siRNAs against the Epstein Barr virus latency replication factor, EBNA1, inhibit its function and growth of EBV-dependent tumor cells

AbstractThe Epstein Barr virus (EBV) plays a role in maintenance of the tumor phenotype in a number of human cancers. The EBV latency replication factor, EBNA1, is required for persistence of the EBV episome, is anti-apoptotic, and is universally expressed in all EBV-associated tumors. Here, we show that EBNA1-specific siRNAs can inhibit EBNA1 expression and function. siRNAs were generated against three target sites in the EBNA1 messenger RNA, and two of these were found to inhibit EBNA1 expression from an ectopic EBNA1 expression cassette. EBNA1 siRNAs also inhibit endogenously expressed EBNA1 in EBV-positive epithelial and B-cell lines. Using a mini-EBV replication model, siRNA-mediated inhibition of EBNA1 expression suppressed the episomal maintenance function of EBNA1. Lastly, introduction of an EBNA1 siRNA into an EBV-positive tumor cell line inhibited tumor cell growth/survival. These data suggest that siRNAs against EBNA1 may have therapeutic value in EBV-associated diseases

Role of c-myc Regulation in Zta-Mediated Induction of the Cyclin-Dependent Kinase Inhibitors p21 and p27 and Cell Growth Arrest

AbstractLatency-associated Epstein–Barr virus (EBV) gene expression induces cell proliferation. Unlike the latency associated genes, lytic gene expression in EBV, as well as other herpesviruses, elicits cell cycle arrest. Previous studies have shown that the EBV immediate early lytic transactivator, Zta, induces a G0/G1 cell cycle arrest through induction of the cyclin-dependent kinase inhibitors, p21 and p27. Here we show that while EBV latency is intimately linked to activation of the protooncogene, c-myc, Zta represses c-myc expression. We also show that inhibition of c-myc expression is required for Zta-mediated growth arrest and for maximal induction of p21 and p27. Nevertheless, induction of p21 and p27 is also influenced by a c-myc-independent mechanism. A detailed genetic analysis of Zta's basic/DNA binding region identified two distinct subregions that contribute to full induction of p21 and p27. One subdomain influences p21 and p27 expression through the c-myc-dependent mechanism and the other subdomain influences p21 and p27 induction through the c-myc-independent pathway. Together, these studies further our understanding of the complex nature of Zta-induced growth arrest

Arsenic trioxide inhibits EBV reactivation and promotes cell death in EBV-positive lymphoma cells

Abstract Background Epstein-Barr Virus (EBV) is associated with hematopoietic malignancies, such as Burkitt’s lymphoma, post-transplantation lymphoproliferative disorder, and diffuse large B-cell lymphoma. The current approach for EBV-associated lymphoma involves chemotherapy to eradicate cancer cells, however, normal cells may be injured and organ dysfunction may occur with currently employed regimens. This research is focused on employing arsenic trioxide (ATO) as EBV-specific cancer therapy takes advantage of the fact the EBV resides within the malignant cells. Methods and results Our research reveals that low ATO inhibits EBV gene expression and genome replication. EBV spontaneous reactivation starts as early as 6 h after re-suspending EBV-positive Mutu cells in RPMI media in the absence of ATO, however this does not occur in Mutu cells cultured with ATO. ATO’s inhibition of EBV spontaneous reactivation is dose dependent. The expression of the EBV immediate early gene Zta and early gene BMRF1 is blocked with low concentrations of ATO (0.5 nM – 2 nM) in EBV latency type I cells and EBV-infected PBMC cells. The combination of ATO and ganciclovir further diminishes EBV gene expression. ATO-mediated reduction of EBV gene expression can be rescued by co-treatment with the proteasome inhibitor MG132, indicating that ATO promotes ubiquitin conjugation and proteasomal degradation of EBV genes. Co-immunoprecipitation assays with antibodies against Zta pulls down more ubiquitin in ATO treated cell lysates. Furthermore, MG132 reverses the inhibitory effect of ATO on anti-IgM-, PMA- and TGF-β-mediated EBV reactivation. Thus, mechanistically ATO’s inhibition of EBV gene expression occurs via the ubiquitin pathway. Moreover, ATO treatment results in increased cell death in EBV-positive cells compared to EBV-negative cells, as demonstrated by both MTT and trypan blue assays. ATO-induced cell death in EBV-positive cells is dose dependent. ATO and ganciclovir in combination further enhances cell death specifically in EBV-positive cells. Conclusion ATO-mediated inhibition of EBV lytic gene expression results in cell death selectively in EBV-positive lymphocytes, suggesting that ATO may potentially serve as a drug to treat EBV-related lymphomas in the clinical setting

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▿

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

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

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, Kaposi's sarcoma-associated herpesvirus and Marek's 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

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

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

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

Bakterizacija tla i leguminoznih biljaka fiksatorima dušika u današnje je vrijeme neizostavan dio poljoprivredne proizvodnje. Simbiotska interakcija između kvržičnih bakterija i biljke omogućava fiksaciju inače nedostupnog, atmosferskog dušika, što je dovelo do formacija raznih inokulanata kao alternative mineralnim gnojivima koja imaju negativan učinak na tlo. Najveći problem se očituje u preživljavanju sojeva simbiotskih bakterija koje ovise o raznim fizikalno – kemijskim uvjetima okoliša. Isključivi čimbenici za njihov razvoj su vlažnost, temperatura i pH sredine. Svrha ovog istraživačkog rada bila je odrediti vijabilnost stanica Bradyrhizobium japonicum pri različitim temperaturama i prilagođenom pH medija in vitro. Rezultati su pokazali najbolju vijabilnost stanica pri neutralnom pH i pri 5 °C. Dokazana je i hipoteza da će se pri različitoj temperaturi skladištenja i smanjenjem pH proprocionalno smanjiti i broj poraslih kolonija bakterije. Rezultati ukazuju kako su tremperatura skladištenja i pH ključni faktori za preživljavanje rizobija.Bacterization of the soil and leguminous plants with nitrogen fixating bacteria is nowadays an indispensable part of agricultural production. The symbiotic interaction between nodulating bacteria and plants allows the fixation of otherwise inaccessible, atmospheric nitrogen, which has led to the formation of various inoculants as an alternative to mineral fertilizers that have a negative effect on the soil. The issue of utmost importance is the survival of strains of symbiotic bacteria that depend on various physico - chemical conditions of the enviroment. Exclusive factors for their development are humidity, temperature and pH of the environment. The purpose of this study was to determine the viability of Bradyrhizobium japonicum cells at different temperatures and adjusted pH of the medium in vitro. The results showed the best cell viability at neutral pH and at 5 ° C. The hypothesis that the number of grown colonies of the bacterium will proportionally decrease at different storage temperature and decrease in pH has also been proven. The results indicate that storage temperature and pH are key factors for rhizobia survival