RNF138‐Mediated Ubiquitination and Degradation of NS5 Restricts Tick‐Borne Encephalitis Virus Infection

ABSTRACT Tick‐borne encephalitis virus (TBEV) causes severe neurological disease. However, whether host restricts its replication and the underlying mechanisms remain incompletely delineated. Here, we identify the E3 ubiquitin ligase RNF138 as an intrinsic restriction factor targeting the viral RNA‐dependent RNA polymerase (RdRp) NS5. Mechanistically, RNF138 directly interacts with the RdRp domain of NS5 through its ubiquitin‐interacting motif, catalyzes K48‐linked polyubiquitination and induces degradation of TBEV NS5. Ubiquitin‐remnant profiling and mutational analyses identify K372, K462, and K470 within NS5 as the ubiquitination sites, and mutating these residues leads to NS5 resistant to RNF138‐induced degradation. Functionally, RNF138 suppresses the replication of TBEV as well as Zika virus, a representative mosquito‐borne flavivirus, through recognizing and degrading NS5 proteins. Ectopic RNF138 reduces viral RNA levels in the brain and peripheral tissues of TBEV‐infected mice, mitigates neuroinflammatory responses, and improves survival. Notably, the antiviral activity is conserved among several mammalian RNF138 orthologs but absent in the arthropod homologs, highlighting a host‐specific antiviral adaptation. Together, these findings reveal RNF138‐mediated ubiquitination and degradation of NS5 as a mechanism of intrinsic defense against TBEV and provide a framework for exploring targeted destabilization of conserved flaviviral replicases.