Dynamically shifting protein-protein interactions (PPIs) regulate cellular responses to viruses and the resulting immune signaling. Here, we use thermal proximity coaggregation (TPCA) mass spectrometry to characterize the on-off behavior of PPIs during infection with herpes simplex virus 1 (HSV-1), a virus with an ancient history of coevolution with hosts. Advancing the TPCA analysis to infer associations de novo, we build a time-resolved portrait of thousands of host-host, virus-host, and virus-virus PPIs. We demonstrate that, early in infection, the DNA sensor IFI16 recruits the active DNA damage response kinase, DNA-dependent protein kinase (DNA-PK), to incoming viral DNA at the nuclear periphery. We establish IFI16 T149 as a substrate of DNA-PK upon viral infection or DNA damage. This phosphorylation promotes IFI16-driven cytokine responses. Together, we characterize the global dynamics of PPIs during HSV-1 infection, uncovering the co-regulation of IFI16 and DNA-PK functions as a missing link in immunity to herpesvirus infection.

动态变化的蛋白质-蛋白质相互作用 (PPI) 调节细胞对病毒的反应和由此产生的免疫信号。在这里，我们使用热邻近共聚集 (TPCA) 质谱来表征 PPI 在感染单纯疱疹病毒 1 (HSV-1) 期间的开关行为，这种病毒具有与宿主共同进化的古老历史。推进 TPCA 分析以从头推断关联，我们构建了数千个宿主-宿主、病毒-宿主和病毒-病毒 PPI 的时间分辨图。我们证明，在感染早期，DNA 传感器 IFI16 将活性 DNA 损伤反应激酶、DNA 依赖性蛋白激酶 (DNA-PK) 募集到核外围的传入病毒 DNA。我们将 IFI16 T149 确定为病毒感染或 DNA 损伤后 DNA-PK 的底物。这种磷酸化促进了 IFI16 驱动的细胞因子反应。