Cellular MicroRNAs 200b and 429 Regulate the Epstein-Barr Virus Switch between Latency and Lytic Replication

The EBNA1 protein of Epstein-Barr virus (EBV) plays multiple roles in EBV latent infection, including altering cellular pathways relevant for cancer. Here we used microRNA (miRNA) cloning coupled with high-throughput sequencing to identify the effects of EBNA1 on cellular miRNAs in two nasopharyngeal carcinoma cell lines. EBNA1 affected a small percentage of cellular miRNAs in both cell lines, in particular, upregulating multiple let-7 family miRNAs, including let-7a. The effects of EBNA1 on let-7a were verified by demonstrating that EBNA1 silencing in multiple EBV-positive carcinomas downregulated let-7a. Accordingly, the let-7a target, Dicer, was found to be partially downregulated by EBNA1 expression (at the mRNA and protein levels) and upregulated by EBNA1 silencing in EBV-positive cells. Reporter assays based on the Dicer 3= untranslated region with and without let-7a target sites indicated that the effects of EBNA1 on Dicer were mediated by let-7a. EBNA1 was also found to induce the expression of let-7a primary RNAs in a manner dependent on the EBNA1 transcriptional activation region, suggesting that EBNA1 induces let-7a by transactivating the expression of its primary transcripts. Consistent with previous reports that Dicer promotes EBV reactivation, we found that a let-7a mimic inhibited EBV reactivation to the lytic cycle, while a let-7 sponge increased reactivation. The results provide a mechanism by which EBNA1 could promote EBV latency by inducing let-7 miRNAs. IMPORTANCE The EBNA1 protein of Epstein-Barr virus (EBV) contributes in multiple ways to the latent mode of EBV infection that leads to lifelong infection.In this study, we identify a mechanism by which EBNA1 helps to maintain EBV infection in a latent state. This involves induction of a family of microRNAs (let-7 miRNAs) that in turn decreases the level of the cellular protein Dicer. We demonstrate that let-7 miRNAs inhibit the reactivation of latent EBV, providing an explanation for our previous observation that EBNA1 promotes latency. In addition, since decreased levels of Dicer have been associated with metastatic potential, EBNA1 may increase metastases by downregulating Dicer. E pstein-Barr virus (EBV) is a gammaherpesvirus that infects most people worldwide and is associated with several types of B-cell lymphomas, as well as nasopharyngeal carcinoma (NPC) and gastric carcinoma (1, 2). The EBV life cycle consists of both latent and lytic modes of infection in B lymphocytes and epithelial cells. Although EBV mainly exists in a latent mode of infection in B cells, it occasionally reactivates to the lytic state for cell-to-cell spread. In addition, lytic reactivation of EBV in epithelial cells of the orthopharynx is necessary for the production of viral particles required for host-to-host transmission of the virus. Lytic infection begins with the expression of BZLF1 (or Zta), followed by the expression of BRLF1 (or Rta). Together these proteins activate the cascade of subsequent lytic gene expression and enable the generation of lin...

Section: Discussionmentioning

confidence: 65%

mentioning

confidence: 99%

Epstein-Barr Virus EBNA1 Protein Regulates Viral Latency through Effects on let-7 MicroRNA and Dicer

Mansouri

Pan

Blencowe

et al. 2014

“…Recently, our laboratory showed that either ZEB1 or ZEB2 can play a central role in the maintenance of EBV latency, doing so in a cell-type-dependent manner (15). We also showed that addition of the cellular microRNAs (miRNAs) 200b and 429 can induce EBV lytic replication in EBV-positive cells by downregulating expression of ZEB1 and ZEB2 (16).…”

mentioning

confidence: 99%

The ZIIR Element of the Epstein-Barr Virus BZLF1 Promoter Plays a Central Role in Establishment and Maintenance of Viral Latency

McCarthy

Lim

et al. 2011

Self Cite

The Epstein-Barr virus (EBV)BZLF1Epstein-Barr virus (EBV), a human gammaherpesvirus, infects approximately 90% of the world's population. It is a causative agent of infectious mononucleosis and is associated with several epithelial and B-cell cancers, including Burkitt's lymphoma (BL), Hodgkin's disease, nasopharygeal carcinoma, and some gastric carcinomas (reviewed in reference 41). EBV can infect primary B lymphocytes, establishing a latent form of infection in which its genome is maintained as an episome, replicating in synchrony with its host's cellular DNA. EBV can also infect epithelial cells, leading to a latent or lytic form of infection, depending upon the specific cell type and state of cellular differentiation (reviewed in reference 28).The switch from EBV latency to lytic replication in B cells is usually initiated via activating expression of the immediateearly (IE) BZLF1 gene (11). The BZLF1 gene encodes a sequence-specific DNA-binding protein, Zta (also called Z, Zebra, and EB1), a member of the bZIP family of leucine-zipper transactivators. The activities of Zta include direct participation in EBV replication via binding to the viral DNA origin of lytic replication, ori-lyt, downregulating the latency-associated promoters Cp and Wp, and serving as a transcriptional transactivator of its own promoter, other IE and early (E) viral promoters, including the BRLF1 promoter, Rp, and several cellular promoters (reviewed in references 26 and 31). The BRLF1 gene encodes a second viral transactivator, Rta (also called R). Acting together, Zta and Rta play multiple roles in lytic replication of EBV (17). While highly quiescent during latency, transcription from the BZLF1 promoter Zp can be activated in some cells by incubation with various inducers, including phorbol esters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), calcium ionophores such as ionomycin, histone deacatylase (HDAC) inhibitors such as valproic acid (VPA) and sodium butyrate, transforming growth factor ␤ (TGF-␤), and anti-immunoglobulins (anti-Igs) (reviewed in references 26 and 31).The cis-acting elements of Zp sufficient for both basal and induced activity lie within the nucleotide (nt) Ϫ221 to ϩ12 region (mini-Zp) relative to Zp's transcription initiation site (reviewed in references 26 and 45) (Fig. 1). Three positive regulatory cis-acting elements, named ZI, ZII, and ZIII, have been identified within mini-Zp. The ubiquitous transcription factors Sp1/Sp3 can bind to the AT-rich ZIA, ZIC, and ZID elements, while the myocyte enhancer factor 2D (MEF2D) can bind to the ZIA, ZIB, and ZID elements (6,33,34). ZII is a cyclic AMP response element (CRE)-like motif that binds CREB, ATF family members, C/EBPs, and the AP-1 family of transcription factors (5,6,19,24,46,50,53). Although the ZIA-ZID and ZII elements have distinct DNA sequences and are bound by different regulatory factors, they both function as sites through which induction can be mediated by chemical inducers such as TPA and ionomycin in reporter assays (20). These inducers ac...

“…The hsa-miR-200 family members miR-200a, -200b, and -429, target ZEB1 and ZEB2 (13,14), suggesting that these miRNAs might induce EBV reactivation. In keeping with this prediction, addition of miR-200b or miR-429 to EBV-positive cells expressing ZEB proteins increased EBV reactivation in a manner dependent on the ZEB binding sites, while inhibition of these miRNAs decreased EBV reactivation (15,16). However, some gastric carcinoma and NPC cell lines do not express ZEB proteins (15) and yet still maintain EBV in a predominantly latent state, indicating that there are additional levels of regulation.…”

Section: Gammaherpesvirusesmentioning

confidence: 59%

Regulation of Herpesvirus Reactivation by Host MicroRNAs

Frappier

2015

The interplay between latent and lytic modes of infection is central to successful infection of all herpesviruses, yet knowledge of the determinants that govern reactivation of these viruses from latent to lytic infection is limited. Recently, several studies have identified roles for specific cellular microRNAs in inhibiting reactivation of various herpesviruses, thereby promoting latent infections. These studies are discussed in the context of current knowledge on mechanisms of regulation of reactivation of specific herpesviruses. Herpesviruses persist for the life of the host due to the interplay between their latent and lytic modes of infection. In latency, viral gene expression is highly restricted such that few or no viral proteins are expressed, enabling the virus to persist under the radar of the host immune system. The latent virus can then be reactivated to the lytic cycle, which involves an ordered expression of most or all of the encoded proteins, starting with one or two transcriptional activators (referred to as lytic-switch proteins) that initiate the cascade of viral gene expression. In lytic infection, viral genomes are amplified for packaging and virions are produced, enabling cell-to-cell and host-to-host spread. The mechanisms that suppress viral expression in latency and cues that trigger viral reactivation have been the subject of many studies and are only partly understood. The fact that latency tends to occur in specific cell types suggests that the cellular environment is key to determining the infectious cycle of the virus. Several studies have pointed to differences in the chromatin states at viral promoters as common determinants of latent versus lytic infection, and hence, levels of host chromatin-modifying enzymes can impact whether or not a virus reactivates (1, 2). Recently, another common mechanism of promoting herpesvirus latency has emerged: suppression of herpesvirus reactivation by cellular microRNAs (miRNAs).miRNAs are ϳ22-nucleotide noncoding RNA molecules that are initially transcribed as long primary RNAs and then are sequentially processed by two RNase III enzymes (Drosha and Dicer) to generate mature miRNAs (3). In complex with an Argonaute protein, the mature miRNA binds through its seed sequence to a complementary sequence mostly in the 3= untranslated region (UTR) of an mRNA, thereby inhibiting its translation or inducing its degradation. miRNAs are encoded both by cellular and viral genomes and can regulate a wide variety of processes. Herpesviruses typically encode multiple miRNAs that are expressed during latent infection. Many studies have indicated that these miRNAs are important for persistent infection, although the precise roles of many of these miRNAs are not yet known (4, 5). In addition, cellular miRNAs are known to impact herpesvirus infections in many different ways, with several recent findings identifying roles in keeping the viruses in a latent state (5). ALPHAHERPESVIRUSESThe alpha subfamily of herpesviruses establish latency in neurons. Herpes simpl...