Molecular Mechanism Of Nuclear Transport Of The DNA Polymerase Of Pseudorabies Virus

Pseudorabies virus(PRV), also known as Suid herpesvirus 1 or Aujeszky’s disease virus, belongs to the genus Varicellovirus in the subfamily Alphaherpesvirinae of the family Herpesviridae. Many livestocks and wild animals are infected with PRV, and the pigs are the major reservoir and infectious source. PRV is prevalent in many countries and poses a great threat to the swine industry, causing tremendous economic losses. PRV DNA replication is a complex process, requiring the synergistic effects of multiple enzymatic proteins encoded by the virus, in which the viral DNA polymerase plays a pivotal role. The PRV DNA polymerase is composed of a catalytic subunit UL30 and an accessory subunit UL42. UL30 has inherent DNA polymerase activity for catalyzing DNA synthesis, and UL42 tethers the DNA polymerase holoenzyme to the DNA template, thus enhancing the processivity of the holoenzyme. The PRV DNA polymerase must be transported into the nucleus after its synthesis in the cytoplasm, a prerequisite for its function in the initiation of the viral DNA replication. However, the molecular mechanism of nuclear transport of the PRV DNA polymerase remains unresolved. As the PRV DNA polymerase accessory subunit, whether UL42 is essential for viral replication remains unclear. The aim of this study was to elucidate the molecular mechanism of nuclear transport of the PRV DNA polymerase UL42 and UL30 subunits, and to clarify the function of UL42 during PRV DNA replication.To elucidate the nuclear import pathways of the PRV DNA polymerase UL42 and UL30 subunits, through immunofluorescence analysis, this study demonstrated that UL42 localizes independently in the nucleus, and UL30 alone predominantly localizes in the cytoplasm, whereas the localization of UL30 was completely shifted to the nucleus when it was coexpressed with UL42, confirming that nuclear transport of UL30 occurs in an UL42-dependent manner. Deletion analysis and site-directed mutagenesis of the two proteins showed that UL42 contains a functional and transferable bipartite nuclear localization signal(NLS) at amino acids 354–370, and that K354, R355, and K367 are important for the integral structure and function of this bipartite NLS, whereas UL30 has no functional NLS. Coimmunoprecipitation assays verified that UL42 interacts with importin α3(Impα3) and Impα4 through its NLS. In vitro nuclear transport assays demonstrated that nuclear localization of UL42 is a temperature-, energy-, and receptor-dependent process, and requires both Impα and Impβ, confirming that UL42 utilizes the Impα/β-mediated pathway for nuclear import. When UL30 was coexpressed with an UL42 NLS-null mutant(UL42ΔNLS), the UL42ΔNLS/UL30 heterodimer was completely confined to the cytoplasm, indicating that UL30 utilizes the NLS function of UL42 for its translocation into the nucleus. These results suggest that nuclear transport of the PRV DNA polymerase holoenzyme complex is dependent on the bipartite NLS in UL42 and is mediated by the Impα/β pathway after its assembly in the cytoplasm.It has been shown that PRV UL42 is able to stimulate the catalytic activity of UL30 in vitro, and it is the nuclear cotransporter of UL30 capable of directing nuclear import of UL30 in vitro. Thus, we speculated that UL42 is an essential gene of PRV replication. This study analyzed the kinetics of UL42 expression after PRV infection, and found that UL42 is an early gene of PRV and can be determined at 5 h after PRV infection. We designed three UL42-specific si RNAs based on UL42 sequence feature, and Western blot assay demonstrated that they can efficiently inhibit UL42 expression in vitro. Following PRV infection of si RNAs-transfected mammalian cells, these three si RNAs induced dose-dependent inhibitory effects on UL42 expression; moreover, downregulation of UL42 expression remarkably reduced PRV replication, indicating that UL42-targeted RNAi can efficiently decrease UL42 expression, thereby inhibiting PRV replication. These results suggest that UL42 is an essential gene of PRV replication.In summary, this study elucidated the molecular mechanism of nuclear transport of the PRV DNA polymerase, and demonstrated that UL42 is an essential gene of PRV replication. It has important scientific significance for further understanding the replication mechanism of PRV.