Abstract / Summary
Pseudorabies virus (PRV), an alphaherpesvirus, establishes lifelong latency in neurons and requires efficient reactivation for pathogenesis. Thymidine kinase (TK), encoded by UL23, is a key virulence factor essential for viral replication in non-dividing cells and for reactivation from latency; however, the regulatory mechanisms governing TK function remain poorly defined. Here, we identify TK as a substrate for small ubiquitin-like modifier (SUMO) modification and investigate its functional significance during PRV infection. We show that TK undergoes endogenous SUMOylation during viral infection is conjugated by SUMO-1, SUMO-2, and SUMO-3, with lysine residues K242 and K267 serving as the principal SUMO acceptor sites. Disruption of these SUMOylation sites does not affect TK stability and kinase activity but markedly alters its subcellular localization by reducing nuclear accumulation. Functionally, SUMOylation of TK is dispensable for viral replication in epithelial PK15 cells but facilitates efficient replication in neuronal N2a cells. Using an in vitro latency/reactivation model, we further show that SUMOylation-deficient TK does not affect the establishment of latent infection but markedly impairs viral reactivation and subsequent productive replication. In a mouse infection model, disruption of TK SUMOylation attenuates PRV pathogenicity, accompanied by reduced viral loads in the brain and milder histopathological lesions. Collectively, our findings demonstrate that SUMOylation regulates nuclear localization and biological function of PRV TK, thereby facilitating efficient neuronal replication, viral reactivation, and pathogenesis. These findings provide new insights into the post-translational regulation of alphaherpesvirus TK and identify TK SUMOylation as a potential target for controlling PRV infection.