Mechanism of virus attenuation by codon pair deoptimization
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA556267 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR9737070.fastq.gz | Fastqsanger.gz | |||
| SRR9737071.fastq.gz | Fastqsanger.gz | |||
| SRR9737072.fastq.gz | Fastqsanger.gz | |||
| SRR9737073.fastq.gz | Fastqsanger.gz | |||
| SRR9737064.fastq.gz | Fastqsanger.gz |
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Project description:Human respiratory syncytial virus (RSV) is the most important viral agent of serious pediatric respiratory-tract disease worldwide. A vaccine or generally effective antiviral drug is not yet available. We designed new live attenuated RSV vaccine candidates by codon-pair deoptimization (CPD). Specifically, viral ORFs were recoded by rearranging existing synonymous codons to increase the content of underrepresented codon pairs. Amino acid coding was completely unchanged. Four CPD RSV genomes were designed in which the indicated ORFs were recoded: Min A (NS1, NS2, N, P, M, and SH), Min B (G and F), Min L (L), and Min FLC (all ORFs except M2-1 and M2-2). Surprisingly, the recombinant CPD viruses were temperature-sensitive for replication in vitro (level of sensitivity: Min FLC > Min L > Min B > Min A). All of the CPD mutants grew less efficiently in vitro than recombinant wild-type (WT) RSV, even at the typically permissive temperature of 32 °C (growth efficiency: WT > Min L > Min A > Min FLC > Min B). CPD of the ORFs for the G and F surface glycoproteins provided the greatest restrictive effect. The CPD viruses exhibited a range of restriction in mice and African green monkeys comparable with that of two attenuated RSV strains presently in clinical trials. This study provided a new type of attenuated RSV and showed that CPD can rapidly generate vaccine candidates against nonsegmented negative-strand RNA viruses, a large and expanding group that includes numerous pathogens of humans and animals.
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Project description:Recent evidence indicates that codon usage bias regulates gene expression. How viruses, such as the emerging mosquito-borne Chikungunya virus (CHIKV), express their genomes at high levels despite an enrichment in rare codons remains a puzzling question. Using ribosome footprinting, here we analysed translational changes that occur upon CHIKV infection at the ER and the cytosol. We show that CHIKV infection induces codon-specific reprogramming of the host translation machinery to favor the translation of viral RNA genomes over host mRNAs with an otherwise optimal codon usage. This reprogramming was mostly apparent at the ER, where CHIKV RNAs are efficiently translated. Mechanistically, it involves CHIKV-induced overexpression of KIAA1456, an enzyme that modifies the wobble U34 position in the anticodon of tRNAs, which we find is required for proper decoding of codons that are highly enriched in CHIKV RNAs. Our findings demonstrate an unprecedented interplay of viruses with the host tRNA epitranscriptome to adapt the host translation machinery to viral production.
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Project description:Zika virus (ZIKV) infection during the large epidemics in the Americas is related to congenital abnormities or fetal demise. To date, there is no vaccine, antiviral drug, or other modality available to prevent or treat Zika virus infection. Here we designed novel live attenuated ZIKV vaccine candidates using a codon pair deoptimization strategy. Three codon pair-deoptimized ZIKVs (Min E, Min NS1, and Min E+NS1) were de novo synthesized and recovered by reverse genetics and contained large amounts of underrepresented codon pairs in the E gene and/or NS1 gene. The amino acid sequence was 100% unchanged. The codon pair-deoptimized variants had decreased replication fitness in Vero cells (Min NS1 ≫ Min E > Min E+NS1), replicated more efficiently in insect cells than in mammalian cells, and demonstrated diminished virulence in a mouse model. In particular, Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer, and a single immunization achieved complete protection against lethal challenge and vertical ZIKV transmission during pregnancy. More importantly, due to the numerous synonymous substitutions in the codon pair-deoptimized strains, reversion to wild-type virulence through gradual nucleotide sequence mutations is unlikely. Our results collectively demonstrate that ZIKV can be effectively attenuated by codon pair deoptimization, highlighting the potential of Min E+NS1 as a safe vaccine candidate to prevent ZIKV infections.IMPORTANCE Due to unprecedented epidemics of Zika virus (ZIKV) across the Americas and the unexpected clinical symptoms, including Guillain-Barré syndrome, microcephaly, and other birth defects in humans, there is an urgent need for ZIKV vaccine development. Here we provided the first attenuated versions of ZIKV with two important genes (E and/or NS1) that were subjected to codon pair deoptimization. Compared to parental ZIKV, the codon pair-deoptimized ZIKVs were mammal attenuated and preferred insect to mammalian cells. Min E+NS1, the most restrictive variant, induced sterilizing immunity with a robust neutralizing antibody titer and achieved complete protection against lethal challenge and vertical virus transmission during pregnancy. More importantly, the massive synonymous mutational approach made it impossible for the variant to revert to wild-type virulence. Our results have proven the feasibility of codon pair deoptimization as a strategy to develop live attenuated vaccine candidates against flaviviruses such as ZIKV, Japanese encephalitis virus, and West Nile virus.
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Project description:A codon usage consonant with the cellular tRNA pool is important for efficient translation. However, Mimivirus, a giant DNA virus, relies on the host translation system of amoeba yet exhibits a codon usage distinct from its host. How this virus copes with the mismatch between the demand and supply of tRNAs remains unknown. Here, we found that the virus generates a subcellular compartment to translate virus mRNAs efficiently. The combination of genome-wide Ribo-Seq and RNA-Seq reveals that ribosomes traversed along viral mRNAs without pausing, despite the codon usage incompatibility with the host mRNAs. Although the virus encodes tRNA genes in the genome, mim-tRNA-Seq shows that the expression does not impact the composition of the cellular tRNA pool during infection. Instead, we observed that through in situ labeling of newly synthesized proteins, viral protein synthesis locally occurs around the periphery region of the viral factory, likely creating a unique environment to facilitate viral translation. Our data provide a perspective on how local translation shapes viral replication overcoming the mismatch of tRNA supply and codon demand.
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Project description:A codon usage consonant with the cellular tRNA pool is important for efficient translation. However, Mimivirus, a giant DNA virus, relies on the host translation system of amoeba yet exhibits a codon usage distinct from its host. How this virus copes with the mismatch between the demand and supply of tRNAs remains unknown. Here, we found that the virus generates a subcellular compartment to translate virus mRNAs efficiently. The combination of genome-wide Ribo-Seq and RNA-Seq reveals that ribosomes traversed along viral mRNAs without pausing, despite the codon usage incompatibility with the host mRNAs. Although the virus encodes tRNA genes in the genome, mim-tRNA-Seq shows that the expression does not impact the composition of the cellular tRNA pool during infection. Instead, we observed that through in situ labeling of newly synthesized proteins, viral protein synthesis locally occurs around the periphery region of the viral factory, likely creating a unique environment to facilitate viral translation. Our data provide a perspective on how local translation shapes viral replication overcoming the mismatch of tRNA supply and codon demand.
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Project description:A codon usage consonant with the cellular tRNA pool is important for efficient translation. However, Mimivirus, a giant DNA virus, relies on the host translation system of amoeba yet exhibits a codon usage distinct from its host. How this virus copes with the mismatch between the demand and supply of tRNAs remains unknown. Here, we found that the virus generates a subcellular compartment to translate virus mRNAs efficiently. The combination of genome-wide Ribo-Seq and RNA-Seq reveals that ribosomes traversed along viral mRNAs without pausing, despite the codon usage incompatibility with the host mRNAs. Although the virus encodes tRNA genes in the genome, mim-tRNA-Seq shows that the expression does not impact the composition of the cellular tRNA pool during infection. Instead, we observed that through in situ labeling of newly synthesized proteins, viral protein synthesis locally occurs around the periphery region of the viral factory, likely creating a unique environment to facilitate viral translation. Our data provide a perspective on how local translation shapes viral replication overcoming the mismatch of tRNA supply and codon demand.
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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.
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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.
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