甲型流感病毒(IAV)仍然对公众健康构成大流行威胁,导致每年和大流行期间的高死亡率。病毒蛋白的翻译后修饰在调节IAV感染中起着重要作用。这里,基于免疫沉淀(IP)的质谱(MS)和纯化的病毒偶联MS,共有89个磷酸化位点分布在10个编码的IAV病毒蛋白中,包括60个新的磷酸化位点。此外,第一次,我们提供的证据表明PB2也可以乙酰化,位点为K187。值得注意的是,在基于IP的MS和纯化的基于病毒的MS中反复鉴定了PB2S181磷酸化位点。S181和K187都暴露在PB2蛋白的表面,在各种IAV菌株中高度保守,表明它们在IAV生命周期中的根本重要性。生物信息学分析结果表明,S181E/A和K187Q/R模拟物突变不会显著改变PB2蛋白的结构。虽然PB2S181E突变模拟的连续磷酸化显著降低了小鼠的病毒适应性,PB2K187Q模拟乙酰化在小鼠中略微增强病毒毒力。机械上,PB2S181E实质上损害病毒聚合酶活性和病毒复制,显著抑制PB2的蛋白稳定性和核积累,并显著减弱IAV诱导的炎症反应。因此,我们的研究进一步丰富了流感病毒蛋白磷酸化和乙酰化位点的数据库,为后续机理研究奠定基础。同时,PB2S181E模拟磷酸化的抗病毒作用可能为后续抗病毒药物的研究提供新的靶点。
Influenza A virus (IAV) continues to pose a pandemic threat to public health, resulting a high mortality rate annually and during pandemic years. Posttranslational modification of viral protein plays a substantial role in regulating IAV infection. Here, based on immunoprecipitation (IP)-based mass spectrometry (MS) and purified virus-coupled MS, a total of 89 phosphorylation sites distributed among 10 encoded viral proteins of IAV were identified, including 60 novel phosphorylation sites. Additionally, for the first time, we provide evidence that PB2 can also be acetylated at site K187. Notably, the PB2 S181 phosphorylation site was consistently identified in both IP-based MS and purified virus-based MS. Both S181 and K187 are exposed on the surface of the PB2 protein and are highly conserved in various IAV strains, suggesting their fundamental importance in the IAV life cycle. Bioinformatic analysis results demonstrated that S181E/A and K187Q/R mimic mutations do not significantly alter the PB2 protein structure. While continuous phosphorylation mimicked by the PB2 S181E mutation substantially decreases viral fitness in mice, PB2 K187Q mimetic acetylation slightly enhances viral virulence in mice. Mechanistically, PB2 S181E substantially impairs viral polymerase activity and viral replication, remarkably dampens protein stability and nuclear accumulation of PB2, and significantly weakens IAV-induced inflammatory responses. Therefore, our study further enriches the database of phosphorylation and acetylation sites of influenza viral proteins, laying a foundation for subsequent mechanistic studies. Meanwhile, the unraveled antiviral effect of PB2 S181E mimetic phosphorylation may provide a new target for the subsequent study of antiviral drugs.