ablation, using an established viral model, bacteriophage T7. Ablating promoters of the two most highly expressed T7 proteins (scaffold and capsid) led to major reductions in transcript abundance of the affected genes, with the effect of the double knockout approximately additive of the effects of single knockouts. Fitness reduction was moderate and also approximately additive; fitness recovery on extended adaptation was partial and did not restore the promoters. The fitness effect of promoter knockouts combined with a previously tested codon deoptimization of the capsid gene was less than additive, as anticipated from their competing mechanisms of action. In one design, the engineering created an unintended consequence that led to further attenuation, the effect of which was studied and understood in hindsight. Overall, the mechanisms and effects of genome engineering on attenuation behaved in a predictable manner. Therefore, this work suggests that the rational design of viral attenuation methods is becoming feasible.
2018-06-07 | GSE115396 | GEO
Project description:Attenuated viruses have numerous applications, in particular in the context of live viral vaccines. However, purposefully designing attenuated viruses remains challenging, in particular if the attenuation is meant to be resistant to rapid evolutionary recovery. Here we develop and analyze a new attenuation method, promoter ablation, using an established viral model, bacteriophage T7. Ablation of promoters of the two most highly expressed T7 proteins (scaffold and capsid) led to major reductions in transcript abundance of the affected genes, with the effect of the double knockout approximately additive of the effects of single knockouts. Fitness reduction was moderate and also approximately additive; fitness recovery on extended adaptation was partial and did not restore the promoters. The fitness effect of promoter knockouts combined with a previously tested codon deoptimization of the capsid gene was less than additive, as anticipated from their competing mechanisms of action. In one design, the engineering created an unintended consequence that led to further attenuation, the effect of which was studied and understood in hindsight. Overall, the mechanisms and effects of genome engineering on attenuation behaved in a predictable manner. Therefore, this work suggests that the rational design of viral attenuation methods is becoming feasible. IMPORTANCE Live viral vaccines rely on attenuated viruses that can successfully infect their host but have reduced fitness or virulence. Such attenuated viruses were originally developed through trial and error, typically by adaptation of the wild-type virus to novel conditions. That method was haphazard, with no way of controlling the degree of attenuation or the number of attenuating mutations or preventing evolutionary reversion. Synthetic biology now enables rational design and engineering of viral attenuation, but rational design must be informed by biological principles to achieve stable, quantitative attenuation. This work shows that in a model system for viral attenuation, bacteriophage T7, attenuation can be obtained from rational design principles, and multiple different attenuation approaches can be combined for enhanced overall effect.
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Project description:Amino acid changes in two viral proteins drive attenuation of the yellow fever 17D vaccine
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Project description:Severe acute respiratory syndrome coronavirus (SARS-CoV) causes a respiratory disease leading to death in 10% of the infected people. A mouse adapted SARS-CoV lacking the envelope (E) protein (rSARS-CoV-MA15-?E) is attenuated in vivo. To identify E protein domains and host responses that contribute to rSARS-CoV-MA15-?E attenuation, several mutants (rSARS-CoV-MA15-E*) containing point mutations or deletions in the amino-terminal or the carboxy-terminal regions of E protein, respectively, were generated. Amino acid substitutions in the amino terminus, or deletion of domains in the internal carboxy terminal region of E protein led to viral attenuation. Attenuated viruses induced minimal lung injury and limited neutrophil influx to the lungs but, interestingly, increased CD4+ and CD8+ T cell counts in BALB/c mice. To analyze the host responses leading to rSARS-CoV-MA15-E* attenuation, the differential gene expression elicited by the native virus and the mutant ones in infected cells was analyzed. The expression levels of a large number of proinflammatory cytokines inducing lung injury was reduced in the lungs of rSARS-CoV-MA15-E* infected mice, whereas the levels of anti-inflammatory cytokines were increased, both at the mRNA and protein levels. These results suggested that the reduction in lung inflammation together with a specific antiviral T cell response, contributed to rSARS-CoV-MA15-E* attenuation. Interestingly, the attenuated viruses completely protected mice against the challenge with the lethal parental virus, being promising vaccine candidates. Three biological replicates were independently hybridized (one channel per slide) for each sample type (rSARS-CoV-MA15-wt, rSARS-CoV-MA15-?E, rSARS-CoV-MA15-?3, rSARS-CoV-MA15-?5, Mock). Slides were Sure Print G3 Agilent 8x60K Mouse (G4852A-028005)
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Project description:Viruses promote infection by hijacking the host ubiquitin machinery to counteract or redirect cellular processes. Adenovirus encodes two early proteins, E1B55K and E4orf6, that together co-opt a cellular ubiquitin ligase complex to overcome host defenses and promote virus production. Adenovirus mutants lacking E1B55K or E4orf6 display defects in viral RNA processing and protein production, but previously identified substrates of the ligase do not explain these phenotypes. Here we used a quantitative proteomics approach to identify substrates of E1B55K/E4orf6 that are ubiquitinated to facilitate RNA processing. While cellular proteins known as substrates of E1B55K/E4orf6 are degraded by the proteasome, we uncovered RNA-binding proteins (RBPs) as high-confidence substrates which are not decreased in overall abundance. We focused on two predominant RBPs, RALY and hnRNP-C, which we confirm are ubiquitinated without degradation. Knockdown of RALY and hnRNP-C rescued levels of viral RNA splicing, protein, and progeny production during infection with E1B55K-deleted virus. Furthermore, deletion of E1B55K resulted in increased interaction of hnRNP-C with viral RNA and attenuation of viral RNA processing. These data suggest viral-mediated ubiquitination of RALY and hnRNP-C relieves a restriction on viral RNA processing, revealing an unexpected role for non-degradative ubiquitination in the manipulation of cellular processes during virus infection.
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Project description:Yellow fever virus, a prototypical member of the Flaviviridae family, is a small single-stranded, positive sense enveloped RNA virus causing viscerotropic, frequently fatal disease. Serial passaging of the virulent YFV Asibi strain in the 1930s yielded the YFV17D strain which remains one of the most effective vaccines ever developed. Remarkably, YFV17D and the virulent parental genome differ only by 68 nucleotides leading to 32 amino acid changes. However, it remains largely unknown which of these sequence differences are responsible for attenuation. Here, we demonstrate that while synonymous mutations are highly conserved across pathogenic YFVs they do not contribute to the characteristic host responses elicited by 17D or virulent viruses. Using a highly modular combinatorial genetic approach we identified key genetic elements in the YFV envelope and non-structural 2A (NS2A) proteins that govern the virus’ ability to spread and suppress host responses.. Introducing these mutations into infectious clones of other YFV genomes results in viral attenuation in vitro and in vivo. Collectively, our results define the genetic basis for YFV17D attenuation and highlight a general approach for creating live-attenuated vaccines for other pathogenic viruses.
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Project description:Yellow fever virus, a prototypical member of the Flaviviridae family, is a small single-stranded, positive sense enveloped RNA virus causing viscerotropic, frequently fatal disease. Serial passaging of the virulent YFV Asibi strain in the 1930s yielded the YFV17D strain which remains one of the most effective vaccines ever developed. Remarkably, YFV17D and the virulent parental genome differ only by 68 nucleotides leading to 32 amino acid changes. However, it remains largely unknown which of these sequence differences are responsible for attenuation. Here, we demonstrate that while synonymous mutations are highly conserved across pathogenic YFVs they do not contribute to the characteristic host responses elicited by 17D or virulent viruses. Using a highly modular combinatorial genetic approach we identified key genetic elements in the YFV envelope and non-structural 2A (NS2A) proteins that govern the virus’ ability to spread and suppress host responses.. Introducing these mutations into infectious clones of other YFV genomes results in viral attenuation in vitro and in vivo. Collectively, our results define the genetic basis for YFV17D attenuation and highlight a general approach for creating live-attenuated vaccines for other pathogenic viruses.
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Project description:Yellow fever virus, a prototypical member of the Flaviviridae family, is a small single-stranded, positive sense enveloped RNA virus causing viscerotropic, frequently fatal disease. Serial passaging of the virulent YFV Asibi strain in the 1930s yielded the YFV17D strain which remains one of the most effective vaccines ever developed. Remarkably, YFV17D and the virulent parental genome differ only by 68 nucleotides leading to 32 amino acid changes. However, it remains largely unknown which of these sequence differences are responsible for attenuation. Here, we demonstrate that while synonymous mutations are highly conserved across pathogenic YFVs they do not contribute to the characteristic host responses elicited by 17D or virulent viruses. Using a highly modular combinatorial genetic approach we identified key genetic elements in the YFV envelope and non-structural 2A (NS2A) proteins that govern the virus’ ability to spread and suppress host responses.. Introducing these mutations into infectious clones of other YFV genomes results in viral attenuation in vitro and in vivo. Collectively, our results define the genetic basis for YFV17D attenuation and highlight a general approach for creating live-attenuated vaccines for other pathogenic viruses.
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Project description:Severe acute respiratory syndrome coronavirus (SARS-CoV) causes a respiratory disease leading to death in 10% of the infected people. A mouse adapted SARS-CoV lacking the envelope (E) protein (rSARS-CoV-MA15-ΔE) is attenuated in vivo. To identify E protein domains and host responses that contribute to rSARS-CoV-MA15-ΔE attenuation, several mutants (rSARS-CoV-MA15-E*) containing point mutations or deletions in the amino-terminal or the carboxy-terminal regions of E protein, respectively, were generated. Amino acid substitutions in the amino terminus, or deletion of domains in the internal carboxy terminal region of E protein led to viral attenuation. Attenuated viruses induced minimal lung injury and limited neutrophil influx to the lungs but, interestingly, increased CD4+ and CD8+ T cell counts in BALB/c mice. To analyze the host responses leading to rSARS-CoV-MA15-E* attenuation, the differential gene expression elicited by the native virus and the mutant ones in infected cells was analyzed. The expression levels of a large number of proinflammatory cytokines inducing lung injury was reduced in the lungs of rSARS-CoV-MA15-E* infected mice, whereas the levels of anti-inflammatory cytokines were increased, both at the mRNA and protein levels. These results suggested that the reduction in lung inflammation together with a specific antiviral T cell response, contributed to rSARS-CoV-MA15-E* attenuation. Interestingly, the attenuated viruses completely protected mice against the challenge with the lethal parental virus, being promising vaccine candidates.
2015-05-19 | GSE59185 | GEO