Role for G-Quadruplex RNA Binding by Epstein-Barr Virus Nuclear Antigen 1 in DNA Replication and Metaphase Chromosome Attachment

Norseen, Julie; Johnson, F. Brad; Lieberman, Paul M.

doi:10.1128/jvi.00747-09

Cited by 141 publications

(157 citation statements)

References 63 publications

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“…Recently, we showed that ICP27 binds specifically to GC-rich sequences but that the N-terminal portion of ICP27 encoding the RGG box does not bind Gquartet structures (5). This finding was surprising because the RGG boxes of two other proteins that bind nucleic acids, FMRP and EBNA1, have been shown to bind to G-quadruplex or G-quartet structures (29,30). G quartets are complex tertiary structures that can form readily in DNA or RNA containing three or more consecutive guanines (4,8,30).…”

Section: Discussionmentioning

confidence: 69%

Three Arginine Residues within the RGG Box Are Crucial for ICP27 Binding to Herpes Simplex Virus 1 GC-Rich Sequences and for Efficient Viral RNA Export

Corbin-Lickfett¹,

Souki²,

Cocco

et al. 2010

J Virol

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ICP27 is a multifunctional protein that is required for herpes simplex virus 1 mRNA export. ICP27 interacts with the mRNA export receptor TAP/NXF1 and binds RNA through an RGG box motif. Unlike other RGG box proteins, ICP27 does not bind G-quartet structures but instead binds GC-rich sequences that are flexible in structure. To determine the contribution of arginines within the RGG box, we performed in vitro binding assays with N-terminal proteins encoding amino acids 1 to 160 of wild-type ICP27 or arginine-to-lysine substitution mutants. The R138,148,150K triple mutant bound weakly to sequences that were bound by the wild-type protein and single and double mutants. Furthermore, during infection with the R138,148,150K mutant, poly(A) ؉ RNA and newly transcribed RNA accumulated in the nucleus, indicating that viral RNA export was impaired. To determine if structural changes had occurred, nuclear magnetic resonance (NMR) analysis was performed on N-terminal proteins consisting of amino acids 1 to 160 from wild-type ICP27 and the R138,148,150K mutant. This region of ICP27 was found to be highly flexible, and there were no apparent differences in the spectra seen with wild-type ICP27 and the R138,148,150K mutant. Furthermore, NMR analysis with the wild-type protein bound to GC-rich sequences did not show any discernible folding. We conclude that arginines at positions 138, 148, and 150 within the RGG box of ICP27 are required for binding to GC-rich sequences and that the N-terminal portion of ICP27 is highly flexible in structure, which may account for its preference for binding flexible sequences.The herpes simplex virus 1 (HSV-1) protein ICP27 is a multifunctional regulatory protein that is required for productive viral infection. ICP27 interacts with a number of cellular proteins, and it binds RNA (35). One of the functions that ICP27 performs is to escort viral mRNAs from the nucleus to the cytoplasm for translation (2,3,5,10,13,21,34). ICP27 binds viral RNAs (5, 34) and interacts directly with the cellular mRNA export receptor TAP/NXF1 (2, 21), which is required for the export of HSV-1 mRNAs (20,21). ICP27 also interacts with the export adaptor proteins Aly/REF (2,3,23) and UAP56 (L. A. Johnson, H. Swesey, and R. M. Sandri-Goldin, unpublished results), which form part of the TREX complex that binds to the 5Ј end of mRNA through an interaction with CBP80 (26, 32, 41). Aly/REF does not appear to bind viral RNA directly (3), and it is not essential for HSV-1 RNA export based upon small interfering RNA (siRNA) knockdown studies (20), but it contributes to the efficiency of viral RNA export (3, 23). ICP27 also interacts with the SR splicing proteins SRp20 and 9G8 (11,36), which have been shown to shuttle between the nucleus and the cytoplasm (1). SRp20 and 9G8 have also been shown to facilitate the export of some cellular RNAs (16,17,27) by binding RNA and interacting with TAP/ NXF1 (14,16,18). The knockdown of SRp20 or 9G8 adversely affects HSV-1 replication and specifically results in a nuclear accumulation of new...

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Section: Discussionmentioning

confidence: 69%

Three Arginine Residues within the RGG Box Are Crucial for ICP27 Binding to Herpes Simplex Virus 1 GC-Rich Sequences and for Efficient Viral RNA Export

Corbin-Lickfett¹,

Souki²,

Cocco

et al. 2010

J Virol

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“…The presence of G-quadruplexes in viral genomes was previously reported (reviewed in reference 67). Interestingly, BRACO-19 negatively affects genome-processing activities, such as replication and transcription of herpes simplex virus 1 (68), Epstein-Barr virus (69), and human immunodeficiency virus type 1 (70). Unlike these viruses, however, in silico analyses of the HHV-6A/B genomes indicate that potential G-quadruplex structures are located exclusively in the viral TMR and not within coding regions (70).…”

Section: Discussionmentioning

confidence: 99%

Stabilization of Telomere G-Quadruplexes Interferes with Human Herpesvirus 6A Chromosomal Integration

Gilbert-Girard

Gravel

Artusi

et al. 2017

J Virol

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Human herpesviruses 6A and 6B (HHV-6A/B) can integrate their genomes into the telomeres of human chromosomes using a mechanism that remains poorly understood. To achieve a better understanding of the HHV-6A/B integration mechanism, we made use of BRACO-19, a compound that stabilizes G-quadruplex secondary structures and prevents telomere elongation by the telomerase complex. First, we analyzed the folding of telomeric sequences into G-quadruplex structures and their binding to BRACO-19 using G-quadruplex-specific antibodies and surface plasmon resonance. Circular dichroism studies indicate that BRACO-19 modifies the conformation and greatly stabilizes the G-quadruplexes formed in G-rich telomeric DNA. Subsequently we assessed the effects of BRACO-19 on the HHV-6A initial phase of infection. Our results indicate that BRACO-19 does not affect entry of HHV-6A DNA into cells. We next investigated if stabilization of G-quadruplexes by BRACO-19 affected HHV-6A's ability to integrate its genome into host chromosomes. Incubation of telomerase-expressing cells with BRACO-19, such as HeLa and MCF-7, caused a significant reduction in the HHV-6A integration frequency (P Ͻ 0.002); in contrast, BRACO-19 had no effect on HHV-6 integration frequency in U2OS cells that lack telomerase activity and elongate their telomeres through alternative lengthening mechanisms. Our data suggest that the fluidity of telomeres is important for efficient chromosomal integration of HHV-6A and that interference with telomerase activity negatively affects the generation of cellular clones containing integrated HHV-6A.IMPORTANCE HHV-6A/B can integrate their genomes into the telomeres of infected cells. Telomeres consist of repeated hexanucleotides (TTAGGG) of various lengths (up to several kilobases) and end with a single-stranded 3= extension. To avoid recognition and induce a DNA damage response, the single-stranded overhang folds back on itself and forms a telomeric loop (T-loop) or adopts a tertiary structure, referred to as a G-quadruplex. In the current study, we have examined the effects of a G-quadruplex binding and stabilizing agent, BRACO-19, on HHV-6A chromosomal integration. By stabilizing G-quadruplex structures, BRACO-19 affects the ability of the telomerase complex to elongate telomeres. Our results indicate that BRACO-19 reduces the number of clones harboring integrated HHV-6A. This study is the first of its kind and suggests that telomerase activity is essential to restore a functional telomere of adequate length following HHV-6A integration.

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“…However, it is unclear whether the effect of the G-quadruplex ligand, PDS, solely targets EBNA1-specific G-quadruplexes in vivo. Notably, it is reported that protein domains within EBNA1 bind G-rich RNAs that are predicted to form G-quadruplex structures 46 , suggesting a further level of regulation whereby EBNA1 protein may also selfmodulate its expression using G-quadruplexes as regulatory elements. Modulation of the translational regulatory effects of these G-quadruplexes has the potential to enhance immune recognition of virally infected cells or inhibit replication of the viral genome, leading to elimination of viral latency.…”

Section: Discussionmentioning

confidence: 99%

G-quadruplexes regulate Epstein-Barr virus–encoded nuclear antigen 1 mRNA translation

et al. 2014

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Viruses that establish latent infections have evolved unique mechanisms to avoid host immune recognition. Maintenance proteins of these viruses regulate their synthesis to levels sufficient for maintaining persistent infection but below threshold levels for host immune detection. The mechanisms governing this finely tuned regulation of viral latency are unknown. Here we show that mRNAs encoding gammaherpesviral maintenance proteins contain within their open reading frames clusters of unusual structural elements, G-quadruplexes, which are responsible for the cisacting regulation of viral mRNA translation. By studying the Epstein-Barr virus-encoded nuclear antigen 1 (EBNA1) mRNA, we demonstrate that destabilization of G-quadruplexes using antisense oligonucleotides increases EBNA1 mRNA translation. In contrast, pretreatment with a G-quadruplex-stabilizing small molecule, pyridostatin, decreases EBNA1 synthesis, highlighting the importance of G-quadruplexes within virally encoded transcripts as unique regulatory signals

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Role for G-Quadruplex RNA Binding by Epstein-Barr Virus Nuclear Antigen 1 in DNA Replication and Metaphase Chromosome Attachment

Cited by 141 publications

References 63 publications

Three Arginine Residues within the RGG Box Are Crucial for ICP27 Binding to Herpes Simplex Virus 1 GC-Rich Sequences and for Efficient Viral RNA Export

Three Arginine Residues within the RGG Box Are Crucial for ICP27 Binding to Herpes Simplex Virus 1 GC-Rich Sequences and for Efficient Viral RNA Export

Stabilization of Telomere G-Quadruplexes Interferes with Human Herpesvirus 6A Chromosomal Integration

G-quadruplexes regulate Epstein-Barr virus–encoded nuclear antigen 1 mRNA translation

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