SUMO Binding by the Epstein-Barr Virus Protein Kinase BGLF4 Is Crucial for BGLF4 Function

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...

Section: E Pstein-barr Virus (Ebv) Is a Ubiquitous Gammaherpesvirus Tmentioning

confidence: 74%

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

Chang

Lee

Huang

et al. 2012

“…In a protein array phosphorylation analysis, SUMO-1 was identified as a BGLF4 substrate (32) and two SUMO binding motifs (SIMs) were identified in BGLF4 (87). Because SUMOylated RanGAP1 can form complexes with Nup358 (88), our observation that BGLF4 induced the RanGAP1 redistribution (Fig.…”

Section: Discussionmentioning

confidence: 91%

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

Chang

Lee

et al. 2015

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 ␤ 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. IMPORTANCEDuring 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.

“…Expression of Z then leads to activation of R expression, and together Z and R induce expression of all the lytic EBV proteins, including the EBV-encoded protein kinase (EBV-PK). Since the EBV protein kinase (encoded by the BGLF4 gene) has been shown to enhance ATM activity through phosphorylation of Tip60 (54,55), this leads to further amplification of ATM kinase activity. Viral DNA replication, which occurs downstream of Z and R activation, may also further activate ATM, although ATM activity is not required for viral DNA replication.…”

Section: Discussionmentioning

confidence: 99%

The Cellular Ataxia Telangiectasia-Mutated Kinase Promotes Epstein-Barr Virus Lytic Reactivation in Response to Multiple Different Types of Lytic Reactivation-Inducing Stimuli

Hagemeier

Barlow

Qiao

et al. 2012

The Epstein-Barr virus (EBV) latent-to-lytic switch is mediated by the viral proteins BZLF1 (Z), BRLF1 (R), and BRRF1 (Na). Since we previously showed that DNA-damaging agents (including chemotherapy and irradiation) can induce EBV lytic reactivation and recently demonstrated that wild-type p53 contributes to lytic reactivation, we investigated the role of the ATM kinase during EBV reactivation. ATM phosphorylates and activates p53, as well as numerous other substrates involved in the cellular DNA damage response. Using an ATM inhibitor (KU55933), we found that ATM activity is required for efficient induction of EBV lytic gene expression by a variety of different stimuli, including a histone deacetylase (HDAC) inhibitor, the transforming growth factor ␤ (TGF-␤) cytokine, a demethylating agent (5-azacytidine), B cell receptor engagement with anti-IgG antibody, hydrogen peroxide, and the proteosome inhibitor bortezomib. In EBV-infected AGS (gastric) cells, knockdown of ATM, or p53, expression inhibits EBV reactivation. Conversely, treatment of these cells with nutlin-3 (which activates p53 and ATM) robustly induces lytic reactivation in a p53-and ATM-dependent manner. The ability of the EBV R and Na proteins to induce lytic reactivation in EBV-infected AGS cells is ATM dependent. However, overexpression of Z induces lytic gene expression in the presence or absence of ATM activity. Our results suggest that ATM enhances Z promoter activity in the context of the intact EBV genome and that p53 contributes to the ATM effect. Nevertheless, since we found that ATM inhibitors also reduce lytic reactivation in Burkitt lymphoma cells that have no p53, additional ATM substrates must also contribute to the ATM effect. E pstein-Barr virus (EBV) is a gammaherpesvirus that is the cause of infectious mononucleosis and is associated with a variety of epithelial and B cell cancers, including nasopharyngeal carcinoma (NPC), a subset of gastric carcinomas, Burkitt lymphoma (BL), and Hodgkin's disease (77,102). Like all herpesviruses, EBV can infect cells in either latent or lytic forms. Following infection of humans, EBV establishes long-term viral latency in the memory B cell compartment but can be reactivated to undergo the lytic form of infection following plasma cell differentiation (51). EBV infection of normal epithelial cells generally results in lytic infection (32,93,94), although EBV ϩ epithelial cell tumors such as NPCs and gastric carcinomas are primarily composed of cells with latent forms of infection (48,77). Both the latent and lytic forms of EBV infection are essential for the long-term success of the virus, and the latent-lytic switch is tightly controlled by both cellular and viral factors.The two EBV immediate-early (IE) proteins, BZLF1 (Z, also known as Zta, ZEBRA, or EB1) and BRLF1 (R), are transcription factors that activate expression of lytic viral promoters, and overexpression of either the Z or R IE protein is sufficient to reactivate the lytic form of EBV infection in many latently infected cell lines (2, ...