Structural and functional characterization of siadenovirus core protein VII nuclear localization demonstrates the existence of multiple nuclear transport pathways

Adenovirus protein VII (pVII) plays a crucial role in the nuclear localization of genomic DNA following viral infection and contains nuclear localization signal (NLS) sequences for the importin (IMP)-mediated nuclear import pathway. However, functional analysis of pVII in adenoviruses to date has failed to fully determine the underlying mechanisms responsible for nuclear import of pVII. Therefore, in the present study, we extended our analysis by examining the nuclear trafficking of adenovirus pVII from a non-human species, psittacine siadenovirus F (PsSiAdV). We identified a putative classical (c)NLS at pVII residues 120-128 ((120)PGGFKRRRL(128)). Fluorescence polarization and electrophoretic mobility shift assays demonstrated direct, high-affinity interaction with both IMP alpha 2 and IMP alpha 3 but not IMP beta. Structural analysis of the pVII-NLS/IMP alpha 2 complex confirmed a classical interaction, with the major binding site of IMP alpha occupied by K-124 of pVII-NLS. Quantitative confocal laser scanning microscopy showed that PsSiAdV pVII-NLS can confer IMP alpha/beta-dependent nuclear localization to GFP. PsSiAdV pVII also localized in the nucleus when expressed in the absence of other viral proteins. Importantly, in contrast to what has been reported for HAdV pVII, PsSiAdV pVII does not localize to the nucleolus. In addition, our study demonstrated that inhibition of the IMP alpha/beta nuclear import pathway did not prevent PsSiAdV pVII nuclear targeting, indicating the existence of alternative pathways for nuclear localization, similar to what has been previously shown for human adenovirus pVII. Further examination of other potential NLS signals, characterization of alternative nuclear import pathways, and investigation of pVII nuclear targeting across different adenovirus species is recommended to fully elucidate the role of varying nuclear import pathways in the nuclear localization of pVII.