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Reduced protein expression in a virus attenuated by codon deoptimization

ABSTRACT: 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.

ORGANISM(S): Escherichia coli

PROVIDER: GSE96573 | GEO | 2017/07/30

SECONDARY ACCESSION(S): PRJNA379044

REPOSITORIES: GEO

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Project description:Terminating protein translation accurately and efficiently is critical for both protein fidelity and ribosome recycling for continued translation. The three bacterial release factors (RFs) play key roles: RF1 and 2 recognize stop codons and terminate translation; and RF3 promotes disassociation of bound release factors. Probing release factors mutations with reporter constructs containing programmed frameshifting sequences or premature stop codons had revealed a propensity for readthrough or frameshifting at these specific sites, but their effects on translation genome-wide have not been examined. We performed ribosome profiling on a set of isogenic strains with well-characterized release factor mutations to determine how they alter translation globally. Consistent with their known defects, strains with increasingly severe release factor defects exhibit increasingly severe accumulation of ribosomes over stop codons, indicative of an increased duration of the termination/release phase of translation. Release factor mutant strains also exhibit increased occupancy in the region following the stop codon at a significant number of genes. Our global analysis revealed that, as expected, translation termination is generally efficient and accurate, but that at a significant number of genes (≥ 50) the ribosome signature after the stop codon is suggestive of translation past the stop codon. Even native E. coli K-12 exhibits the ribosome signature suggestive of protein extension, especially at UGA codons, which rely exclusively on the reduced function RF2 allele of the K-12 strain for termination. Deletion of RF3 increases the severity of the defect. We unambiguously demonstrate readthrough and frameshifting protein extensions and their further accumulation in mutant strains for a few select cases. In addition to enhancing recoding, ribosome accumulation over stop codons disrupts attenuation control of biosynthetic operons, and may alter expression of some overlapping genes. Together, these functional alterations may either augment the protein repertoire or produce deleterious proteins.

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Reduced protein expression in a virus attenuated by codon deoptimization

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