Telomere Repeat Binding Factors TRF1, TRF2, and hRAP1 Modulate Replication of Epstein-Barr Virus OriP

Abstract

Epstein-Barr virus OriP confers cell cycle-dependent DNA replication and stable maintenance on plasmids in EBNA1-positive cells. The dyad symmetry region of OriP contains four EBNA1 binding sites that are punctuated by 9-bp repeats referred to as nonamers. Previous work has shown that the nonamers bind to cellular factors associated with human telomeres and contribute to episomal maintenance of OriP. In this work, we show that substitution mutation of all three nonamer sites reduces both DNA replication and plasmid maintenance of OriP-containing plasmids by 2.5-to 5-fold. The nonamers were required for high-affinity binding of TRF1, TRF2, and hRap1 to the dyad symmetry element but were not essential for the binding of EBNA1 as determined by DNA affinity purification from nuclear extracts. Chromatin immunoprecipitation assays indicated that TRF1, TRF2, and hRap1 bound OriP in vivo. Cell cycle studies indicate that TRF2 binding to OriP peaks in G 1 /S while TRF1 binding peaks in G 2 /M. OriP replication was inhibited by transfection of full-length TRF1 but not by deletion mutants lacking the myb DNA binding domain. In contrast, OriP replication was not affected by transfection of full-length TRF2 or hRap1 but was potently inhibited by dominant-negative TRF2 or hRap1 amino-terminal truncation mutants. Knockdown experiments with short interfering RNAs (siRNAs) directed against TRF2 and hRap1 severely reduced OriP replication, while TRF1 siRNA had a modest stimulatory effect on OriP replication. These results indicate that TRF2 and hRap1 promote, while TRF1 antagonizes, OriP-dependent DNA replication and suggest that these telomeric factors contribute to the establishment of replication competence at OriP.Epstein-Barr virus (EBV) is a lymphotropic gammaherpesvirus that can be cultured in latently infected B-cell lines as a multicopy extrachromosomal plasmid (reviewed in reference 27). The latent viral genome can be isolated from a variety of tumor tissues and is causally linked with Burkitt's lymphoma, nasopharyngeal carcinoma, and lymphoproliferative disorders in the immunosuppressed population (reviewed in reference 52). In most, if not all, latent infections the virus-encoded EBV nuclear antigen 1 (EBNA1) can be detected. Genetic and biochemical experiments have established that EBNA1 is essential for the maintenance of the viral genome during latency (34, 74). EBNA1 binds with high avidity to three regions of the viral genome, namely, the family of 30-bp repeats (FR), the dyad symmetry (DS) region, and the Q promoter (49). The FR and DS together comprise the viral origin of plasmid replication (OriP). The DS is required for DNA replication initiation, while FR stimulates DS replication and provides plasmid maintenance activity (3,11,29,55,73). The Q promoter has no known replication activity but is thought to autoregulate EBNA1 RNA transcription (62). EBNA1 binding to OriP is sufficient to confer plasmid replication and maintenance in most human and primate cell types but may be restricted in murine and hamster c...

Shelterin complex and associated factors at human telomeres

“…The inspection of the EBV origin of replication reveals a TRF1 or TRF2 binding site (TTA GGG TTA) repeated three times, and TRF1, TRF2 and RAP1 were found to bind there, in a search for binding factors by affinity chromatography. 106,107 In this system, TRF2 was shown to be important for the recruitment of specific replication factors, in particular the viral origin binding protein EBNA1, themselves essential for the effective replication of the virus. Further work revealed that interactions between TRF2 and the ORC complex, essential for the initial recognition of DNA replication origins, were important in this context, 108 and were also at play at telomeres.…”

The plasmid replicon of Epstein–Barr virus: Mechanistic insights into efficient, licensed, extrachromosomal replication in human cells

“…The replication of oriP plasmids is significantly inhibited in cells hypomorphic for ORC2 or overexpressing the cellular protein geminin that inhibits full assembly of the pre-RC, confirming EBV's reliance on these cellular pathways for the replication of its own genome (Dhar, Yoshida et al 2001). While a pair of EBNA1-binding sites is sufficient for DNA synthetic function, the presence of the nonamer elements flanking the origin and the binding of TRF2 to them stimulates the initiation of DNA synthesis from that origin (Deng, Lezina et al 2002;Deng, Atanasiu et al 2003;Atanasiu, Deng et al 2006). Some of this stimulation likely results from TRF2's increasing EBNA1's apparent affinity for DS; in addition, some may also result from the direct interaction of TRF2 with the BAH domain of ORC1, allowing increased recruitment of ORC1 to DS at the G1/S border of the cell cycle (Ritzi, Tillack et al 2003;Atanasiu, Deng et al 2006).…”

“…Flanking the pairs of EBNA1-binding sites of DS are three 9bp elements that resemble telomeric repeats, and have been termed nonamers (Niller, Glaser et al 1995), (Yates, Camiolo et al 2000). Lieberman and colleagues have shown in several studies that these elements are bound by cellular proteins associated with the telomeres, including TTAGGG-Repeat binding Factors (TRF1, TRF2), the human repressor activator protein 1 (hRap1), and tankyrase (Deng, Lezina et al 2002), (Deng, Atanasiu et al 2003), (Deng, Atanasiu et al 2005). Deletion or mutation of the nonamer elements decreases by twofold the average number of plasmids maintained per cell (Deng, Lezina et al 2002), (Deng, Atanasiu et al 2003).…”

“…One protein found to bind these elements, TRF2, exhibits cooperative binding with a truncated form of EBNA1 at DS as measured in gel shift assays, and may contribute to the enhanced replication of DNA by increasing the apparent affinity of EBNA1 for its binding sites (Deng, Lezina et al 2002). Despite this two-fold effect, the nonamer elements are not necessary for replicative function (Julien, Polonskaya et al 2004), (Yates, Camiolo et al 2000), (Koons, Van Scoy et al 2001), (Deng, Lezina et al 2002), (Deng, Atanasiu et al 2003;Wang, Lindner et al 2006). Genetic dissections of DS indicate that the minimal requirement for its replicative function is one pair of EBNA1-binding sites (Harrison, Fisenne et al 1994), (Shirakata and Hirai 1998), (Yates, Camiolo et al 2000), (Koons, Van Scoy et al 2001), (Bashaw and Yates 2001).…”

“…Lieberman and colleagues have shown in several studies that these elements are bound by cellular proteins associated with the telomeres, including TTAGGG-Repeat binding Factors (TRF1, TRF2), the human repressor activator protein 1 (hRap1), and tankyrase (Deng, Lezina et al 2002), (Deng, Atanasiu et al 2003), (Deng, Atanasiu et al 2005). Deletion or mutation of the nonamer elements decreases by twofold the average number of plasmids maintained per cell (Deng, Lezina et al 2002), (Deng, Atanasiu et al 2003). One protein found to bind these elements, TRF2, exhibits cooperative binding with a truncated form of EBNA1 at DS as measured in gel shift assays, and may contribute to the enhanced replication of DNA by increasing the apparent affinity of EBNA1 for its binding sites (Deng, Lezina et al 2002).…”