Uracil in Oris of herpes simplex 1 alters its specific recognition by origin binding protein (OBP): does virus induced uracil-DNA glycosylase play a key role in viral reactivation and replication?

Focher, Federico; Verri, Annalisa; Verzeletti, Simona; Mazzarello, Paolo; Spadari, Silvio

doi:10.1007/bf02451788

Cited by 39 publications

(40 citation statements)

References 19 publications

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“…The absence of the 5-methyl group in uracil has the potential to alter DNA structure and dynamic features that might be important for recognition by transcription factors or other DNA binding proteins (23,25). For example, uracils localized in the origin of replication in HSV-1 were found to perturb the binding of the HSV-1 origin binding protein (27), and incorporated uracils in the cAMP response element (CRE) of rat stomatostatin gene led to a two-to fourfold decrease in binding of CRE binding proteins from HeLa extracts (26). Our observation of reduced GFP expression from the exogenously uracilated DNA in the HT29-Ugi cell line supports the proposal that the presence of uracil in DNA can lead to reduced gene transcription.…”

Section: Discussionmentioning

confidence: 99%

Uracil DNA glycosylase initiates degradation of HIV-1 cDNA containing misincorporated dUTP and prevents viral integration

Weil

Ghosh²,

Zhou

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

119

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Significance Misincorporation of dUTP into DNA is detrimental to eukaryotes, prokaryotes, and viruses. This study reveals the fate of uracilated HIV-1 DNA in human cells. Early stages of the viral life cycle are unaffected, but integration of uracilated viral DNA into the host genome is prevented. This effect is wholly dependent on the presence of UNG2, a nuclear enzyme that excises uracil from DNA. This study establishes that UNG2 is a restriction factor for uracilated HIV-1 DNA and explains why this pathway is not fully engaged in CD4+ T cells and macrophages.

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Section: Discussionmentioning

confidence: 99%

Uracil DNA glycosylase initiates degradation of HIV-1 cDNA containing misincorporated dUTP and prevents viral integration

Weil

Ghosh²,

Zhou

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

119

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“…In both EBV and HSV, the binding of OBP to the origin of lytic replication is absolutely required for viral DNA replication. In HSV, it has been demonstrated that uracil replacing either cytosine (mimicking cytosine deamination) or thymine (mimicking dUTP misincorporation) on site I of OriS diminishes the interaction between OBP and OriS in a gel shift assay (20). Thus, experiments to determine the effects of the binding of Zta to the uracil-containing Zta responsive element are awaited.…”

Section: Discussionmentioning

confidence: 99%

“…To express His-BKRF3 protein, pET15a-BKRF3 was transformed into BL21(DE3), and the bacteria were induced in the exponential phase with 1 mM IPTG at 37°C for 2 h. The cell extracts were homogenized in lysis buffer (20 Antibodies. To generate BKRF3-specific polyclonal antibodies, 6-week-old BALB/c mice were immunized with 50 g His-BKRF3 protein in complete Freund's adjuvant subcutaneously, followed by three boosters with incomplete Freund's adjuvant at 2-week intervals.…”

Section: Constructs and Strainsmentioning

confidence: 99%

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

Huang

Wang

et al. 2007

J Virol

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Uracil-DNA glycosylases (UDGs) of the uracil-N-glycosylase (UNG) family are the primary DNA repair enzymes responsible for removal of inappropriate uracil from DNA. Recent studies further suggest that the nuclear human UNG2 and the UDGs of large DNA viruses may coordinate with their DNA polymerase accessory factors to enhance DNA replication. Based on its amino acid sequence, the putative UDG of Epstein-Barr virus (EBV), BKRF3, belongs to the UNG family of proteins, and it was demonstrated previously to enhance oriLyt-dependent DNA replication in a cotransfection replication assay. However, the expression and enzyme activity of EBV BKRF3 have not yet been characterized. In this study, His-BKRF3 was expressed in bacteria and purified for biochemical analysis. Similar to the case for the Escherichia coli and human UNG enzymes, His-BKRF3 excised uracil from single-stranded DNA more efficiently than from double-stranded DNA and was inhibited by the purified bacteriophage PBS1 inhibitor Ugi. In addition, BKRF3 was able to complement an E. coli ung mutant in rifampin and nalidixic acid resistance mutator assays. The expression kinetics and subcellular localization of BKRF3 products were detected in EBV-positive lymphoid and epithelial cells by using BKRF3-specific mouse antibodies. Expression of BKRF3 is regulated mainly by the immediateearly transcription activator Rta. The efficiency of EBV lytic DNA replication was slightly affected by BKRF3 small interfering RNA (siRNA), whereas cellular UNG2 siRNA or inhibition of cellular and viral UNG activities by expressing Ugi repressed EBV lytic DNA replication. Taking these results together, we demonstrate the UNG activity of BKRF3 in vitro and in vivo and suggest that UNGs may participate in DNA replication or repair and thereby promote efficient production of viral DNA.

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“…Hence, it has been shown that uracil substitutions in the viral origins of replication alters their recognition by the viral initiator protein (5). Moreover, whereas UL2 may be dispensable for viral replication in fibroblast (6), UL2 mutants exhibit reduced neurovirulence and a decreased frequency of reactivation from latency (7).…”

mentioning

confidence: 99%

The replicative DNA polymerase of herpes simplex virus 1 exhibits apurinic/apyrimidinic and 5′-deoxyribose phosphate lyase activities

Bogani

Boehmer

2008

Proc. Natl. Acad. Sci. U.S.A.

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Base excision repair (BER) is essential for maintaining genome stability both to counter the accumulation of unusual bases and to protect from base loss in the DNA. Herpes simplex virus 1 (HSV-1) is a large dsDNA virus that encodes its own DNA replication machinery, including enzymes involved in nucleotide metabolism. We report on a replicative family B and a herpesvirus-encoded DNA Pol that possesses DNA lyase activity. We have discovered that the catalytic subunit of the HSV-1 DNA polymerase (Pol) (UL30) exhibits apurinic/apyrimidinic (AP) and 5 -deoxyribose phosphate (dRP) lyase activities. These activities are integral to BER and lead to DNA cleavage on the 3 side of abasic sites and 5 -dRP residues that remain after cleavage by 5 -AP endonuclease. The UL30-catalyzed reaction occurs independently of divalent cation and proceeds via a Schiff base intermediate, indicating that it occurs via a lyase mechanism. Partial proteolysis of the Schiff base shows that the DNA lyase activity resides in the Pol domain of UL30. These observations together with the presence of a virus-encoded uracil DNA glycosylase indicates that HSV-1 has the capacity to perform critical steps in BER. These findings have implications on the role of BER in viral genome maintenance during lytic replication and reactivation from latency.base excision repair ͉ abasic DNA ͉ uracil DNA glycosylase H erpes simplex virus 1 (HSV-1) is a dsDNA virus with a genome of Ϸ152 kbp (1). HSV-1 switches between lytic replication in epithelial cells and a state of latency in sensory neurons during which there is no detectable DNA replication (1). Replication of the genome is mediated by seven essential virus-encoded factors with the following functions: a DNA polymerase (Pol) catalytic subunit (UL30), its associated processivity factor (UL42), a ssDNA binding protein (UL29), a heterotrimeric helicase-primase (UL5, UL8, and UL52) and an initiator protein (UL9) that binds to and unwinds the viral replication origins (1-4). HSV-1 also encodes several other enzymes that are dispensable for replication in cell culture, which are involved in nucleotide metabolism and perform other important roles in maintaining the viral genome including a thymidine kinase (UL23), a ribonucleotide reductase (UL39 and UL40), a dUTPase (UL50), an exonuclease (UL12), and notably a uracil DNA glycosylase (UDG) (UL2) (4).The presence of a virus-encoded UDG suggests that excision of uracil may be important during viral replication. Hence, it has been shown that uracil substitutions in the viral origins of replication alters their recognition by the viral initiator protein (5). Moreover, whereas UL2 may be dispensable for viral replication in fibroblast (6), UL2 mutants exhibit reduced neurovirulence and a decreased frequency of reactivation from latency (7). Thus, UDG action in HSV-1 may be important for viral reactivation after quiescence in neuronal cells during which the genome may accumulate uracil as a result of spontaneous deamination of cytosine. In cytomegalovirus, the viral UDG ...

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Uracil in Oris of herpes simplex 1 alters its specific recognition by origin binding protein (OBP): does virus induced uracil-DNA glycosylase play a key role in viral reactivation and replication?

Cited by 39 publications

References 19 publications

Uracil DNA glycosylase initiates degradation of HIV-1 cDNA containing misincorporated dUTP and prevents viral integration

Uracil DNA glycosylase initiates degradation of HIV-1 cDNA containing misincorporated dUTP and prevents viral integration

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

The replicative DNA polymerase of herpes simplex virus 1 exhibits apurinic/apyrimidinic and 5′-deoxyribose phosphate lyase activities

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