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Protein array identification of substrates of the Epstein-Barr virus protein kinase BGLF4.

ABSTRACT: A conserved family of herpesvirus protein kinases plays a crucial role in herpesvirus DNA replication and virion production. However, despite the fact that these kinases are potential therapeutic targets, no systematic studies have been performed to identify their substrates. We generated an Epstein-Barr virus (EBV) protein array to evaluate the targets of the EBV protein kinase BGLF4. Multiple proteins involved in EBV lytic DNA replication and virion assembly were identified as previously unrecognized substrates for BGLF4, illustrating the broad role played by this protein kinase. Approximately half of the BGLF4 targets were also in vitro substrates for the cellular kinase CDK1/cyclin B. Unexpectedly, EBNA1 was identified as a substrate and binding partner of BGLF4. EBNA1 is essential for replication and maintenance of the episomal EBV genome during latency. BGLF4 did not prevent EBNA1 binding to sites in the EBV latency origin of replication, oriP. Rather, we found that BGLF4 was recruited by EBNA1 to oriP in cells transfected with an oriP vector and BGLF4 and in lytically induced EBV-positive Akata cells. In cells transfected with an oriP vector, the presence of BGLF4 led to more rapid loss of the episomal DNA, and this was dependent on BGLF4 kinase activity. Similarly, expression of doxycycline-inducible BGLF4 in Akata cells led to a reduction in episomal EBV genomes. We propose that BGLF4 contributes to effective EBV lytic cycle progression, not only through phosphorylation of EBV lytic DNA replication and virion proteins, but also by interfering with the EBNA1 replication function.

SUBMITTER: Zhu J

PROVIDER: S-EPMC2682057 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Protein array identification of substrates of the Epstein-Barr virus protein kinase BGLF4.

Zhu Jian J Liao Gangling G Shan Liang L Zhang Jun J Chen Mei-Ru MR Hayward Gary S GS Hayward S Diane SD Desai Prashant P Zhu Heng H

Journal of virology 20090225 10

A conserved family of herpesvirus protein kinases plays a crucial role in herpesvirus DNA replication and virion production. However, despite the fact that these kinases are potential therapeutic targets, no systematic studies have been performed to identify their substrates. We generated an Epstein-Barr virus (EBV) protein array to evaluate the targets of the EBV protein kinase BGLF4. Multiple proteins involved in EBV lytic DNA replication and virion assembly were identified as previously unrec ...[more]

PMID: 19244323

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Project description:UnlabelledBGLF4 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.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.

Dataset Information

Protein array identification of substrates of the Epstein-Barr virus protein kinase BGLF4.

Publications

Protein array identification of substrates of the Epstein-Barr virus protein kinase BGLF4.

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