The Epstein-Barr Virus Polymerase Accessory Factor BMRF1 Adopts a Ring-shaped Structure as Visualized by Electron Microscopy

Makhov, Alexander M.; Subramanian, Deepa; Holley-Guthrie, Elizabeth; Kenney, Shannon C.; Griffith, Jack D.

doi:10.1074/jbc.m408733200

Cited by 11 publications

(9 citation statements)

References 27 publications

(16 reference statements)

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“…Although BMRF1 also possesses DNA binding activity (18), its stoichiometry for DNA binding remains unknown. Electron microscopy observations revealed that BMRF1 adopts a ring-shaped structure, which may contain six monomers (19). This is almost twice as large as the previously reported PCNA ring structure.…”

supporting

confidence: 62%

“…According to an electron microscopy study (19), a ring structure with a 55-Å diameter was observed for intact BMRF1, which includes six molecules within the ring. The crystal structure of BMRF1-WT⌬C showed an elliptic ring (long axis ϳ50 Å, short axis ϳ40 Å).…”

Section: Discussionmentioning

confidence: 99%

“…This is almost twice as large as the previously reported PCNA ring structure. Glutathione S-transferase pulldown assays also revealed that BMRF1 can form homo-oligomers (19).…”

mentioning

confidence: 99%

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

Murayama

Nakayama

Kato-Murayama

et al. 2009

Journal of Biological Chemistry

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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...

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supporting

confidence: 62%

Section: Discussionmentioning

confidence: 99%

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

Murayama

Nakayama

Kato-Murayama

et al. 2009

Journal of Biological Chemistry

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“…A recent electron microscopy study indicated that the UL44 homolog from the ␥-herpesvirus Epstein-Barr virus assembles in a higher ordered structure that is reminiscent of a lock washer or ring-like arrangement (60). Furthermore, the inner diameter of the rings in these electron microscope images have widths similar to that of the central cavity of the infinite "lock washer" helical arrangement of dimers observed in the UL44-UL54 crystal lattice.…”

Section: Ul44 May Be Capable Of Forming Highermentioning

confidence: 65%

“…Although the side chain of Ile 135 forms some van der Waals interactions with the peptide, including the sulfur of Cys 1241 and C-␤ of Ala 1238 , these interactions are less extensive than the hydrophobic interactions that Ile 135 makes with other side chains of UL44. Thus, the side chain of Ile 135 is positioned as a hydrophobic "anchor" underneath the hydrogen-bonding network, where it makes favorable van der Waals contacts with several important residues from the central ␤-sheet of UL44, including Val 41 , Ile 49 , and the aliphatic portions of Gln 51 and Lys 60 . The second set of interactions involves the packing of a hydrophobic "plug" into a crevice along the central ␤-sheet of UL44 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Crystal Structure of the Cytomegalovirus DNA Polymerase Subunit UL44 in Complex with the C Terminus from the Catalytic Subunit

Appleton

Brooks

Loregian

et al. 2006

Journal of Biological Chemistry

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The human cytomegalovirus DNA polymerase is composed of a catalytic subunit, UL54, and an accessory protein, UL44, which has a structural fold similar to that of other processivity factors, including herpes simplex virus UL42 and homotrimeric sliding clamps such as proliferating cell nuclear antigen. Several specific residues in the C-terminal region of UL54 and in the "connector loop" of UL44 are required for the association of these proteins. Here, we describe the crystal structure of residues 1-290 of UL44 in complex with a peptide from the extreme C terminus of UL54, which explains this interaction at a molecular level. The UL54 peptide binds to structural elements similar to those used by UL42 and the sliding clamps to associate with their respective binding partners. However, the details of the interaction differ from those of other processivity factor-peptide complexes. Crucial residues include a three-residue hydrophobic "plug" from the UL54 peptide and Ile 135 of UL44, which forms a critical intramolecular hydrophobic anchor for interactions between the connector loop and the peptide. As was the case for the unliganded UL44 structure, the UL44-peptide complex forms a head-tohead dimer that could potentially form a C-shaped clamp on DNA. However, the peptide-bound structure displays subtle differences in the relative orientation of the two subdomains of the protein, resulting in a more open clamp, which we predicted would affect its association with DNA. Indeed, filter binding assays revealed that peptide-bound UL44 binds DNA with higher affinity. Thus, interaction with the catalytic subunit appears to affect both the structure and function of UL44.The replication of DNA requires a number of multiprotein assemblies, including a DNA polymerase that synthesizes tens of thousands of nucleotides without dissociating from the primer-template. Most replicative DNA polymerases require not only catalytic subunits, but also accessory subunits known as processivity factors to remain tethered to the template during replication. The well characterized processivity factors known as sliding clamps, which include proliferating cell nuclear antigen (PCNA) 5 from eukaryotes and archaebacteria, ␤-subunits from prokaryotes, and gp45 from T4 and RB69 bacteriophage, display no intrinsic affinity for DNA. They tether their corresponding catalytic subunits to DNA after they are loaded onto DNA as toroidal homomultimers via clamp loader complexes in ATP-dependent processes (reviewed in Ref. 1).Herpesviruses, including herpes simplex virus (HSV) and human cytomegalovirus (HCMV), encode a DNA polymerase consisting of two proteins that are essential for viral DNA replication (2-4). The DNA polymerase is composed of a 1242-residue catalytic protein, UL54 (5, 6), and a 433-residue accessory protein, UL44 or ICP36 (7). UL54, a member of the polymerase ␣ family, displays DNA-dependent DNA polymerase and 3Ј-5Ј exonuclease activities (8 -10). UL44 is analogous to the processivity factor UL42 (11), as it binds double-stranded DNA, spec...

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Epstein-Barr Viral Productive Amplification Reprograms Nuclear Architecture, DNA Replication, and Histone Deposition

Chiu

Sugden

2013

Cell Host & Microbe

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Summary The spontaneous transition of Epstein-Barr Virus (EBV) from latency to productive infection is infrequent, making its analysis in the resulting mixed cell populations difficult. We engineered cells to support this transition efficiently and developed EBV DNA variants that could be visualized and measured as fluorescent signals over multiple cell cycles. This approach revealed that EBV’s productive replication began synchronously for viral DNAs within a cell but asynchronously between cells. EBV DNA amplification was delayed until early S-phase and occurred in factories characterized by the absence of cellular DNA and histones, by a sequential redistribution of PCNA, and by localization away from the nuclear periphery. The earliest amplified DNAs lacked histones accompanying a decline in four histone chaperones. Thus, EBV transitions from being dependent on the cellular replication machinery during latency to commandeering both that machinery and nuclear structure for its own reproductive needs.

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The Epstein-Barr Virus Polymerase Accessory Factor BMRF1 Adopts a Ring-shaped Structure as Visualized by Electron Microscopy

Cited by 11 publications

References 27 publications

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

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

Crystal Structure of the Cytomegalovirus DNA Polymerase Subunit UL44 in Complex with the C Terminus from the Catalytic Subunit

Epstein-Barr Viral Productive Amplification Reprograms Nuclear Architecture, DNA Replication, and Histone Deposition

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