The Pseudorabies Virus DNA Polymerase Accessory Subunit UL42 Directs Nuclear Transport of the Holoenzyme

文献类型： 外文期刊

第一作者： Wang, Yi-Ping

作者： Wang, Yi-Ping;Du, Wen-Juan;Huang, Li-Ping;Wei, Yan-Wu;Wu, Hong-Li;Feng, Li;Liu, Chang-Ming

作者机构：

关键词： pseudorabies virus;DNA polymerase;accessory subunit;UL42;nuclear transport

期刊名称：FRONTIERS IN MICROBIOLOGY （ 影响因子：5.64； 五年影响因子：6.32 ）

ISSN： 1664-302X

年卷期： 2016 年 7 卷

页码：

收录情况： SCI

摘要： Pseudorabies virus (PRV) DNA replication occurs in the nuclei of infected cells and requires the viral DNA polymerase. The PRV DNA polymerase comprises a catalytic subunit, UL3O, and an accessory subunit, UL42, that confers processivity to the enzyme. Its nuclear localization is a prerequisite for its enzymatic function in the initiation of viral DNA replication. However, the mechanisms by which the PRV DNA polymerase holoenzyme enters the nucleus have not been determined. In this study, we characterized the nuclear import pathways of the PRV DNA polymerase catalytic and accessory subunits. Immunofluorescence analysis showed that UL42 localizes independently in the nucleus, whereas UL3O alone predominantly localizes in the cytoplasm. Intriguingly, the localization of UL3O was completely shifted to the nucleus when it was coexpressed with UL42, demonstrating that nuclear transport of UL3O 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 K-354, R-355, and K-367 are important for the NLS function, whereas UL3O has no NLS. Coimmunoprecipitation assays verified that UL42 interacts with importins alpha 3 and alpha 4 through its NLS. In vitro nuclear import assays demonstrated that nuclear accumulation of UL42 is a temperature-and energy-dependent process and requires both importins alpha and beta, confirming that UL42 utilizes the importin alpha/beta-mediated pathway for nuclear entry. In an UL42 NLS-null mutant, the UL42/UL3O heterodimer was completely confined to the cytoplasm when UL42 was coexpressed with UL3O, indicating that UL3O utilizes the NLS function of UL42 for its translocation into the nucleus. Collectively, these findings suggest that UL42 contains an importin alpha/beta-mediated bipartite NLS that transports the viral DNA polymerase holoenzyme into the nucleus in an in vitro expression system.

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