New Insights on the Role of the γ-Herpesvirus Uracil-DNA Glycosylase Leucine Loop Revealed by the Structure of the Epstein-Barr Virus Enzyme in Complex with an Inhibitor Protein

Géoui, Thibault; Buisson, Marlyse; Tarbouriech, Nicolas; Burmeister, W.P.

doi:10.1016/j.jmb.2006.11.007

Cited by 29 publications

(37 citation statements)

References 55 publications

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“…To this end, two Flag-BKRF3 mutants were generated, one mutated at the catalytic site (Q90L,D91N) and the other at the leucine loop (H213L) (Fig. 8A) (37). As indicated by the immunoblot, viral lytic protein expression was comparable in cells complemented with plasmids expressing wild-type or mutant BKRF3 (Fig.…”

Section: Bkrf3 Was Translocated Into the Nucleus During Ebv Lytic Repmentioning

confidence: 96%

“…8C, lanes 9 and 10). According to the 3D structural analyses of BKRF3 and human UNG (37,60), the leucine loop of UDG is within its DNAcontacting region (resides 213 to 229 of BKRF3). Therefore, single-stranded DNA cellulose chromatography was performed to determine whether the replacement of histidine 213 with leucine affects the general DNA binding ability of BKRF3.…”

Section: Bkrf3 Was Translocated Into the Nucleus During Ebv Lytic Repmentioning

confidence: 99%

“…Crystal structure analysis of BKRF3 in complex with the UDG inhibitor protein (Ugi) revealed that BKRF3 shares considerable similarity in overall structure with proteins in the family 1 UNGs. Four out of the five catalytic motifs are conserved completely, whereas the fifth domain carries a seven-residue insertion in the leucine loop, indicating that the leucine loop of BKRF3 plays a role in viral replication (37). Previously, we characterized the biochemical properties of BKRF3 with DNA glycosylase and mutator assays.…”

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confidence: 99%

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Uracil DNA Glycosylase BKRF3 Contributes to Epstein-Barr Virus DNA Replication through Physical Interactions with Proteins in Viral DNA Replication Complex

Liu

et al. 2014

J Virol

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Epstein-Barr virus (EBV) BKRF3 shares sequence homology with members of the uracil-N-glycosylase (UNG) protein family and has DNA glycosylase activity. Here, we explored how BKRF3 participates in the DNA replication complex and contributes to viral DNA replication. Exogenously expressed Flag-BKRF3 was distributed mostly in the cytoplasm, whereas BKRF3 was translocated into the nucleus and colocalized with the EBV DNA polymerase BALF5 in the replication compartment during EBV lytic replication. The expression level of BKRF3 increased gradually during viral replication, coupled with a decrease of cellular UNG2, suggesting BKRF3 enzyme activity compensates for UNG2 and ensures the fidelity of viral DNA replication. In immunoprecipitation-Western blotting, BKRF3 was coimmunoprecipitated with BALF5, the polymerase processivity factor BMRF1, and the immediate-early transactivator Rta. Coexpression of BMRF1 appeared to facilitate the nuclear targeting of BKRF3 in immunofluorescence staining. Residues 164 to 255 of BKRF3 were required for interaction with Rta and BALF5, whereas residues 81 to 166 of BKRF3 were critical for BMRF1 interaction in glutathione S-transferase (GST) pulldown experiments. Viral DNA replication was defective in cells harboring BKRF3 knockout EBV bacmids. In complementation assays, the catalytic mutant BKRF3(Q90L,D91N) restored viral DNA replication, whereas the leucine loop mutant BKRF3(H213L) only partially rescued viral DNA replication, coupled with a reduced ability to interact with the viral DNA polymerase and Rta. Our data suggest that BKRF3 plays a critical role in viral DNA synthesis predominantly through its interactions with viral proteins in the DNA replication compartment, while its enzymatic activity may be supplementary for uracil DNA glycosylase (UDG) function during virus replication. IMPORTANCECatalytic activities of both cellular UDG UNG2 and viral UDGs contribute to herpesviral DNA replication. To ensure that the enzyme activity executes at the right time and the right place in DNA replication forks, complex formation with other components in the DNA replication machinery provides an important regulation for UDG function. In this study, we provide the mechanism for EBV UDG BKRF3 nuclear targeting and the interacting domains of BKRF3 with viral DNA replication proteins. Through knockout and complementation approaches, we further demonstrate that in addition to UDG activity, the interaction of BKRF3 with viral proteins in the replication compartment is crucial for efficient viral DNA replication.

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Section: Bkrf3 Was Translocated Into the Nucleus During Ebv Lytic Repmentioning

confidence: 96%

Section: Bkrf3 Was Translocated Into the Nucleus During Ebv Lytic Repmentioning

confidence: 99%

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confidence: 99%

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Uracil DNA Glycosylase BKRF3 Contributes to Epstein-Barr Virus DNA Replication through Physical Interactions with Proteins in Viral DNA Replication Complex

Liu

et al. 2014

J Virol

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“…Conserved domain analysis (see Figure 5) also indicated that UL2 obviously contains the conserved active site, ligand-binding site, UDG inhibitor interface, and catalytic site of the UDG enzyme within the UDG_F1 domain (Dean and Cheung, 1993;Pearl, 2000;Geoui et al, 2007). Consequently, UL2 might have a close relationship with the UDG family and high similarity with its counterparts encoded by UDG_F1 genes.…”

Section: Discussionmentioning

confidence: 99%

Molecular characterization of the pseudorabies virus UL2 gene

Cai

Wang²,

Cui³

et al. 2013

Genet. Mol. Res.

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ABSTRACT.A 948-bp sequence of the UL2 gene was amplified from the pseudorabies virus (PRV) Becker strain genome using polymerase chain reaction, and the gene identity was confirmed through further cloning and sequencing. Bioinformatic analysis indicated that the PRV UL2 gene encodes a putative polypeptide with 315-amino acid residues. Its encoding protein, designated UL2, has a conserved uracil-DNA glycosylase (UDG)_F1 domain, which is closely related to the herpesvirus UDG family and is highly conserved among its counterparts encoded by UDG genes. Multiple nucleic acid and amino acid sequence alignments suggested that the product of PRV UL2 has a relatively higher homology with UL2-like proteins of Alphaherpesvirinae than that of other subfamilies of Herpesviridae. In addition, phylogenetic analysis showed that PRV UL2 had a close evolutionary relationship with members of Alphaherpesvirinae, especially members of the genus Varicellovirus of bovine herpesvirus 1 and bovine herpesvirus 5. Antigen prediction indicated the presence of several potential B-cell epitopes in PRV UL2. In addition, secondary structure and 3-dimensional structure prediction revealed that PRV UL2 consisted predominantly of an α-helix. Taken together, these results provide molecular biological insight for the further study of the function and mechanism of UL2 during PRV infection.

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“…Generally, the biochemical properties of viral UNGs are very similar to those of their cellular counterparts [225,226]. Structures of the viral enzymes reveal the conserved UNG superfamily fold [16,227,228]. Unlike in the case of cellular organisms, where UNG plays an important but non-vital role, deletions of poxviral UNG severely suppress viral replication [221].…”

Section: Ber and Immunoglobulin Gene Diversificationmentioning

confidence: 99%

Base excision DNA repair

Zharkov

2008

Cell. Mol. Life Sci.

256

215

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DNA repair is a collection of several multienzyme, multistep processes keeping the cellular genome intact against genotoxic insults. One of these processes is base excision repair, which deals with the most ubiquitous lesions in DNA: oxidative base damage, alkylation, deamination, sites of base loss and single-strand breaks, etc. Individual enzymes acting in base excision repair have been identified. The recent years were marked with many advances in understanding of their structure and many interactions that make base excision repair a functional, versatile system. This review describes the current knowledge of structural biology and biochemistry of individual steps of base excision repair, several subpathways of the common base excision repair pathway, and interactions of the repair process with other cellular processes.

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New Insights on the Role of the γ-Herpesvirus Uracil-DNA Glycosylase Leucine Loop Revealed by the Structure of the Epstein-Barr Virus Enzyme in Complex with an Inhibitor Protein

Cited by 29 publications

References 55 publications

Uracil DNA Glycosylase BKRF3 Contributes to Epstein-Barr Virus DNA Replication through Physical Interactions with Proteins in Viral DNA Replication Complex

Uracil DNA Glycosylase BKRF3 Contributes to Epstein-Barr Virus DNA Replication through Physical Interactions with Proteins in Viral DNA Replication Complex

Molecular characterization of the pseudorabies virus UL2 gene

Base excision DNA repair

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