Cooperation of EBV DNA Polymerase and EA-D(BMRFl)in vitro and colocalization in nuclei of infected cells

Kiehl, Anita; Dorsky, David I.

doi:10.1016/0042-6822(91)90849-7

Cited by 64 publications

(70 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In EBV, PPF (BMRF1), which is thought to be similar to the cellular PCNA sliding clamp, is known to physically interact with POL (BALF5) and serve as an accessory protein to activate and modulate the catalytic activity of POL (34,35,38,69). In HSV, the affinity of POL (UL30) for a synthetic primertemplate junction is increased 10-fold by the presence of PPF (UL42), consistent with the notion that herpesvirus PPF acts as a sliding clamp for the herpesvirus POL (25).…”

Section: Discussionmentioning

confidence: 99%

Origin-Independent Assembly of Kaposi's Sarcoma-Associated Herpesvirus DNA Replication Compartments in Transient Cotransfection Assays and Association with the ORF-K8 Protein and Cellular PML

Ahn

Alcendor³

et al. 2001

J Virol

150

183

View full text Add to dashboard Cite

Six predicted Kaposi's sarcoma virus herpesvirus (KSHV) proteins have homology with other well-characterized herpesvirus core DNA replication proteins and are expected to be essential for viral DNA synthesis. Intact Flag-tagged protein products from all six were produced from genomic expression vectors, although the ORF40/41 transcript encoding a primase-helicase component proved to be spliced with a 127-bp intron. The intracellular localization of these six KSHV replication proteins and the mechanism of their nuclear translocation were investigated. SSB (single-stranded DNA binding protein, ORF6) and PPF (polymerase processivity factor, ORF59) were found to be intrinsic nuclear proteins, whereas POL (polymerase, ORF9), which localized in the cytoplasm on its own, was translocated to the nucleus when cotransfected with PPF. PAF (primaseassociated factor, ORF40/41), a component of the primase-helicase tripartite subcomplex together with PRI (primase, ORF56) and HEL (helicase, ORF44), required the presence of all five other replication proteins for efficient nuclear translocation. Surprisingly, even in the absence of a lytic cycle replication origin (ori-Lyt) and any known initiator or origin binding protein, the protein products of all six KSHV core replication genes cooperated in a transient cotransfection assay to form large globular shaped pseudo-replication compartments (pseudo-RC), which excluded cellular DNA. These pseudo-RC structures were confirmed to include POL, SSB, PRI, and PAF but did not contain any newly synthesized DNA. Similar to the human cytomegalovirus system, the peripheries of these KSHV pre-RC were also found to be surrounded by punctate PML oncogenic domains (PODs). Furthermore, by transient cotransfection, the six KSHV core replication machinery proteins successfully replicated a plasmid containing EBV ori-Lyt in the presence of the Epstein-Barr virus-encoded DNA binding initiator protein, ZTA. The KSHV-encoded K8 (ORF-K8) protein, which is a distant evolutionary homologue to ZTA, was incorporated into pseudo-RC structures formed by transient cotransfection with the six core KSHV replication genes. However, unlike ZTA, K8 displayed a punctate nuclear pattern both in transfected cells and at early stages of lytic infection and colocalized with the cellular PML proteins in PODs. Finally, K8 was also found to accumulate in functional viral RC, detected by incorporation of pulse-labeled bromodeoxyuridine into newly synthesized DNA in both tetradecanoyl phorbol acetate-induced JSC-1 primary effusion lymphoblasts and in KSHV lytically infected endothelial cells.

show abstract

Section: Discussionmentioning

confidence: 99%

Origin-Independent Assembly of Kaposi's Sarcoma-Associated Herpesvirus DNA Replication Compartments in Transient Cotransfection Assays and Association with the ORF-K8 Protein and Cellular PML

Ahn

Alcendor³

et al. 2001

J Virol

150

183

View full text Add to dashboard Cite

show abstract

“…The physical interaction requires the amino-terminal region of the former and the basic leucine zipper domain (aa 175-220) of the latter (21,37). It is reported that the amino-terminal 45 amino acids in the BMRF1 protein are not required for DNA binding activity or polymerase processivity function (15). To test whether the interaction between the BMRF1 and BZLF1 gene products is important for enhancement of the BZLF1-mediated transcriptional activation by the BMRF1 protein, we prepared amino-terminally truncated mutants, BMRF1 ⌬1-10 and ⌬1-30.…”

Section: Bzlf1 Protein Binding To the Promoter Is Necessary And Suffimentioning

confidence: 99%

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

Nakayama

Murata

Murayama

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

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 singlestranded 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 singlestranded DNA-binding protein.

show abstract

“…Additionally, BMRF1 appears to increase the processivity of the viral polymerase, suggesting a model in which the accessory protein acts as a sliding clamp that prevents dissociation of the polymerase from the active template. The herpes simplex virus type 1 UL42 polymerase accessory protein does not share extensive amino acid homology with BMRF1, but in both proteins the aminoterminal two-thirds of the molecule contains a DNA-binding domain and a region of interaction with the catalytic subunit (15). Both BMRF1 and UL42 bind to double strand DNA with high affinity in contrast to the well characterized sliding clamps Escherichia coli ␤, T4 gene 45 protein, and PCNA, which lack an intrinsic DNA-binding activity (16 -18).…”

Section: From the ‡Department Of Microbiology And Immunology Linebermentioning

confidence: 99%

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

Makhov

Subramanian

Holley-Guthrie

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

Epstein-Barr virus (EBV) encodes a set of core replication factors used during lytic infection in human cellsthat parallels the factors used in many other systems. These include a DNA polymerase and its accessory factor, a helicase/primase, and a single strand binding protein. The EBV polymerase accessory factor has been identified as the product of the BMRF1 gene and has been shown by functional assays to increase the activity and processivity of the polymerase. Unlike other members of this class of factors, BMRF1 is also a transcription factor regulating certain EBV genes. Although several polymerase accessory factors, including eukaryotic proliferating cell nuclear antigen, Escherichia coli ␤ protein, and T4 gene 45 protein have been shown to form oligomeric rings termed sliding clamps, nothing is known about the oligomeric state of BMRF1 or whether it forms a ring. In this work, BMRF1 was purified directly from human cells infected with an adenovirus vector expressing the BMRF1 gene product. The protein was purified to near homogeneity, and examination by negative staining electron microscopy revealed large, flat, ring-shaped molecules with a diameter of 15.5 ؎ 0.8 nm and a distinct 5.3-nm diameter hole in the center. The size of these rings is consistent with an oligomer of 6 monomers, nearly twice as large as the trimeric proliferating cell nuclear antigen ring. Unlike the herpes simplex virus UL42 homologue, BMRF1 was found to self-associate in solution. These findings extend the theme of polymerase accessory factors adopting ringshaped structures and provide an example in which the ring is significantly larger than any previously described sliding clamp.

show abstract

Cooperation of EBV DNA Polymerase and EA-D(BMRFl)in vitro and colocalization in nuclei of infected cells

Cited by 64 publications

References 41 publications

Origin-Independent Assembly of Kaposi's Sarcoma-Associated Herpesvirus DNA Replication Compartments in Transient Cotransfection Assays and Association with the ORF-K8 Protein and Cellular PML

Origin-Independent Assembly of Kaposi's Sarcoma-Associated Herpesvirus DNA Replication Compartments in Transient Cotransfection Assays and Association with the ORF-K8 Protein and Cellular PML

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

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

Contact Info

Product

Resources

About