Identification of nuclear and nucleolar localization signals of pseudorabies virus (PRV) early protein UL54 reveals that its nuclear targeting is required for efficient production of PRV.

The pseudorabies virus (PRV) early protein UL54 is a homologue of herpes simplex virus 1 (HSV-1) immediate-early protein ICP27, which is a multifunctional protein that is essential for HSV-1 infection. In this study, the subcellular localization and nuclear import signals of PRV UL54 were characterized. UL54 was shown to predominantly localize to the nucleolus in transfected cells. By constructing a series of mutants, a functional nuclear localization signal (NLS) and a genuine nucleolar localization signal (NoLS) of UL54 were for the first time identified and mapped to amino acids (61)RQRRR(65) and (45)RRRRGGRGGRAAR(57), respectively. Additionally, three recombinant viruses with mutations of the NLS and/or the NoLS in UL54 were constructed based on PRV bacterial artificial chromosome (BAC) pBecker2 to test the effect of UL54 nuclear targeting on viral replication. In comparison with the wild-type virus, a recombinant virus harboring an NLS or NoLS mutation of UL54 reduced viral production to different extents. However, mutations of both the NLS and NoLS targeted UL54 to the cytoplasm in recombinant virus-infected cells and significantly impaired viral replication, comparable to the UL54-null virus. In addition, a virus lacking the NLS or the NoLS displayed modest defects in viral gene expression and DNA synthesis. However, deletion of both the NLS and the NoLS resulted in severe defects in viral gene expression and DNA synthesis, as well as production of infectious progeny. Thus, we have identified a classical NLS and a genuine NoLS in UL54 and demonstrate that the nuclear targeting of UL54 is required for efficient production of PRV.