Epstein-Barr Virus Polymerase Processivity Factor Enhances BALF2 Promoter Transcription as a Coactivator for the BZLF1 Immediate-Early Protein

Sanae Nakayama,Teru Kanda,Yoshihiro Yasui,Takayuki Murata,Yoshitaka Sato,Ayumi Kudoh,Hiroki Isomura,Satoko Iwahori,Tatsuya Tsurumi,Kazutaka Murayama

doi:10.1074/jbc.m109.015685

Sanae Nakayama, Teru Kanda + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m109.015685

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Abstract

The Epstein-Barr virus (EBV) BMRF1 protein is an essential replication protein acting at viral replication forks as a viral DNA polymerase processivity factor, whereas the BALF2 protein is a single-stranded DNA-binding protein that also acts at replication forks and is most abundantly expressed during viral productive replication. Here we document that the BMRF1 protein evidently enhances viral BZLF1 transcription factor-mediated transactivation of the BALF2 gene promoter. Mutagenesis and electrophoretic mobility shift assays demonstrated the BALF2 promoter to harbor two BZLF1 protein-binding sites (BZLF1-responsive elements). Direct binding of the BZLF1 protein to BZLF1-responsive elements and physical interaction between BZLF1 and BMRF1 proteins are prerequisite for the BMRF1 protein up-regulation of the BALF2 gene promoter. A monomeric mutant, C95E, which is defective in homodimerization, could still interact and enhance BZLF1-mediated transactivation. Furthermore although EBV protein kinase phosphorylates BMRF1 protein extensively, it turned out that phosphorylation of the protein by the kinase is inhibitory to the enhancement of the BZLF1-mediated transactivation of BALF2 promoter. Exogenous expression of BMRF1 protein augmented BALF2 expression in HEK293 cells harboring the EBV genome but lacking BMRF1 and BALF5 genes, demonstrating functions as a transcriptional regulator in the context of viral infection. Overall the BMRF1 protein is a multifunctional protein that cannot only act as a DNA polymerase processivity factor but also enhances BALF2 promoter transcription as a coactivator for the BZLF1 protein, regulating the expression level of viral single-stranded DNA-binding protein.

Highlights

Several human cancers, including Burkitt lymphoma and nasopharyngeal carcinoma [1]
In the viral productive cycle, the Epstein-Barr virus (EBV) genome is amplified more than 100-fold utilizing viral replication machinery consisting of the BZLF1 protein, an oriLyt-binding protein; the BALF5 protein, a DNA polymerase (Pol); the BMRF1 protein, a Pol processivity factor; the BALF2 protein, a singlestranded DNA-binding protein; and the BBLF4, BSLF1, and BBLF2/3 proteins, which are predicted to be helicase, primase, and helicase-primase-associated proteins, respectively [5]
BZLF1-mediated Transactivation of the BALF2 Promoter Is Enhanced by BMRF1 Protein Expression—When EBV lytic replication was induced in B95-8 cells with chemical compounds, viral early proteins, such as BALF2 or BMRF1, appeared at 12 h postinduction and reached plateaus at around 24 h (Fig. 1)

Summary

Introduction

Several human cancers, including Burkitt lymphoma and nasopharyngeal carcinoma [1]. EBV possesses two alternative life styles: latent and lytic. To generate a double deletion mutant BAC of BMRF1 and BALF5 genes, EBV BAC⌬M⌬Pol, double-stranded DNAs for recombination were prepared by PCR as described above. BZLF1-mediated Transactivation of the BALF2 Promoter Is Enhanced by BMRF1 Protein Expression—When EBV lytic replication was induced in B95-8 cells with chemical compounds, viral early proteins, such as BALF2 or BMRF1, appeared at 12 h postinduction and reached plateaus at around 24 h (Fig. 1).

Results

Conclusion