Qinyan Yin

Epstein-Barr Virus Latent Membrane Protein 1 Induces Cellular MicroRNA miR-146a, a Modulator of Lymphocyte Signaling Pathways

ABSTRACT The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is a functional homologue of the tumor necrosis factor receptor family and contributes substantially to the oncogenic potential of EBV through activation of nuclear factor κB (NF-κB). MicroRNAs (miRNAs) are a class of small RNA molecules that are involved in the regulation of cellular processes such as growth, development, and apoptosis and have recently been linked to cancer phenotypes. Through miRNA microarray analysis, we demonstrate that LMP1 dysregulates the expression of several cellular miRNAs, including the most highly regulated of these, miR-146a. Quantitative reverse transcription-PCR analysis confirmed induced expression of miR-146a by LMP1. Analysis of miR-146a expression in EBV latency type III and type I cell lines revealed substantial expression of miR-146a in type III (which express LMP1) but not in type I cell lines. Reporter studies demonstrated that LMP1 induces miR-146a predominantly through two NF-κB binding sites in the miR-146a promoter and identified a role for an Oct-1 site in conferring basal and induced expression. Array analysis of cellular mRNAs expressed in Akata cells transduced with an miR-146a-expressing retrovirus identified genes that are directly or indirectly regulated by miR-146a, including a group of interferon-responsive genes that are inhibited by miR-146a. Since miR-146a is known to be induced by agents that activate the interferon response pathway (including LMP1), these results suggest that miR-146a functions in a negative feedback loop to modulate the intensity and/or duration of the interferon response.

MicroRNA-155 Is an Epstein-Barr Virus-Induced Gene That Modulates Epstein-Barr Virus-Regulated Gene Expression Pathways

ABSTRACT The cellular microRNA miR-155 has been shown to be involved in lymphocyte activation and is expressed in Epstein-Barr virus (EBV)-infected cells displaying type III latency gene expression but not type I latency gene expression. We show here that the elevated levels of miR-155 in type III latency cells is due to EBV gene expression and not epigenetic differences in cell lines tested, and we show that expression in EBV-infected cells requires a conserved AP-1 element in the miR-155 promoter. Gene expression analysis was carried out in a type I latency cell line transduced with an miR-155-expressing retrovirus. This analysis identified both miR-155-suppressed and -induced cellular mRNAs and suggested that in addition to direct targeting of 3′ untranslated regions (UTRs), miR-155 alters gene expression in part through the alteration of signal transduction pathways. 3′ UTR reporter analysis of predicted miR-155 target genes identified the transcriptional regulatory genes encoding BACH1, ZIC3, HIVEP2, CEBPB, ZNF652, ARID2, and SMAD5 as miR-155 targets. Western blot analysis of the most highly suppressed of these, BACH1, showed lower expression in cells transduced with a miR-155 retrovirus. Inspection of the promoters from genes regulated in EBV-infected cells and in cells infected with an miR-155 retrovirus identified potential binding sequences for BACH1 and ZIC3. Together, these experiments suggest that the induction of miR-155 by EBV contributes to EBV-mediated signaling in part through the modulation of transcriptional regulatory factors.

B-cell Receptor Activation Induces BIC/miR-155 Expression through a Conserved AP-1 Element

microRNA-155 is an oncogenic microRNA that has been shown to be critical for B-cell maturation and immunoglobulin production in response to antigen. In line with its function in B-cell activation, miR-155, and its primary transcript, B-cell integration cluster (BIC), is induced by B-cell receptor (BCR) cross-linking. Using pharmacological inhibitors in the human B-cell line, Ramos, we show that activation of BIC and miR-155 expression by BCR signaling occurs through the extracellular signaling-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways but not the p38 pathway. BCR activation results in the induction of c-Fos, FosB, and JunB, and expression of these are suppressed by ERK and JNK inhibitors. Reporter analysis established a key role for a conserved AP-1 site ∼40 bp upstream from the site of initiation but not an upstream NF-κB site or a putative c-Ets located at the site of initiation. Lastly, chromatin immunoprecipitation analysis demonstrated the recruitment of FosB and JunB to the miR-155 promoter following BCR activation. These results identify key determinants of BCR-mediated signaling that lead to the induction of BIC/miR-155.

Epstein-Barr virus growth/latency III program alters cellular microRNA expression

The Epstein-Barr virus (EBV) is associated with lymphoid and epithelial cancers. Initial EBV infection alters lymphocyte gene expression, inducing cellular proliferation and differentiation as the virus transitions through consecutive latency transcription programs. Cellular microRNAs (miRNAs) are important regulators of signaling pathways and are implicated in carcinogenesis. The extent to which EBV exploits cellular miRNAs is unknown. Using micro-array analysis and quantitative PCR, we demonstrate differential expression of cellular miRNAs in type III versus type I EBV latency including elevated expression of miR-21, miR-23a, miR-24, miR-27a, miR-34a, miR-146a and b, and miR-155. In contrast, miR-28 expression was found to be lower in type III latency. The EBV-mediated regulation of cellular miRNAs may contribute to EBV signaling and associated cancers.

MicroRNA miR-155 Inhibits Bone Morphogenetic Protein (BMP) Signaling and BMP-Mediated Epstein-Barr Virus Reactivation

ABSTRACT MicroRNA miR-155 is expressed at elevated levels in human cancers including cancers of the lung, breast, colon, and a subset of lymphoid malignancies. In B cells, miR-155 is induced by the oncogenic latency gene expression program of the human herpesvirus Epstein-Barr virus (EBV). Two other oncogenic herpesviruses, Kaposis sarcoma-associated herpesvirus and Mareks disease virus, encode functional homologues of miR-155, suggesting a role for this microRNA in the biology and pathogenesis of these viruses. Bone morphogenetic protein (BMP) signaling is involved in an array of cellular processes, including differentiation, growth inhibition, and senescence, through context-dependent interactions with multiple signaling pathways. Alteration of this pathway contributes to a number of disease states including cancer. Here, we show that miR-155 targets the 3′ untranslated region of multiple components of the BMP signaling cascade, including SMAD1, SMAD5, HIVEP2, CEBPB, RUNX2, and MYO10. Targeting of these mediators results in the inhibition of BMP2-, BMP6-, and BMP7-induced ID3 expression as well as BMP-mediated EBV reactivation in the EBV-positive B-cell line, Mutu I. Further, miR-155 inhibits SMAD1 and SMAD5 expression in the lung epithelial cell line A549, it inhibits BMP-mediated induction of the cyclin-dependent kinase inhibitor p21, and it reverses BMP-mediated cell growth inhibition. These results suggest a role for miR-155 in controlling BMP-mediated cellular processes, in regulating BMP-induced EBV reactivation, and in the inhibition of antitumor effects of BMP signaling in normal and virus-infected cells.

Differential Expression of the miR-200 Family MicroRNAs in Epithelial and B Cells and Regulation of Epstein-Barr Virus Reactivation by the miR-200 Family Member miR-429

ABSTRACT The miR-200 microRNA family is important for maintaining the epithelial phenotype, partially through suppressing ZEB1 and ZEB2. Since ZEB1 inhibits Epstein-Barr virus (EBV) reactivation, we hypothesized that expression of miR-200 family members in epithelial cells may partly account for higher levels of EBV reactivation in this tissue (relative to nonplasma B cells). Here we show that, whereas miR-200 family members are expressed in epithelial cells, their expression is low in latently infected B cells. Furthermore, the miR-200 family member miR-429 shows elevated expression in plasma cell lines and is induced by B-cell-receptor activation in Akata cells. Lastly, expression of miR-429 can break latency.

OncomiR Addiction Is Generated by a miR-155 Feedback Loop in Theileria-Transformed Leukocytes

The intracellular parasite Theileria is the only eukaryote known to transform its mammalian host cells. We investigated the host mechanisms involved in parasite-induced transformation phenotypes. Tumour progression is a multistep process, yet ‘oncogene addiction’ implies that cancer cell growth and survival can be impaired by inactivating a single gene, offering a rationale for targeted molecular therapies. Furthermore, feedback loops often act as key regulatory hubs in tumorigenesis. We searched for microRNAs involved in addiction to regulatory loops in leukocytes infected with Theileria parasites. We show that Theileria transformation involves induction of the host bovine oncomiR miR-155, via the c-Jun transcription factor and AP-1 activity. We identified a novel miR-155 target, DET1, an evolutionarily-conserved factor involved in c-Jun ubiquitination. We show that miR-155 expression led to repression of DET1 protein, causing stabilization of c-Jun and driving the promoter activity of the BIC transcript containing miR-155. This positive feedback loop is critical to maintain the growth and survival of Theileria-infected leukocytes; transformation is reversed by inhibiting AP-1 activity or miR-155 expression. This is the first demonstration that Theileria parasites induce the expression of host non-coding RNAs and highlights the importance of a novel feedback loop in maintaining the proliferative phenotypes induced upon parasite infection. Hence, parasite infection drives epigenetic rewiring of the regulatory circuitry of host leukocytes, placing miR-155 at the crossroads between infection, regulatory circuits and transformation.

Global Bidirectional Transcription of the Epstein-Barr Virus Genome during Reactivation

ABSTRACT Epstein-Barr virus (EBV) reactivation involves the ordered induction of approximately 90 viral genes that participate in the generation of infectious virions. Using strand-specific RNA-seq to assess the EBV transcriptome during reactivation, we found extensive bidirectional transcription extending across nearly the entire genome. In contrast, only 4% of the EBV genome is currently bidirectionally annotated. Most of the newly identified transcribed regions show little evidence of coding potential, supporting noncoding roles for most of these RNAs. Based on previous cellular long noncoding RNA size calculations, we estimate that there are likely hundreds more EBV genes expressed during reactivation than was previously known. Limited 5′ and 3′ rapid amplification of cDNA ends (RACE) experiments and findings of novel splicing events by RNA-seq suggest that the complexity of the viral genome during reactivation may be even greater. Further analysis of antisense transcripts at some of the EBV latency gene loci showed that they are “late” genes, they are nuclear, and they tend to localize in areas of the nucleus where others find newly synthesized viral genomes. This raises the possibility that these transcripts perform functions such as new genome processing, stabilization, organization, etc. The finding of a significantly more complex EBV transcriptome during reactivation changes our view of the viral production process from one that is facilitated and regulated almost entirely by previously identified viral proteins to a process that also involves the contribution of a wide array of virus encoded noncoding RNAs. IMPORTANCE Epstein-Barr virus (EBV) is a herpesvirus that infects the majority of the worlds population, in rare cases causing serious disease such as lymphoma and gastric carcinoma. Using strand-specific RNA-seq, we have studied viral gene expression during EBV reactivation and have discovered hundreds more viral transcripts than were previously known. The finding of alternative splicing and the prevalence of overlapping transcripts indicate additional complexity. Most newly identified transcribed regions do not encode proteins but instead likely function as noncoding RNA molecules which could participate in regulating gene expression, gene splicing or even activities such as viral genome processing. These findings broaden the scope of what we need to consider to understand the viral manufacturing process. As more detailed studies are undertaken they will likely change the way we view this process as a whole.

Latent Expression of the Epstein-Barr Virus (EBV)-Encoded Major Histocompatibility Complex Class I TAP Inhibitor, BNLF2a, in EBV-Positive Gastric Carcinomas

ABSTRACT The Epstein-Barr virus (EBV) BNLF2a gene product provides immune evasion properties to infected cells through inhibition of transporter associated with antigen processing (TAP)-mediated transport of antigen peptides. Although BNLF2a is considered to be a lytic gene, we demonstrate that it is expressed in nearly half of the EBV-associated gastric carcinomas analyzed. Further, we show that BNLF2a expression is dissociated from lytic gene expression. BNLF2a is therefore expressed in this latency setting, potentially helping protect the infected tumor cells from immunosurveillance.

Identification of a Negative Regulatory Element in the Epstein-Barr Virus Zta Transactivation Domain That Is Regulated by the Cell Cycle Control Factors c-Myc and E2F1

ABSTRACT Reactivation in Epstein-Barr virus (EBV) is closely associated with a G0/G1 cell cycle arrest which can be induced either by lytic cycle-inducing agents or by the immediate-early gene product Zta. Accumulating evidence shows that in epithelial cells, downregulation of the proto-oncogene, c-myc, plays an important role in lytic cycle-associated cell growth arrest. Here, we provide evidence that c-Myc provides a gatekeeper function to ensure that certain cell cycle inhibitory events have been capitulated prior to full progression into the lytic cycle. Specifically, we show that reconstitution of c-Myc expression during the lytic cycle to levels observed in cycling uninduced cells inhibits the transactivation function of Zta. Nuclear localization studies show that c-Myc does not grossly alter the nuclear localization of Zta or its association with the insoluble nuclear fraction. Enforced expression of another transcription factor that promotes cell cycle progression, E2F1, also inhibits Zta transactivation. Analysis of c-Myc- and E2F1-mediated inhibition of a panel of Zta mutants shows parallel genetics and inhibition maps to a small bipartite sequence located between amino acids 29 and 53 of Zta, containing homology to the proline-rich domain of the tumor suppressor protein p53. Mutation of a conserved tryptophan residue located at amino acid 49 of Zta largely prevents inhibition by both c-Myc and E2F1. These studies identify a negative regulatory element within the Zta activation domain that is regulated by the cell cycle-promoting factors c-Myc and E2F1.