Combinitorial Approaches to Viral Attenuation

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. Examination of promoter knockouts, stop codon deletion, and codon-deoptimization on viral gene expression.减毒病毒（attenuated viruses）具有诸多应用场景，尤其在活病毒疫苗（live viral vaccines）领域。然而，定向设计减毒病毒仍颇具挑战，若要求减毒状态能够抵御快速的进化回复（evolutionary recovery），这一难题尤为突出。本研究以经典的噬菌体T7（bacteriophage T7）为病毒模型，开发并分析了一种全新的减毒方法——启动子敲除（promoter ablation）。敲除两种高表达T7蛋白（支架蛋白与衣壳蛋白）的启动子后，靶基因的转录本丰度（transcript abundance）显著降低，双敲除（double knockout）的效应近似于两个单敲除（single knockout）效应的叠加。病毒适合度（fitness）的降幅较为温和，且同样呈现近似加性效应；经过长期传代适应后，病毒的适合度仅得到部分恢复，且并未修复被敲除的启动子。将启动子敲除与此前已验证的衣壳蛋白基因密码子去优化（codon deoptimization）相结合，其对适合度的影响低于加性效应，这与二者作用机制相互竞争的预期相符。在某一工程设计中，该操作产生了意料之外的附加减毒效应，我们对该效应进行了回溯性研究与阐释。总体而言，基因组工程用于减毒的作用机制与效应呈现出可预测性。因此，本研究表明，病毒减毒方法的理性设计正逐渐具备可行性。本研究还考察了启动子敲除、终止密码子缺失（stop codon deletion）以及密码子去优化对病毒基因表达的影响。