Epstein-Barr Virus Protein Kinase BGLF4 Targets the Nucleus through Interaction with Nucleoporins

Abstract

bBGLF4 of Epstein-Barr virus (EBV) encodes a serine/threonine protein kinase that phosphorylates multiple viral and cellular substrates to optimize the cellular environment for viral DNA replication and the nuclear egress of viral nucleocapsids. BGLF4 is expressed predominantly in the nucleus at early and late stages of virus replication, while a small portion of BGLF4 is distributed in the cytoplasm at the late stage of virus replication and packaged into the virion. Here, we analyzed systematically the functional domains crucial for nuclear localization of BGLF4 and found that both the N and C termini play important modulating roles. Analysis of amino acid substitution mutants revealed that the C terminus of BGLF4 does not contain a conventional nuclear localization signal (NLS). Additionally, deletion of the C-terminal putative helical regions at amino acids 386 to 393 and 410 to 419 diminished the nuclear translocation of BGLF4, indicating that the secondary structure of the C terminus is important for the localization of BGLF4. The green fluorescent protein-fused wild-type or C-terminal helical regions of BGLF4 associate with phenylalanine/glycine repeat-containing nucleoporins (Nups) in nuclear envelope fractionation. Both coimmunoprecipitation and in vitro pull-down assays further demonstrated that BGLF4 binds to Nup62 and Nup153. Remarkably, nuclear import assay with permeabilized HeLa cells demonstrated that BGLF4 translocated into nucleus independent of cytosolic factors. Data presented here suggest that BGLF4 employs a novel mechanism through direct interactions with nucleoporins for its nuclear targeting. E pstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus that infects most of the human population worldwide (64).Upon stimulation of various kinds, the latent virus may be reactivated through expression of the viral immediate-early transactivators Zta and Rta, which then turn on the expression cascade of viral genes (39, 41). BGLF4 kinase is a virion-associated serine/ threonine kinase expressed during the early and late stages of the lytic cycle (55). Several viral proteins expressed at different stages of virus replication have been shown to be phosphorylated by BGLF4, including EBNA2, EBNA-LP, BMRF1, BZLF1, viral DNA replication proteins, and structural components (3,26,27,61,65,66). In cells replicating EBV, BGLF4 colocalizes with the viral DNA polymerase processivity factor BMRF1 in the viral DNA replication compartment and phosphorylates BMRF1 at multiple sites in vitro and in vivo (55,61). BGLF4 phosphorylates the cellular replication origin binding complex MCM4-MCM6-MCM7, leading to inhibition of its helicase activities (31). Our study also indicated that BGLF4 recruits the cellular nucleotide excision repair protein XPC to the viral replication compartment to enhance viral DNA replication (40). Expression of BGLF4 alone in transfected cells induces premature chromosome condensation through the activation of condensin and topoisomerase II (33). Most importantly, small interfering RNA expe...

BGLF4 Kinase Modulates the Structure and Transport Preference of the Nuclear Pore Complex To Facilitate Nuclear Import of Epstein-Barr Virus Lytic Proteins

“…The plasmid expressing green fluorescent protein (GFP)-tagged BGLF4 (GFP-BGLF4) (pCPL4) was generated in a previous study (41). GFP-K102I was generated using a single-primer mutagenesis protocol with pCPL4 as the template (38). pGEX-2T-HA-PTAC97 (glutathione S-transferase [GST]-importin ␤) was a gift from Y. Yoneda (Osaka University, Osaka, Japan).…”

“…HeLa cell lysates were harvested and immunoprecipitated with MAb414 or Nup62 antibody, and the pulled down immunocomplexes served as the substrates for immunoprecipitation (IP) kinase assays. Purified BGLF4 linked to GST (GST-BGLF4) or the BGLF4 K102I mutant linked to GST (GST-K102I) from Saccharomyces cerevisiae yeast (38) was incubated with the immunoprecipitated proteins in the presence of 10 Ci [␥-…”

“…Nuclear envelope fractionation was performed as described previously with slight modifications (38,49). To isolate nuclei, HeLa cells (3 ϫ 10 6 ) transfected with BGLF4, the BGLF4 kinase-dead K102I mutant, or the vector control were incubated with 700 l hypotonic buffer (10 mM Tris HCl, pH 8, 60 mM KCl, 1.5 mM MgCl 2 , 0.5% NP-40, 1 mM phenylmethylsulfonyl fluoride [PMSF]) on ice for 1.5 h. After centrifugation at 200 ϫ g for 5 min, the pellet was harvested as the nuclear fraction.…”

“…Nuclear import assays were performed on the basis of a previously described protocol with slight modification (38,50). For the nuclear import of GSTimportin ␤, HeLa cells expressing BGLF4, the BGLF4 kinase-dead K102I mutant, or the vector control were grown on slides and permeabilized at RT with 40 g/ml digitonin (Sigma) in transport buffer (TB; 20 mM HEPES, pH 7.3, 110 mM potassium acetate, 2 mM magnesium acetate, 1 mM EGTA, 2 mM DTT, 1ϫ protease inhibitor) for 5 min, followed by 5 min on ice, and washed with ice-cold TB twice.…”

“…Simultaneously, BGLF4 induces the dissociation of a portion of FG-Nups from the nuclear rim into the nucleus, revealed by confocal microscopy (38). In this study, we aimed to identify the mechanism involved in the structural and functional changes of the NPC during EBV replication.…”