DNA-PK and ATM drive phosphorylation signatures that antagonistically regulate cytokine responses to herpesvirus infection or DNA damage.

The DNA-dependent protein kinase, DNA-PK, is an essential regulator of DNA damage repair. DNA-PK-driven phosphorylation events and the activated DNA damage response (DDR) pathways are also components of antiviral intrinsic and innate immune responses. Yet, it is not clear whether and how the DNA-PK response differs between these two forms of nucleic acid stress-DNA damage and DNA virus infection. Here, we define DNA-PK substrates and the signature cellular phosphoproteome response to DNA damage or infection with the nuclear-replicating DNA herpesvirus, HSV-1. We establish that DNA-PK negatively regulates the ataxia-telangiectasia-mutated (ATM) DDR kinase during viral infection. In turn, ATM blocks the binding of DNA-PK and the nuclear DNA sensor IFI16 to viral DNA, thereby inhibiting cytokine responses. However, following DNA damage, DNA-PK enhances ATM activity, which is required for IFN-β expression. These findings demonstrate that the DDR autoregulates cytokine expression through the opposing modulation of DDR kinases.