UCHL3 Regulates Subgenomic Flaviviral RNA Condensates to Promote Virus Propagation

ABSTRACT Flavivirus subgenomic RNAs (sfRNAs) antagonise antiviral defences, yet how sfRNAs are organized and maintained in cells remains poorly understood. Here we identify ubiquitin C‐terminal hydrolase L3 (UCHL3) as a post‐translational regulator of flavivirus sfRNA stability and function. Using activity‐based protein profiling during ZIKV and DENV infections, we discovered that UCHL3 is activated upon flavivirus infection. CRISPR‐Cas9 knockout of UCHL3 significantly impaired viral replication and reduced viral protein expression across multiple cell models; reconstitution with wild‐type UCHL3 rescued these defects, whereas a catalytically inactive mutant (C95A) failed to restore replication, confirming the requirement for deubiquitylase activity. Through a proximity‐biotinylation sfRNA‐interactome capture assay, we show that UCHL3 physically interacts with sfRNA complexes. Importantly, UCHL3 deletion accelerates RNase L activation, causing enhanced sfRNA relocalization from protective P‐bodies to PABPC1‐positive RNase L‐induced bodies (RLBs), resulting in viral RNA decay. RNase L knockdown restores viral replication in UCHL3‐deficient cells, confirming RNase L‐dependent antiviral effects. This pro‐viral effect of UCHL3 operates through interferon‐independent mechanisms, as replication defects persist despite exogenous interferon treatment. This work therefore identifies UCHL3 as a regulator of sfRNA fate between pro‐viral and antiviral RNA condensates, identifying a post‐translational mechanism governing viral RNA stability and a potential therapeutic target for flavivirus infections.