Dual roles of USP39 in stabilizing PB2 and orchestrating ribonucleoprotein assembly drive H5 influenza virus replication and pathogenicity

Host cells combat avian influenza virus (AIV) infection by targeting viral polymerase PB2 for degradation, yet how the virus counteracts this remains elusive. In this study, we analyze the host proteins interacting with H5 AIV PB2 and identify USP39 as a deubiquitinase with dual functions in viral replication. Catalytically, USP39 directly deubiquitinates PB2 at lysine 660 (K660), preventing its degradation and sustaining polymerase activity. In parallel, independently of enzymatic activity, USP39 promotes PB2-PB1 association, facilitating formation of ribonucleoprotein (RNP) complexes. These complementary functions amplify viral RNA synthesis, dampen host antiviral responses, and drive efficient viral replication. Consistently, a PB2 K660R substitution enhances viral replication in vitro and increases pathogenicity in mice. Our findings reveal a mechanism by which AIV hijacks USP39 to circumvent host ubiquitination, facilitate RNP biogenesis, and identify USP39 as a promising therapeutic target for broadly effective antivirals against pandemic-prone H5 viruses.