Functional characterization of nuclear trafficking signals in Pseudorabies Virus pUL31

The herpesviral nuclear egress complex (NEC), consisting of pUL31 and pUL34 homologs, mediates efficient translocation of newly synthesized capsids from the nucleus to the cytosol. The tail-anchored membrane protein pUL34 is autonomously targeted to the nuclear envelope, while pUL31 is recruited to the inner nuclear membrane (INM) by interaction with pUL34. A nuclear localization signal (NLS) in several pUL31 homologs suggests importin-mediated translocation of the protein. Here we demonstrate that deletion or mutation of the NLS in pseudorabies virus (PrV) pUL31 resulted in an exclusive cytosolic localization indicating active nuclear export. Deletion or mutation of a predicted nuclear export signal (NES) in mutants lacking a functional NLS resulted in diffuse nuclear and cytosolic localization indicating that both signals are functional. pUL31 molecules lacking the complete NLS or NES were not recruited to the INM by pUL34, while the site-specifically mutated proteins formed the NEC and partially complemented the defect of the UL31-deletion mutant. Our data demonstrate that the N-terminus of pUL31 encompassing the NLS is required for efficient nuclear targeting but not for pUL34 interaction, while the C-terminus containing the NES, but not necessarily the NES itself, is required for complex formation and efficient budding of viral capsids at the INM. Moreover, pUL31-ΔNLS displayed a dominant negative effect on PrV-wild type replication probably by diverting pUL34 to cytoplasmic membranes.