Impact of viral and cellular protein phosphorylation on the regulation of innate antiviral immune responses against influenza A virus infections

流感病毒是编码能力有限的小RNA病毒。因此，病毒蛋白必须在受感染的细胞中发挥多种功能。这意味着必须有允许以时空方式控制多功能的机制。我们假设，病毒和细胞蛋白的动态磷酸化是迄今为止被低估的病毒繁殖和对感染的先天免疫反应的调节层。为了解决这个问题，我们遵循了一种策略，将重点放在一组单独的功能相关的病毒和细胞磷蛋白上，这些蛋白是由我们自己的磷酸蛋白质组筛选确定的，这些蛋白在先天性免疫反应和病毒复制中具有可疑功能。在之前的资助期间，我们不仅能够描述一系列病毒和细胞蛋白磷酸化的调节功能，而且还确定了可能用于新的宿主定向抗感染策略的靶点。我们现在已经进行了额外的磷酸化蛋白质组和动态组筛选，发现了病毒干扰素拮抗蛋白NS1的更多新的磷酸化位点，以及在病毒相关功能网络分析中起核心作用的激酶和激酶调节因子。我们现在希望在这些发现的基础上，通过对NS1中新发现的三个位点的功能表征来加强我们的研究，这一次还考虑了与邻近磷酸受体位点的动态相互作用。此外，我们将揭开两个特殊的激酶，CDK1和PKA的作用，它们在病毒复制周期中被差异地磷酸化和激活，也与先天性免疫反应有关，并被描述为磷酸化NS1。这项研究将提供关于调节宿主-病毒相互作用的更深层次的知识，并是发现药物干预的新线索的关键。

Influenza viruses are small RNA viruses with a limited coding capacity. Therefore, viral proteins have to fulfill multiple functions in the infected cell. This implies that there must be mechanisms allowing for the control of multi-functionality in a spatio-temporal manner. We hypothesized that dynamic phosphorylation of viral and cellular proteins is a so far underestimated layer of regulation of virus propagation and innate immune responses to infection. To address this, we followed a strategy to focus for in depth functional analysis on a set of individual functionally related viral and cellular phosphoproteins identified by our own phospho-proteomic screen with a suspected function in the innate immune response and viral replication. In the previous funding period, we were not only able to describe the regulatory function of phosphorylation of a range of viral and cellular proteins, but also identified targets that may be used for novel host-directed anti-infective strategies. We have now performed additional phospho-proteome and kinome screenings, which identified further novel phospho-sites of the viral IFN-antagonistic protein NS1 as well as kinases and kinase regulators that are central in a virusrelated functional network analysis. We now want to build on these findings and enhance our studies by functional characterization of the three newly identified sites in NS1, this time also considering dynamic interaction with neighboring phospho-acceptor sites. Furthermore, we will unravel the role of two particular kinases, Cdk1 and PKA, that are differentially phosphorylated and activated during the virus replication cycle and also are linked to innate immune responses and described to phosphorylate NS1. This study will provide deeper knowledge about the regulatory host-virus interplay and is key to uncover new leads for pharmaceutical intervention.