title: BGLF4 kinase modulates the structure and transport preference of the nuclear pore complex to facilitate nuclear import of Epstein-Barr virus lytic proteins.
abstract: BGLF4 kinase, the only Ser/Thr protein kinase encoded by the Epstein-Barr virus (EBV) genome, phosphorylates multiple viral and cellular substrates to optimize the cellular environment for viral DNA replication and the nuclear egress of nucleocapsids. Previously, we found that nuclear targeting of BGLF4 is through direct interaction with the FG repeat-containing nucleoporins (FG-Nups) Nup62 and Nup153 independently of cytosolic transport factors. Here, we investigated the regulatory effects of BGLF4 on the structure and biological functions of the nuclear pore complex (NPC). In EBV-positive NA cells, the distribution of FG-Nups was modified during EBV reactivation. In transfected cells, BGLF4 changed the staining pattern of Nup62 and Nup153 in a kinase activity-dependent manner. Detection with anti-phospho-Ser/Thr-Pro MPM-2 antibody demonstrated that BGLF4 induced the phosphorylation of Nup62 and Nup153. The nuclear targeting of importin β was attenuated in the presence of BGLF4, leading to inhibition of canonical nuclear localization signal (NLS)-mediated nuclear import. An in vitro nuclear import assay revealed that BGLF4 induced the nuclear import of larger molecules. Notably, we found that BGLF4 promoted the nuclear import of several non-NLS-containing EBV proteins, including the viral DNA-replicating enzymes BSLF1, BBLF2/3, and BBLF4 and the major capsid protein (VCA), in cotransfected cells. The data presented here suggest that BGLF4 interferes with the normal functions of Nup62 and Nup153 and preferentially helps the nuclear import of viral proteins for viral DNA replication and assembly. In addition, the nuclear import-promoting activity was found in cells expressing the BGLF4 homologs of another two gammaherpesviruses but not those from alpha- and betaherpesviruses.
  
  
  OBJECTIVE
  During lytic replication, many EBV genome-encoded proteins need to be transported into the nucleus, not only for viral DNA replication but also for the assembly of nucleocapsids. Because nuclear pore complexes are effective gateways that control nucleocytoplasmic traffic, most EBV proteins without canonical NLSs are retained in the cytoplasm until they form complexes with their NLS-containing partners for nuclear targeting. In this study, we found that EBV BGLF4 protein kinase interacts with the Nup62 and Nup153 and induces the redistribution of FG-Nups. BGLF4 modulates the function of the NPC to inhibit the nuclear import of host NLS-containing proteins. Simultaneously, the nuclear import of non-NLS-containing EBV lytic proteins was enhanced, possibly through phosphorylation of Nup62 and Nup153, nuclear pore dilation, or microtubule reorganization. Overall, our data suggest that BGLF4-induced modification of nuclear pore transport may block nuclear targeting of cellular proteins and increase the import of viral proteins to promote viral lytic replication.
authors.affiliation: Graduate Institute and Department of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Chou-Wei
authors.initials: CW
authors.lastname: Chang
authors.affiliation: General Education Center, National Taipei University of Nursing and Health Sciences, Taipei, Taiwan.
authors.forename: Chung-Pei
authors.initials: CP
authors.lastname: Lee
authors.affiliation: Graduate Institute and Department of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Mei-Tzu
authors.initials: MT
authors.lastname: Su
authors.affiliation: Graduate Institute and Department of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Ching-Hwa
authors.initials: CH
authors.lastname: Tsai
authors.affiliation: Graduate Institute and Department of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan mrc@ntu.edu.tw.
authors.forename: Mei-Ru
authors.initials: MR
authors.lastname: Chen
mesh_terms: D021581:Active Transport, Cell Nucleus; D002460:Cell Line; D004854:Herpesvirus 4, Human / Q000502:physiology*; D054884:Host-Pathogen Interactions; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D022022:Nuclear Pore / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*; D019065:Virus Assembly; D014779:Virus Replication
chemical_list: D008562:Membrane Glycoproteins; C079377:NUP153 protein, human; D028861:Nuclear Pore Complex Proteins; D014764:Viral Proteins; C049312:nuclear pore protein p62; C406063:BGLF4 protein, Epstein-Barr virus; D017346:Protein Serine-Threonine Kinases