Reduced protein expression in a virus attenuated by codon deoptimization. Escherichia coli

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. Overall design: mRNA profiles of T7-infected E. coli from three different strains of T7, across three distinct time points post-infection, with three replicates of each strain-time combination.

病毒减毒的通用策略之一，是对病毒基因组内的同义密码子进行大规模重编码。然而，该策略的分子机制仍未明确。本研究借助定量蛋白质组学（quantitative proteomics）与RNA测序（RNA sequencing）技术，针对一株经基因工程改造、在主要衣壳蛋白基因中引入182个次优密码子的T7噬菌体（bacteriophage T7）菌株，探究其减毒的分子机制。
研究未检测到重编码对转录过程产生的影响。蛋白质组学分析结果显示，经重编码的主要衣壳蛋白基因的蛋白表达量降低了一半，而多个共表达的下游基因的蛋白水平则出现了小幅下降。未观察到上游编码的其他共表达基因的蛋白丰度发生变化。与衣壳蛋白丰度的变化一致，病毒裂解量（viral burst size）同样降低了一半。综合上述实验结果，本研究提示：在该噬菌体中，必需多顺反子转录本（polycistronic transcript）的翻译效率下降与病毒粒子组装受损共同构成了该毒株减毒的分子基础。
总体实验设计：针对三种不同T7菌株感染大肠杆菌的样本，于感染后三个不同时间点采集mRNA转录组谱，每个菌株-时间组合设置三次生物学重复。