Project description:How viruses, such as the emerging mosquito-borne Chikungunya virus (CHIKV), express their genomes at high levels despite an enrichment in suboptimal codons remains a puzzling question. By integrating subcellular fractionation and transcriptome-wide analyses of translation in CHIKV-infected human cells, we demonstrate an unanticipated virus-induced reprogramming of the host translation machinery to favor translation of viral RNA over cellular genes featuring optimal codon usage. This reprogramming was specifically apparent at the endoplasmic reticulum (ER), the preferred translation compartment of CHIKV RNA, and it is mediated by the wobble uridine 34 tRNA modification enzyme KIAA1456 whose expression is enhanced upon viral infection. Since KIAA1456 itself is encoded by a CHIKV-like codon usage, infection triggers a positive feed-back loop that ensures efficient virus protein production. Our findings demonstrate an unprecedented interplay of viruses with the host tRNA epitranscriptome to favor viral protein expression.
Project description:A general means of viral attenuation involves the extensive recoding of synonymous codons in the viral genome. The mechanistic underpinnings of this approach remain unclear, however. Using quantitative proteomics and RNA sequencing, we explore the molecular basis of attenuation in a strain of bacteriophage T7 whose major capsid gene was engineered to carry 182 suboptimal codons. We do not detect transcriptional effects from recoding. Proteomic observations reveal that translation is halved for the recoded major capsid gene, and a more modest reduction applies to several co-expressed downstream genes. We observe no changes in protein abundances of other co-expressed genes that are encoded upstream. Viral burst size, like capsid protein abundance, is also decreased by half. Together, these observations suggest that, in this virus, reduced translation of an essential polycistronic transcript and diminished virion assembly form the molecular basis of attenuation.
Project description:A general means of viral attenuation involves the extensive recoding of synonymous codons in the viral genome. The mechanistic underpinnings of this approach remain unclear, however. Using quantitative proteomics and RNA sequencing, we explore the molecular basis of attenuation in a strain of bacteriophage T7 whose major capsid gene was engineered to carry 182 suboptimal codons. We do not detect transcriptional effects from recoding. Proteomic observations reveal that translation is halved for the recoded major capsid gene, and a more modest reduction applies to several co-expressed downstream genes. We observe no changes in protein abundances of other co-expressed genes that are encoded upstream. Viral burst size, like capsid protein abundance, is also decreased by half. Together, these observations suggest that, in this virus, reduced translation of an essential polycistronic transcript and diminished virion assembly form the molecular basis of attenuation.
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.
Project description:Protein translation depends on mRNA-specific initiation, elongation, and termination rates. While the regulation of ribosome elongation is well studied in bacteria and yeast, less is known in higher eukaryotes. Here, we combined ribosome and tRNA profiling to investigate the relations between ribosome elongation rates, (aminoacyl-) tRNA levels and codon usage in mammals. We modeled codon-specific ribosome dwell times and translation fluxes from ribosome profiling, considering pair-interactions between ribosome sites. In mouse liver, the model revealed site and codon specific dwell times, as well as codon pair-interactions clustering by amino acids. While translation fluxes varied significantly across diurnal time and feeding regimen, codon dwell times were highly stable, and conserved in human. Fasting had no effect on codon dwell times in mouse liver. Profiling of total and aminoacylated tRNAs revealed highly heterogeneous levels with specific isoacceptor patterns and a correlation with codon usage. tRNAs for isoleucine, asparagine, aspartate and arginine were lowly loaded and conserved in fasted mice. Finally, codons with low levels of charged tRNAs and high codon usage relative to tRNA abundance exhibited long dwell times. Together, these analyses pave the way towards understanding the complex relation between tRNA loading, codon usage and ribosome dwell times in mammals.