RAD18 Knockout Cell Line (HEK293)

The transcription factor interferon regulatory factor 3 (IRF3) initiates type I interferon transcription, which is required for host defense. Here, we identify RAD18 as a central E3 ubiquitin ligase that selectively targets phosphorylated IRF3 (p-IRF3) for autophagic degradation. RAD18 specifically promotes the dissociation of p-IRF3 from the IFNB promoter and in turn terminates its transcriptional activity. Mechanistically, RAD18 binds the p-IRF3 dimer located on the IFNB promoter and triggers K63 polyubiquitylation of p-IRF3 at Lys 193. The ubiquitylated p-IRF3 dimer consequently dissociates from the IFNB promoter, translocates out of the nucleus and undergoes OPTN-mediated autophagic degradation. Rad18 Lysm-cre mice resist lethal vesicular stomatitis virus infection in vivo due to IFNβ overproduction. In H1N1-infected human macrophages or monocytes from individuals with active systemic lupus erythematosus, RAD18 protein levels negatively correlate with p-IRF3 and IFNB1 mRNA levels. Thus, RAD18 functions as a break to terminate IRF3-driven IFNB1 transcription and may be a potential therapeutic target for RNA virus infection or autoimmune diseases.

The nuclear-cytoplasmic trafficking of matrix proteins (M) is essential for henipavirus budding, with M protein ubiquitination playing a pivotal role in this dynamic process. Despite its importance, the intricacies of the M ubiquitination cascade have remained elusive. In this study, we elucidate a novel mechanism by which Nipah virus (NiV), a highly pathogenic henipavirus, utilizes a ubiquitination complex involving the E2 ubiquitin-conjugating enzyme RAD6A and the E3 ubiquitin ligase RAD18 to ubiquitinate the virus's M protein, thereby facilitating its nuclear-cytoplasmic trafficking. We demonstrate that RAD18 interacts with RAD6A, enabling the latter to supply ubiquitins for the RAD18-mediated transfer of ubiquitin to M through RAD18-M interactions. Specifically, M is ubiquitinated by the RAD6A-RAD18 complex at lysine (K) 258 through a K63-linked ubiquitination, a modification crucial for M's function. This ubiquitination drives M's relocation to the cytoplasm, directing it to plasma membranes for effective viral egress. Conversely, disrupting the RAD6A-RAD18-M axis, mutating RAD18's E3 ligase activity, or inhibiting RAD6A activity with TZ9 (a RAD6-ubiquitin thioester formation inhibitor) impairs M ubiquitination, resulting in defective nuclear export and budding of NiV. Significantly, live NiV and Hendra virus infection is attenuated in RAD18 knockout cells or in cells treated with TZ9, highlighting the critical physiological role of RAD6A-RAD18-mediated M ubiquitination in the henipavirus life cycle. Our findings not only reveal how NiV manipulates a nucleus-localized ubiquitination complex to promote virus's M protein ubiquitination and nuclear export, but also suggest that the small molecule inhibitor TZ9 could serve as a potential therapeutic against henipavirus infection.