The dynamic proteome of influenza A virus infection identifies M segment splicing as a host range determinant

A century ago, influenza A virus (IAV) infection caused the 1918 flu pandemic and killed an estimated 20-40 million people. Pandemic IAV outbreaks occur when strains from animal reservoirs acquire the ability to infect and spread among humans. The molecular details of this species barrier are incompletely understood. We combined metabolic pulse labeling and quantitative shotgun proteomics to globally monitor protein synthesis upon infection of human cells with a human- and a bird-adapted IAV strain. While production of host proteins was remarkably similar, we observed striking differences in the kinetics of viral protein synthesis over the course of infection. Most importantly, the matrix protein M1 was inefficiently produced by the bird-adapted strain at later stages. We show that impaired production of M1 from bird-adapted strains is caused by increased splicing of the M segment RNA to alternative isoforms. Experiments with reporter constructs and recombinant influenza viruses revealed that strain-specific M segment splicing is controlled by the 3’ splice site and functionally important for permissive infection. Independent in silico and biochemical evidence shows that avian-adapted M segments have evolved different conserved RNA structure features than human-adapted sequences. Thus, our data identifies M segment RNA splicing as a viral determinant of host range.

一个世纪前，甲型流感病毒（influenza A virus, IAV）感染引发了1918年流感大流行，据估计造成2000万至4000万人死亡。甲型流感病毒大流行的暴发，源于来自动物宿主的毒株获得了感染人类并实现人际传播的能力。目前学界对这一种属屏障的分子机制尚未完全阐明。本研究结合代谢脉冲标记（metabolic pulse labeling）技术与定量鸟枪法蛋白质组学（quantitative shotgun proteomics），对人类细胞感染人源适应性与禽源适应性甲型流感病毒毒株后的蛋白质合成情况进行了全局监测。尽管宿主蛋白的整体合成水平极为相近，但研究团队观察到，在感染进程中，两种毒株的病毒蛋白合成动力学特征存在显著差异。尤为关键的是，禽源适应性毒株在感染后期无法高效合成基质蛋白M1。本研究证实，禽源适应性毒株的M1蛋白合成受损，是由其M片段RNA的可变剪接比例升高所导致的。通过报告基因构建体（reporter constructs）与重组流感病毒（recombinant influenza viruses）开展的实验表明，毒株特异性的M片段剪接受3’剪接位点（3’ splice site）调控，且该调控过程对病毒实现允许性感染具有重要功能意义。独立的计算机模拟（in silico）与生化实验证据显示，相较于人源适应性序列，禽源适应性M片段已演化出不同的保守RNA结构特征。综上，本研究结果表明，M片段RNA剪接是决定病毒宿主范围的病毒因子。