Structural features of the single-stranded DNA-binding protein of Epstein–Barr virus

Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA-1) plays key roles in both the regulation of gene expression and the replication of the EBV genome in latently infected cells. To characterize the RNA-binding activity of EBNA-1, it was demonstrated that EBNA-1 binds efficiently to RNA homopolymers that are composed of poly(G) and weakly to those composed of poly(U). All three RGG boxes of EBNA-1 contributed additively to poly(G)-binding activity and could mediate RNA binding when attached to a heterologous protein in an RNA gel mobility-shift assay. In vitro-transcribed EBV and non-EBV RNA probes revealed that EBNA-1 bound to most RNAs examined and the affinity increased as the content of G and U increased, as demonstrated in competition assays. Among these probes, the 5′ non-coding region (NCR) (nt 131–278) of hepatitis C virus RNA appeared to be the strongest competitor for EBNA-1 binding to the EBV-encoded small nuclear RNA 1 (EBER1) probe, whereas a mutant 5′ NCR RNA with partially disrupted secondary structure was a weak competitor. Furthermore, the interaction of endogenous EBNA-1 and EBER1 in EBV-infected cells was demonstrated by a ribonucleoprotein immunoprecipitation assay. These results revealed that EBNA-1 is a DNA-binding protein with strong binding activity to a relatively broad spectrum of RNA and suggested an additional biological impact of EBNA-1 through its ability to bind to RNA.

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Characterization of Epstein-Barr Virus DNase and Its Interaction with the Major DNA Binding Protein

Virology ◽

10.1006/viro.1995.1203 ◽

1995 ◽

Vol 208 (2) ◽

pp. 712-722 ◽

Cited By ~ 12

Author(s):

Su-Fang Lin ◽

Tsuey-Ying Hsu ◽

Mei-Ying Liu ◽

Lung-Shen Lin ◽

Huey-Lang Yang ◽

...

Keyword(s):

Dna Binding ◽

Epstein Barr Virus ◽

Binding Protein ◽

Dna Binding Protein ◽

Barr Virus ◽

Epstein Barr

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Two Domains of the Epstein-Barr Virus Origin DNA-binding Protein, EBNA1, Orchestrate Sequence-specific DNA Binding

Journal of Biological Chemistry ◽

10.1074/jbc.m001414200 ◽

2000 ◽

Vol 275 (29) ◽

pp. 22273-22277 ◽

Cited By ~ 31

Author(s):

Jennifer Cruickshank ◽

Kathy Shire ◽

Alan R. Davidson ◽

Aled M. Edwards ◽

Lori Frappier

Keyword(s):

Dna Binding ◽

Epstein Barr Virus ◽

Binding Protein ◽

Dna Binding Protein ◽

Barr Virus ◽

Virus Origin ◽

Epstein Barr

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Epstein-Barr Virus Immediate-Early Protein Zta Co-Opts Mitochondrial Single-Stranded DNA Binding Protein To Promote Viral and Inhibit Mitochondrial DNA Replication

Journal of Virology ◽

10.1128/jvi.02198-07 ◽

2008 ◽

Vol 82 (9) ◽

pp. 4647-4655 ◽

Cited By ~ 37

Author(s):

Andreas Wiedmer ◽

Pu Wang ◽

Jing Zhou ◽

Andrew J. Rennekamp ◽

Valeria Tiranti ◽

...

Keyword(s):

Mitochondrial Dna ◽

Dna Binding ◽

Binding Protein ◽

Dna Binding Protein ◽

Lytic Replication ◽

Single Stranded Dna ◽

Barr Virus ◽

Early Protein ◽

Associated Proteins ◽

Epstein Barr

ABSTRACT Disruption of cellular metabolic processes and usurpation of host proteins are hallmarks of herpesvirus lytic infection. Epstein-Barr virus (EBV) lytic replication is initiated by the immediate-early protein Zta. Zta is a multifunctional DNA binding protein that stimulates viral gene transcription, nucleates a replication complex at the viral origin of lytic replication, and inhibits cell cycle proliferation. To better understand these functions and identify cellular collaborators of Zta, we purified an epitope-tagged version of Zta in cells capable of supporting lytic replication. FLAG-tagged Zta was purified from a nuclear fraction using FLAG antibody immunopurification and peptide elution. Zta-associated proteins were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by mass spectrometry. The Zta-associated proteins included members of the HSP70 family and various single-stranded DNA and RNA binding proteins. The nuclear replication protein A subunits (RPA70 and RPA32) and the human mitochondrial single-stranded DNA binding protein (mtSSB) were confirmed by Western blotting to be specifically enriched in the FLAG-Zta immunopurified complex. mtSSB coimmunoprecipitated with endogenous Zta during reactivation of EBV-positive Burkitt lymphoma and lymphoblastoid cell lines. Small interfering RNA depletion of mtSSB reduced Zta-induced lytic replication of EBV but had only a modest effect on transcription activation function. A point mutation in the Zta DNA binding domain (C189S), which is known to reduce lytic cycle replication, eliminated mtSSB association with Zta. The predominantly mitochondrial localization of mtSSB was shifted to partly nuclear localization in cells expressing Zta. Mitochondrial DNA synthesis and genome copy number were reduced by Zta-induced EBV lytic replication. We conclude that Zta interaction with mtSSB serves the dual function of facilitating viral and blocking mitochondrial DNA replication.

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