A transgenic approach to investigate the role of Epstein-Barr virus encoded RNA1 in lymphomagenesis

Epstein-Barr Virus (EBV) is associated with several human cancers including Burkitt's lymphoma (BL), Hodgkin's disease and nasopharyngeal carcinoma amongst others. In these cancers, a different subset of the viral latent genes are expressed, but all express the EBV small encoded RNAs: EBERl and EBER2. The EBERs are polymerase III (pol III) genes but do not fall neatly into any of the 3 promoter types as they combine both pol II (Spl, ATF and TATA-like box) and pol III elements (A and B boxes). The EBERs have been shown to confer resistance to interferon (IFN)-alpha-induced apoptosis via binding of the IFN-inducible, double-stranded (ds) RNA-activated protein kinase PKR and inhibition of its activation by phosphorylation. Evidence has also suggested an oncogenic role of the EBERs in BL cells, indicating their possible contribution to the disease process of EBV-associated tumours. In order to investigate the potential role of EBER1 as an oncogenic RNA in vivo, 13 lines of transgenic mice designed to express EBER1 in lymphoid cells using three variant transgenes were generated. The transgenes incorporate a novel combination of tissue-specific RNA pol II and pol III elements. The efficacy of transgene expression was confirmed in culture. Mice of 10 of the transgenic lines were shown to express EBER1 in lymphoid tissues and the expression varied between the lines. The phenotypic consequences of EBER1 expression in vivo were examined and lymphoid expansion in mice of several lines was observed at a young age as well as the development of B-cell lymphoma in one of the lines. Cross-breeding programmes were undertaken and have shown that EBER1 does not cooperate in lymphomagenesis with EBNAl. However cooperation was observed in B-cell lymphomagenesis between EBER1 and N-myc although not with c-Myc. This might suggest that the oncogenic mechanism is elicited through cell survival. The role of EBER1 in response to dsRNA stimulation was analysed in vivo and results indicate an inhibition of Statl expression and activation by EBER1, This might reflect downstream actions of blockade of the IFN pathway or a new pathway. The results in this study support the hypothesis that EBER1 has oncogenic properties, the first pol III RNA described as such. This implicates the RNA in the pathogenesis of EBV associated lymphoma in addition to its role in immune evasion

Lymphoid Hyperplasia and Lymphoma in Transgenic Mice Expressing the Small Non-Coding RNA, EBER1 of Epstein-Barr Virus

Non-coding RNAs have critical functions in diverse biological processes, particularly in gene regulation. Viruses, like their host cells, employ such functional RNAs and the human cancer associated Epstein-Barr virus (EBV) is no exception. Nearly all EBV associated tumours express the EBV small, non-coding RNAs (EBERs) 1 and 2, however their role in viral pathogenesis remains largely obscure.To investigate the action of EBER1 in vivo, we produced ten transgenic mouse lines expressing EBER1 in the lymphoid compartment using the mouse immunoglobulin heavy chain intronic enhancer Emicro. Mice of several of these EmicroEBER1 lines developed lymphoid hyperplasia which in some cases proceeded to B cell malignancy. The hallmark of the transgenic phenotype is enlargement of the spleen and mesenteric lymph nodes and in some cases enlargement of the thymus, liver and peripheral lymph nodes. The tumours were found to be of B cell origin and showed clonal IgH rearrangements. In order to explore if EBER1 would cooperate with c-Myc (deregulated in Burkitt's lymphoma) to accelerate lymphomagenesis, a cross-breeding study was undertaken with EmicroEBER1 and EmicroMyc mice. While no significant reduction in latency to lymphoma onset was observed in bi-transgenic mice, c-Myc induction was detected in some EmuEBER1 single transgenic tumours, indicative of a functional cooperation.This study is the first to describe the in vivo expression of a polymerase III, non-coding viral gene and demonstrate its oncogenic potential. The data suggest that EBER1 plays an oncogenic role in EBV associated malignant disease

Sorting nexin 9 differentiates ligand-activated Smad3 from Smad2 for nuclear import and transforming growth factor ß signaling

Transforming growth factor ß (TGFß) is a pleiotropic protein secreted from essentially all cell types and primary tissues. While TGFß's actions reflect the activity of a number of signaling networks, the primary mediator of TGFß responses are the Smad proteins. Following receptor activation, these cytoplasmic proteins form hetero-oligomeric complexes that translocate to the nucleus and affect gene transcription. Here, through biological, biochemical, and immunofluorescence approaches, sorting nexin 9 (SNX9) is identified as being required for Smad3-dependent responses. SNX9 interacts with phosphorylated (p) Smad3 independent of Smad2 or Smad4 and promotes more rapid nuclear delivery than that observed independent of ligand. Although SNX9 does not bind nucleoporins Nup153 or Nup214 or some ß importins (Imp7 or Impß), it mediates the association of pSmad3 with Imp8 and the nuclear membrane. This facilitates nuclear translocation of pSmad3 but not SNX9

The Human Papillomavirus Type 31 Late 3′ Untranslated Region Contains a Complex Bipartite Negative Regulatory Element

The papillomavirus life cycle is tightly linked to epithelial cell differentiation. Production of virus capsid proteins is restricted to the most terminally differentiated keratinocytes in the upper layers of the epithelium. However, mRNAs encoding the capsid proteins can be detected in less-differentiated cells, suggesting that late gene expression is controlled posttranscriptionally. Short sequence elements (less than 80 nucleotides in length) that inhibit gene expression in undifferentiated epithelial cells have been identified in the late 3′ untranslated regions (UTRs) of several papillomaviruses, including the high-risk mucosal type human papillomavirus type 16 (HPV-16). Here we show that closely related high-risk mucosal type HPV-31 also contains elements that can act to repress gene expression in undifferentiated epithelial cells. However, the HPV-31 negative regulatory element is surprisingly complex, comprising a major inhibitory element of approximately 130 nucleotides upstream of the late polyadenylation site and a minor element of approximately 110 nucleotides mapping downstream. The first 60 nucleotides of the major element have 68% identity to the negative regulatory element of HPV-16, and these elements bind the same cellular proteins, CstF-64, U2AF(65), and HuR. The minor inhibitory element binds some cellular proteins in common with the major inhibitory element, though it also binds certain proteins that do not bind the upstream element