The BTK-DDX41 axis of the STING pathway is activated during cytomegalovirus lytic infection.

The innate immune response is the first line of defense against invading pathogens, including the betaherpesvirus human cytomegalovirus (CMV). The host's innate response acts as the first line of defense, and CMV, like other viruses, has consequently evolved multiple mechanisms to manipulate host interferon (IFN) responses. DEAD-box helicase 41 (DDX41) is an intracellular dsDNA sensor that, upon activation by Bruton's tyrosine kinase (BTK), triggers type I IFN production through the stimulator of interferon genes (STING) signaling pathway. Here, we show the activation of this signaling pathway during lytic CMV infection, wherein BTK, DDX41, and STING are activated through tyrosine phosphorylation, and both DDX41 and BTK interact with STING. Furthermore, CMV infection re-localizes a fraction of DDX41 from the nucleus to the cytoplasm. Here, DDX41 phosphorylation is attenuated, suggesting that cytoplasmic redistribution leads to a less active or inactive form. Additionally, a pool of DDX41 co-localizes in the virus assembly compartment (vAC) with the CMV tegument proteins, pp65 and pp71, each of which interact with DDX41 in immunoprecipitation assays. We further demonstrate the protective role of this signaling pathway, as treatment with the BTK inhibitor orelabrutinib attenuates DDX41 phosphorylation/activation and supports increased expression of viral proteins and virus replication. In sum, our work highlights the important role of BTK-DDX41-STING signaling in the innate immune response against CMV, which the virus subverts by attenuating its cytoplasmic activity, thereby diverting it from its typically protective function. IMPORTANCE: Human cytomegalovirus (CMV) is an ubiquitous pathogen that poses a significant threat to immunocompromised individuals, highlighting the critical role of innate immunity in controlling this viral infection. Despite extensive research, the complex mechanisms underlying innate immunity against CMV and the virus's strategies for evading immune detection remain only partially understood. This study identifies the activation of the cellular BTK-DDX41-STING innate signaling axis during lytic CMV infection, which ultimately results in protective interferon responses. Our findings show that CMV infection triggers the cytoplasmic redistribution of the cellular protein, DDX41, leading to reduced phosphorylation and activity, thereby undermining its protective function. Additionally, pharmacological inhibition of BTK enhances viral protein expression and replication, highlighting the importance of this pathway in immune defense. Our work identifies BTK- and DDX41-dependent STING signaling as important for innate immune responses against CMV and further advances understanding of CMV's manipulation of these responses.