Structural Insights Into the Nuclear Import of Marek's Disease Virus Large Tegument Protein.

Marek's disease (MD) is a highly contagious neoplastic disorder of poultry caused by MD virus (MDV; gallid alphaherpesvirus 2 [GaAHV2]). Infection results in profound immunosuppression, neurological dysfunction, and the development of malignant T-cell lymphomas. Continued viral evolution has produced increasingly virulent strains capable of partially or fully evading current vaccines, leaving few options for controlling emerging variants. This highlights the importance of identifying new antiviral targets, particularly those involved in the nuclear trafficking events essential for GaAHV2 replication. The UL36 large tegument protein of alphaherpesviruses contains N-terminal nuclear localization signals (NLSs) thought to guide capsid transport to the nuclear pore complex. However, the specific mechanism by which GaAHV2 UL36 engages the host nuclear import machinery remains unclear. In this work, we defined the NLS within the N-terminal region of GaAHV2 UL36 and characterized its interaction with importin proteins. Through high-resolution crystallography and quantitative binding assays, we pinpointed the residues and structural motifs within UL36 that mediate recognition by importin-α (IMPα) and compared their affinities across different IMPα isoforms. Our structural and biochemical data show that the predicted N-terminal NLS of GaAHV2 UL36 is essential for IMPα binding. These findings provide a detailed molecular framework for host-virus interactions during GaAHV2 nuclear entry and offer potential avenues for the development of targeted antiviral strategies.