Crystal Structure of Epstein-Barr Virus DNA Polymerase Processivity Factor BMRF1

Abstract

The DNA polymerase processivity factor of the Epstein-Barr virus, BMRF1, associates with the polymerase catalytic subunit, BALF5, to enhance the polymerase processivity and exonuclease activities of the holoenzyme. In this study, the crystal structure of C-terminally truncated BMRF1 (BMRF1-⌬C) was solved in an oligomeric state. The molecular structure of BMRF1-⌬C shares structural similarity with other processivity factors, such as herpes simplex virus UL42, cytomegalovirus UL44, and human proliferating cell nuclear antigen. However, the oligomerization architectures of these proteins range from a monomer to a trimer. PAGE and mutational analyses indicated that BMRF1-⌬C, like UL44, forms a C-shaped head-to-head dimer. DNA binding assays suggested that basic amino acid residues on the concave surface of the C-shaped dimer play an important role in interactions with DNA. The C95E mutant, which disrupts dimer formation, lacked DNA binding activity, indicating that dimer formation is required for DNA binding. These characteristics are similar to those of another dimeric viral processivity factor, UL44. Although the R87E and H141F mutants of BMRF1-⌬C exhibited dramatically reduced polymerase processivity, they were still able to bind DNA and to dimerize. These amino acid residues are located near the dimer interface, suggesting that BMRF1-⌬C associates with the catalytic subunit BALF5 around the dimer interface. Consequently, the monomeric form of BMRF1-⌬C probably binds to BALF5, because the steric consequences would prevent the maintenance of the dimeric form. A distinctive feature of BMRF1-⌬C is that the dimeric and monomeric forms might be utilized for the DNA binding and replication processes, respectively.The Epstein-Barr virus (EBV), 4 a human herpesvirus harboring a 172-kbp dsDNA genome, is associated with several B-cell and epithelial cell malignancies and can choose between two alternative life cycles, latent and lytic infection (1). The EBV genomes are replicated as circular plasmid molecules, using the cellular replication machinery of the host in the latent phase of the viral life cycle. On the other hand, after the induction of lytic viral replication, the EBV genome is amplified 100 -1,000-fold by the viral replication machinery. The replication intermediates are large head-to-tail concatemers resulting from rolling-circle DNA replication initiated from oriLyt (2). The EBV replication machinery consists of seven viral gene products (3) as follows: the BZLF1 protein, an oriLytbinding protein; the BALF5 protein, a DNA polymerase catalytic subunit; the BMRF1 protein, a polymerase processivity factor; the BALF2 protein, a single-stranded DNA-binding protein; and the BBLF4, BSLF1, and BBLF2/3 proteins, putative helicase, primase, and helicase-primase-associated proteins, respectively. It has been suggested that all of the proteins, except for the BZLF1 protein, work together at replication forks to synthesize the leading and lagging strands of the concatemeric EBV genome (2). The EBV DNA polymerase holoenzy...

Nuclear Transport of Epstein-Barr Virus DNA Polymerase Is Dependent on the BMRF1 Polymerase Processivity Factor and Molecular Chaperone Hsp90

“…BMRF1 is a major phosphoprotein demonstrating abundant expression in lytic replication-induced cells (8,9) when the expression level of the BALF5 protein is low. The BMRF1 can form head-to-head homodimer or tetrameric ring in solution (10). Judging from the finding that almost all expressed BMRF1 proteins bind to viral genome DNA (2,11), the factor could not only act as a polymerase processivity factor but also perform other unknown functions (2).…”

Tetrameric Ring Formation of Epstein-Barr Virus Polymerase Processivity Factor Is Crucial for Viral Replication

“…The mutant, however, retained polymerase processivity activity (38) and could still enhance BZLF1-mediated transactivation of the BALF2 promoter (39). The C206E mutant, whose mutation should prevent the tailto-tail contact, still formed a head-to-head dimer retaining DNA binding activity and polymerase processivity (38). Although the H141F mutant exhibited reduced polymerase processivity, it was still able to bind DNA and to dimerize.…”

“…A monomeric C95E mutant, which is impaired in head-to-head homodimerization, showed decreased DNA binding activity in vitro, suggesting that dimer formation enhances its DNA binding. The mutant, however, retained polymerase processivity activity (38) and could still enhance BZLF1-mediated transactivation of the BALF2 promoter (39). The C206E mutant, whose mutation should prevent the tailto-tail contact, still formed a head-to-head dimer retaining DNA binding activity and polymerase processivity (38).…”

“…From our recent resolution of the crystal structure of Cterminally truncated BMRF1 protein (38), the molecular structure shares structural similarity with other processivity factors, such as herpes simplex virus type 1 (HSV-1) UL42, human cytomegalovirus (HCMV) UL44, and human proliferating cell nuclear antigen (PCNA). Most BMRF1 proteins form a Cshaped head-to-head homodimer, but some form ring-shaped tetramers through tail-to-tail association (Fig.…”

“…In our previous study (38), several BMRF1 mutants were prepared: the C95E, H141F, and C206E mutations are predicted to affect the dimer interface, and the K19E, K29E, R87E, K99E, and R256E mutations are in the putative DNA binding region. Some were mapped on the molecular surface, as shown in Fig.…”