Characterization of the Uracil-DNA Glycosylase Activity of Epstein-Barr Virus BKRF3 and Its Role in Lytic Viral DNA Replication

Lu, Cheng‐Hsun; Huang, Ho-Ting; Wang, Jiin-Tarng; Slupphaug, Geir; Li, Tsai-Kun; Wu, Mao S.; Chen, Yi‐Chun; Lee, Chung-Pei; Chen, Mei‐Ru

doi:10.1128/jvi.01518-06

Cited by 33 publications

(45 citation statements)

References 79 publications

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“…However, HSV-1 reactivation was measured post-explant from trigeminal ganglia: a tissue composed of terminally differentiated adult neurons with low levels of host UNG (18,36). Reactivation is also reduced in human gammaherpesvirus latency cell systems lacking the EBV viral UNG (BKRF3) alone or in the absence of both the vUNG and the host UNG (13,18,(37)(38)(39). Interestingly, Su et al (13) recently reported that host UNG2 levels decrease as EBV reactivation proceeds, indicating that the viral UNG is needed to promote replication under conditions of low host UNG levels (13).…”

Section: Discussionmentioning

confidence: 99%

Absence of the Uracil DNA Glycosylase of Murine Gammaherpesvirus 68 Impairs Replication and Delays the Establishment of Latency In Vivo

Minkah

Macaluso

Oldenburg

et al. 2015

J Virol

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Uracil DNA glycosylases (UNG) are highly conserved proteins that preserve DNA fidelity by catalyzing the removal of mutagenic uracils. All herpesviruses encode a viral UNG (vUNG), and yet the role of the vUNG in a pathogenic course of gammaherpesvirus infection is not known. First, we demonstrated that the vUNG of murine gammaherpesvirus 68 (MHV68) retains the enzymatic function of host UNG in an in vitro class switch recombination assay. Next, we generated a recombinant MHV68 with a stop codon in ORF46/UNG (⌬UNG) that led to loss of UNG activity in infected cells and a replication defect in primary fibroblasts. Acute replication of MHV68⌬UNG in the lungs of infected mice was reduced 100-fold and was accompanied by a substantial delay in the establishment of splenic latency. Latency was largely, yet not fully, restored by an increase in virus inoculum or by altering the route of infection. MHV68 reactivation from latent splenocytes was not altered in the absence of the vUNG. A survey of host UNG activity in cells and tissues targeted by MHV68 indicated that the lung tissue has a lower level of enzymatic UNG activity than the spleen. Taken together, these results indicate that the vUNG plays a critical role in the replication of MHV68 in tissues with limited host UNG activity and this vUNG-dependent expansion, in turn, influences the kinetics of latency establishment in distal reservoirs. IMPORTANCEHerpesviruses establish chronic lifelong infections using a strategy of replicative expansion, dissemination to latent reservoirs, and subsequent reactivation for transmission and spread. We examined the role of the viral uracil DNA glycosylase, a protein conserved among all herpesviruses, in replication and latency of murine gammaherpesvirus 68. We report that the viral UNG of this murine pathogen retains catalytic activity and influences replication in culture. The viral UNG was impaired for productive replication in the lung. This defect in expansion at the initial site of acute replication was associated with a substantial delay of latency establishment in the spleen. The levels of host UNG were substantially lower in the lung compared to the spleen, suggesting that herpesviruses encode a viral UNG to compensate for reduced host enzyme levels in some cell types and tissues. These data suggest that intervention at the site of initial replicative expansion can delay the establishment of latency, a hallmark of chronic herpesvirus infection.A ll herpesvirus genomes encode a homolog of the host uracil DNA glycosylase. If not repaired, uracil misincorporation during DNA replication (1, 2) or conversion upon cytosine deamination (2, 3) will lead to a C:G-to-T:A transition mutation that may have severe consequences for genomic integrity (1). In the host, the recognition and removal of uracils is mediated by members of the uracil DNA glycosylase (UDG) superfamily. UNG2 is the predominant nuclear enzyme responsible for initiating repair (2-6). The role of a seemingly redundant herpesvirus UNG in viral replication is ...

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Section: Discussionmentioning

confidence: 99%

Absence of the Uracil DNA Glycosylase of Murine Gammaherpesvirus 68 Impairs Replication and Delays the Establishment of Latency In Vivo

Minkah

Macaluso

Oldenburg

et al. 2015

J Virol

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“…Western blotting was performed as described previously (47). To detect the EBV lytic proteins, the primary antibodies used were laboratory-made mouse anti-BKRF3 serum 3, mouse anti-Zta 1B4, anti-Rta 467, anti-BGLF4 2616, and anti-BMRF 88A9, as described previously (4,48). The BALF2 antibody (OT13B) was kindly provided by J. M. Middeldorp (49).…”

Section: Methodsmentioning

confidence: 99%

“…Subcellular fractionation. The subcellular fractionation protocol was modified from a previous study (4). Briefly, trypsinized cells were harvested, washed, and incubated with 1 ml hypotonic buffer (5 mM TrisHCl, pH 7.4, 5 mM KCl, 1.5 mM MgCl 2 , 0.1 mM EGTA, 1 mM dithiothreitol [DTT], and 1 mM phenylmethylsulfonyl fluoride [PMSF]) with gentle shaking at 4°C for 1 h. The cell suspension then was passed 15 times through a 26-gauge needle and centrifuged at 500 ϫ g at 4°C for 5 min.…”

Section: Methodsmentioning

confidence: 99%

“…BKRF3 was able to complement the phenotype of an Escherichia coli ung mutant and showed a higher efficiency in removing uracil from an artificial single-stranded DNA (ssDNA) probe than double-stranded DNA (dsDNA) in vitro. Using short interfering RNA (siRNA) to knock down BKRF3 expression caused an approximately 20% decrease in viral DNA replication in EBV-positive NA cells (4). In this study, we aimed to examine further the role and functional domains of BKRF3 in EBV lytic replication.…”

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confidence: 99%

“…The core viral DNA replication complex contains eight virus-encoded proteins, including BZLF1 (oriLyt binding protein), BRLF1 (immediate-early transactivator), BALF5 (DNA polymerase), BMRF1 (also called EA-D; polymerase processivity factor), BALF2 (single-stranded-DNA binding protein), BBLF4 (helicase), BSLF1 (primase), and BBLF2/3 (helicase-primase-associated protein) (2,3). In addition, EBV encodes a viral uracil DNA glycosylase (UDG), BKRF3, which is a homolog of the human uracil-N-glycosylase (UNG) family (4).…”

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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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Human genetic variants of homologous recombination repair genes first found to be associated with Epstein–Barr virus antibody titers in healthy Cantonese

Shen

Pan

Hong

et al. 2011

Intl Journal of Cancer

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Epstein-Barr virus (EBV) infection is a major risk factor for nasopharyngeal carcinoma (NPC). Despite high prevalence of infection among the general population worldwide, only a small proportion of infected individuals presents with seropositivity for EBV-specific IgA antibodies. This seropositive subgroup of EBV carriers has an elevated cumulative risk for NPC during their lifetime. Previous studies reported that the host homologous recombination repair (HRR) system participates in EBV lytic replication, suggesting a potential mechanism to influence EBV reactivation status and thus seropositivity. To investigate whether genetic variants of HRR genes are associated with the serostatus in a healthy population, we investigated the association between seropositivity for anti-VCA-IgA and 156 tagging SNPs in 35 genes connected with HRR in an observational study among 755 healthy Cantonese speakers in southern China. Six variant alleles of MDC1, RAD54L, TP53BP1, RPA1, LIG3 and RFC1 exhibited associations with seropositivity (p trend from 0.0085 to 0.00027). Our study provides evidence that genetic variation within the HRR might affect an individual's propensity for EBV seropositive status of anti-VCA IgA antibody.Epstein-Barr virus (EBV) is a member of the herpes virus family. More than 90% of humans worldwide have been infected with EBV.

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Characterization of the Uracil-DNA Glycosylase Activity of Epstein-Barr Virus BKRF3 and Its Role in Lytic Viral DNA Replication

Cited by 33 publications

References 79 publications

Absence of the Uracil DNA Glycosylase of Murine Gammaherpesvirus 68 Impairs Replication and Delays the Establishment of Latency In Vivo

Absence of the Uracil DNA Glycosylase of Murine Gammaherpesvirus 68 Impairs Replication and Delays the Establishment of Latency In Vivo

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

Human genetic variants of homologous recombination repair genes first found to be associated with Epstein–Barr virus antibody titers in healthy Cantonese

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