Abstract

Alphaviruses are mosquito-borne RNA viruses that pose a significant threat to humans, with risks worsened by global warming. Non-structural protein 3 (nsP3) is critical for alphavirus infection, yet its precise roles remain poorly defined. Using Venezuelan equine encephalitis virus (VEEV) as a model, this study reveals that alphavirus unique domain (AUD) of nsP3 is essential for its condensation and viral replication. nsP3 forms gel-like condensates to bind to the second Tudor domain of Fragile X-related protein 1 (FXR1), forming replication supporting complexes. FXR1 bridges nsP3 and viral RNA within these condensates, which compartmentalize viral replication independent of protein interactions. Furthermore, UBAP2L was identified as a condensate-resident restriction factor against VEEV. Based on conserved AUD across alphaviruses, we developed a "21 R-AUD" antiviral strategy by fusing TRIM21's RING domain to VEEV's AUD. This approach induces proteasomal degradation of nsP3 condensates and inhibits replication of multiple alphaviruses, establishing conserved nsP3 condensation as a broad-spectrum antiviral target.