Two phenylalanines in the C-terminus of Epstein–Barr virus Rta protein reciprocally modulate its DNA binding and transactivation function

The Epstein-Barr virus (EBV) lytic transactivator Rta activates promoters through direct binding to cognate DNA sites termed Rta response elements (RREs). Rta also activates promoters that apparently lack Rta binding sites, notably Zp and Rp. Chromatin immunoprecipitation (ChIP) of endogenous Rta expressed during early replication in B95-8 cells was performed to identify Rta binding sites in the EBV genome. Quantitative PCR (qPCR) analysis showed strong enrichment for known RREs but little or no enrichment for Rp or Zp, suggesting that the Rta ChIP approach enriches for direct Rta binding sites. Rta ChIP combined with deep sequencing (ChIP-seq) identified most known RREs and several novel Rta binding sites. Rta ChIP-seq peaks were frequently upstream of Rta-responsive genes, indicating that these Rta binding sites are likely functioning as RREs. Unexpectedly, the BALF5 promoter contained an Rta binding peak. To assess whether BALF5 might be activated by an RRE-dependent mechanism, an Rta mutant (Rta K156A), deficient for DNA binding and RRE activation but competent for Zp/Rp activation, was used. Rta K156A failed to activate BALF5p, suggesting this promoter can be activated by an RRE-dependent mechanism. Rta binding to late gene promoters was not seen at early time points but was specifically detected at later times within the Rta-responsive BLRF2 and BFRF3 promoters, even when DNA replication was inhibited. Our results represent the first characterization of Rta binding to the EBV genome during replication, identify previously unknown RREs, such as one in BALF5p, and highlight the complexity of EBV late gene promoter activation by Rta.

Section: Discussionmentioning

confidence: 54%

Genome-Wide Analysis of Epstein-Barr Virus Rta DNA Binding

Heilmann

Calderwood

Portal

et al. 2012

“…(F) Lysates were prepared from P3HR1 cells that had been treated with TPA and sodium butyrate and from 293T cells that had been transfected with pCMV-R. Rta and tubulin (Tub) in the lysates were analyzed by immunoblotting. domain, which eliminates Rta's transactivation activity; mutant F600/605A, while binding strongly to RRE, exhibits lower transactivation activity than does the wild-type Rta (44). The ability of Rta and its mutants to activate the BZLF1 promoter was confirmed in a transient-reporter assay (Fig.…”

Section: Resultsmentioning

confidence: 84%

“…Plasmids pCMV-Rta and pCMV-Zta were used to express Rta and Zta as described elsewhere (14). Plasmids pR550 and pRtaF600/ 605A carry Rta mutated genes for R550 and RtaF600/605A (44). Plasmid pGFP-LC3 has been described elsewhere (45).…”

Section: Methodsmentioning

confidence: 99%

Regulation of Autophagic Activation by Rta of Epstein-Barr Virus via the Extracellular Signal-Regulated Kinase Pathway

Hung

Chen

Wang

et al. 2014

Self Cite

Autophagy is an intracellular degradation pathway that provides a host defense mechanism against intracellular pathogens. However, many viruses exploit this mechanism to promote their replication. This study shows that lytic induction of EpsteinBarr virus (EBV) increases the membrane-bound form of LC3 (LC3-II) and LC3-containing punctate structures in EBV-positive cells. Transfecting 293T cells with a plasmid that expresses Rta also induces autophagy, revealing that Rta is responsible for autophagic activation. The activation involves Atg5, a key component of autophagy, but not the mTOR pathway. The expression of Rta also activates the transcription of the genes that participate in the formation of autophagosomes, including LC3A, LC3B, and ATG9B genes, as well as those that are involved in the regulation of autophagy, including the genes TNF, IRGM, and TRAIL. Additionally, treatment with U0126 inhibits the Rta-induced autophagy and the expression of autophagy genes, indicating that the autophagic activation is caused by the activation of extracellular signal-regulated kinase (ERK) signaling by Rta. Finally, the inhibition of autophagic activity by an autophagy inhibitor, 3-methyladenine, or Atg5 small interfering RNA, reduces the expression of EBV lytic proteins and the production of viral particles, revealing that autophagy is critical to EBV lytic progression. This investigation reveals how an EBV-encoded transcription factor promotes autophagy to affect viral lytic development. IMPORTANCEAutophagy is a cellular process that degrades and recycles nutrients under stress conditions to promote cell survival. Although autophagy commonly serves as a defense mechanism against viral infection, many viruses exploit this mechanism to promote their replication. This study finds that a transcription factor that is encoded by Epstein-Barr virus (EBV), Rta, activates autophagy, and the inhibition of autophagy reduces the ability of the virus to express viral lytic proteins and to generate progeny. Unlike other virus-encoded proteins that modulate autophagy by interacting with proteins that are involved in the autophagic pathway, Rta activates the transcription of the autophagy-related genes via the ERK pathway. The results of this study reveal how the virus manipulates autophagy to promote its lytic development.

“…The proline rich region can be divided into two subregions, aa 352 to 410 and aa 450 to 500. In addition, a DNA binding-inhibitory sequence (DBIS) spans the region between aa 555 and 605 (25). Deletion of this region enhanced the capacity of Rta to bind to its corresponding response elements.…”

Section: Role Of Rta In Activating Expression Of Genes Encoding the Ementioning

confidence: 99%

“…The DNA binding activity of Rta is regulated by an intrinsic 55-amino-acid sequence (positions 555 to 605) referred to as the DNA binding-inhibitory sequence (DBIS). The DBIS blocks the capacity of wild-type (wt) Rta or the minimal DNA binding region of Rta (positions 1 to 350) to interact with DNA (25). Deletion of the last 10 amino acids in Rta or alanine substitution of phenylalanines 600 and 605 disrupted the action of the DBIS.…”

mentioning

confidence: 99%

Essential Role of Rta in Lytic DNA Replication of Epstein-Barr Virus

El-Guindy

Ghiassi-Nejad

Golden

et al. 2013

Self Cite

f Two transcription factors, ZEBRA and Rta, switch Epstein-Barr virus (EBV) from the latent to the lytic state. While ZEBRA also plays an obligatory role as an activator of replication, it is not known whether Rta is directly required for replication. Rta is dispensable for amplification of an oriLyt-containing plasmid in a transient-replication assay. Here, we assessed the requirement for Rta in activation of viral DNA synthesis from the endogenous viral genome, a function that has not been established. Initially, we searched for a ZEBRA mutant that supports viral replication but not transcription. We found that Z(S186A), a mutant of ZEBRA unable to activate transcription of Rta or viral genes encoding replication proteins, is competent to bind to oriLyt and to function as an origin recognition protein. Ectopic expression of the six components of the EBV lytic replication machinery failed to rescue replication by Z(S186A). However, addition of Rta to Z(S186A) and the mixture of replication factors activated viral replication and late gene expression. Deletion mutagenesis of Rta indicated that the C-terminal 10 amino acids (aa) were essential for the function of Rta in replication. In vivo DNA binding studies revealed that Rta interacted with the enhancer region of oriLyt. In addition, expression of Rta and Z(S186A) together, but not individually, activated synthesis of the BHLF1 transcript, a lytic transcript required for the process of viral DNA replication. Our findings demonstrate that Rta plays an indispensable role in the process of lytic DNA replication. L ytic infection is intrinsic to Epstein-Barr virus (EBV) pathogenesis. Viral particles are synthesized and assembled exclusively during the lytic state. Activation of the lytic cycle gene expression program is the only route for virus propagation. Activation of the lytic program is mediated by two transcription factors, ZEBRA and Rta, encoded by the viral BZLF1 and BRLF1 genes (1-4). Both proteins are required to trigger sequential events that include expression of replication proteins (RPs), viral genome amplification, and synthesis of late structural proteins (1,2,(5)(6)(7)(8)(9)(10)(11)(12)(13)(14).A complete linear viral genome contains two copies of the origin of lytic DNA replication (oriLyt), which regulate the process of genome amplification. These oriLyt sequences are ϳ105 kbp apart and are located in the left and right duplicated sequences of the genome (DS L and DS R ) (15). Naturally occurring EBV strains with deletions of one copy of either origin, such as the B95-8 and P3HR1 virus strains, still maintain the capacity to replicate the entire viral genome (16,17). Each origin of replication contains the DNA regulatory elements sufficient to replicate a surrogate oriLyt plasmid in cis (15). Previous studies with oriLyt plasmids characterized three important cis elements present in the DS L origin, also known as BamHI-H oriLyt (18). These cis elements are the upstream and downstream elements, which are essential for genome amplification, and a ...