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The present study aimed to elucidate the mechanisms by which leucine impacts the secretion of pancreatic enzymes, especially amylase, by studying the proteomics profiles of pancreatic acinar (PA) cells from dairy cows. PA cells, the experimental model, were treated with four concentrations of leucine (0, 0.23, 0.45, and 0.90 mM). The abundance of different proteins in the four leucine treatment groups was detected. Label-free proteomic analysis enabled the identification of 1,906 proteins in all four treatment groups, and 1,350 of these proteins showed common expression across the groups. The primary effects of leucine supplementation were increased ( P < 0.05) citrate synthase and ATPase activity, which enlarged the cytosolic ATP pool, and the upregulation of secretory protein 61 (Sec61) expression, which promoted protein secretion. In summary, these results suggest that leucine increases citrate synthase in the TCA cycle and ATPase activity and promotes the Sec signaling pathway to increase the exocrine function of PA cells.

Histone deacetylase 6 (HDAC6) regulates cytoplasmic signaling networks through deacetylation of various cytoplasmic substrates and serves as a key member of the ubiquitin proteasome system (UPS). This study is focused on HDAC6 regulation of the Notch1 receptor that plays a crucial role in tumor growth in NSCLC. A series of cell culture experiments were employed using A549, Lewis lung carcinoma 2 (LL2), and H1299 NSCLC cell lines to investigate HDAC6-mediated regulation of the Notch1 receptor through the UPS. HDAC6 was inhibited with small molecule inhibitors tubacin and ACY1215 in vitro and in vivo. Inhibition of HDAC6 led to reduced levels of Notch1 receptor in a dose-dependent manner in all three NSCLC cell lines tested. HDAC6 inhibition with ACY1215 led to G2 arrest, increased apoptosis, and increased levels of cleaved PARP1 in A549, LL2, and H1299 cell lines. In vivo inhibition of HDAC6 with ACY1215 significantly reduced LL2 tumor growth rate. Our data show that HDAC6 in NSCLC cells supports Notch1 signaling and promotes cell survival and proliferation. Our results support clinical investigation of HDAC6 inhibitors as a potential therapeutic option for treatment of NSCLC patients.

BackgroundNumerous publications suggest an association between herpes virus infection and idiopathic pulmonary fibrosis (IPF). These reports have employed immunohistochemistry, in situ hybridization and/or PCR, which are susceptible to specificity artifacts.MethodsWe investigated the possible association between IPF and viral RNA expression using next-generation sequencing, which has the potential to provide a high degree of both sensitivity and specificity. We quantified viral RNA expression for 740 viruses in 28 IPF patient lung biopsy samples and 20 controls. Key RNA-seq results were confirmed using Real-time RT-PCR for select viruses (EBV, HCV, herpesvirus saimiri and HERV-K).ResultsWe identified sporadic low-level evidence of viral infections in our lung tissue specimens, but did not find a statistical difference for expression of any virus, including EBV, herpesvirus saimiri and HERV-K, between IPF and control lungs.ConclusionsTo the best of our knowledge, this is the first publication that employs RNA-seq to assess whether viral infections are linked to the pathogenesis of IPF. Our results do not address the role of viral infection in acute exacerbations of IPF, however, this analysis patently did not support an association between herpes virus detection and IPF.

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.

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 world's 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.

The set of proteins within a cell varies as a function of the stage of development and conditions, such as wound repair, and alternate splicing is a means of modulating the protein set. RNA splicing may result in gain or loss of protein function. Thus, next generation sequencing has the …

The relationship between Epstein Barr Virus (EBV) and miR-155 is well established. EBV infection induces miR-155 expression, which is expressed at higher levels in EBV latency type III cells compared to EBV latency type I cells. However, the mechanism by which EBV latency genes activate miR-155 expression is still unclear. Here we present data showing that DNA methylation regulates miR-155 expression. We also provide evidence that the AP1 signaling pathway is involved in EBV-mediated miR-155 activation, and that Bay11 influences signaling of the miR-155 promoter AP1 element. Lastly, we show that LMP2A, LMP1 and EBNAs cannot activate miR-155 expression alone, indicating that the regulation of miR-155 by EBV is dependent on more than one EBV gene or cell signaling pathway. We conclude that the regulation of miR-155 in EBV-positive cells occurs through multiple cell signaling processes involving EBV-mediated chromatin remodeling, cell signaling regulation and transcription factor activation.

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.

Previous studies have demonstrated the utility of microarray expression analysis to identify potential microRNA targets. Nevertheless, technical limitations intrinsic to this platform constrain its ability to fully exploit the potential of assessing transcript level changes to explore microRNA targetomes. High-throughput multiplexed Illumina-based next-generation sequencing (NGS) provides a digital readout of absolute transcript levels and imparts a higher level of accuracy and dynamic range than microarray platforms. We used Illumina NGS to analyze transcriptome changes induced by the human microRNA MIR155. This analysis resulted in a larger inferred targetome than similar studies carried out using microarray platforms. A comparison with 3′ UTR reporter data demonstrated general concordance between NGS and corresponding 3′ UTR reporter results. Nonharmonious results were investigated more deeply using transcript structure information assembled from the NGS data. This analysis revealed that transcript structure plays a substantial role in mitigated targeting and in frank targeting failures. With its high level of accuracy, its broad dynamic range, its utility in assessing transcript structure, and its capacity to accurately interrogate global direct and indirect transcriptome changes, NGS is a useful tool for investigating the biology and mechanisms of action of microRNAs.

Reactivation in Epstein-Barr virus (EBV) is closely associated with a G 0 /G 1 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.

Background The Epstein-Barr virus (EBV) is a causal agent in a number of malignancies in humans including hematopoietic tumors and non-hematopoietic tumors. Burkitt’s lymphoma cell lines containing the Epstein-Barr virus have been shown to form tumors in nude mice while clonal derivatives of such cell lines in which the viral genome has been lost do not (JID 177: 1194-1201, 1998; JV 72: 9150-9156, 1998; JV 68: 6069-6073, 1994). The re-introduction of EBV into these EBV negative BLs reconstitutes the tumor phenotype. Thus, EBV-induced cellular genes play critical role in EBV-related tumors. Methods and results In an attempt to identify cellular genes regulated by EBV that may contribute to its tumorigenic properties, we have enforced genome loss in the Burkitt’s lymphoma (BL) line, MutuI, by introducing a dominant negative form of the episomal replication factor, EBNA1 and carried out gene array analysis. One of the genes identified by this analysis is PLAC1, a gene originally identified as being expressed exclusively in placental tissue. Real time RT-PCR analysis verified higher expression in EBV positive vs. EBV negative Mutu clones. Analysis of a panel of RNAs from 20 normal tissues demonstrated the highest level of expression in placenta but significant expression was also observed in testis and brain cerebellum. PLAC1 expression was also observed in non-BL tumor cell lines derived from breast, ovary, and prostate. Lastly, expression of PLAC1 was found to be higher in some primary breast tumors compared to normal adjacent tissues. Conclusion This data suggests that the EBV-induced PLAC1 is a member of the cancer/testis group of tumor antigens.

Latency-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.

Tomato yetlow leaf curl viruses betong to Begomoviruses of geminiviruses. In this work, we first found and demonstrated that the small circular DNA molecules were derived from Chinese tomato yetlow leaf curl viruses (TYLCV-CHI). These small circular DNA molecules are about 1,3 kb, which are half the full-length of TYLCV-CHI DNA A. It was shown by sequence determination and analysis that there was unknown-origin sequence insertion in the middle of the small molecules. These sequences of unknown-origin were neither homologous to DNA A nor to DNA B, and were formed by recombination of virus DNA and plant DNA. Although various defective molecules contained different unknown-origin sequence insertion, all the molecules contained the intergenic region and part of the AC1 (Rep) gene. But they did not contain full ORF.

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.

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 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 approximately 40 bp upstream from the site of initiation but not an upstream NF-kappaB 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.

Transcription of the immediate early gene BZLF1 is mediated initially through the activation of cellular transcription factors. Reporter-based studies have provided evidence that following this initial activation, the BZLF1 gene product Zta may be involved in an autoactivation loop through binding to its promoter Zp. In contrast, other reports have shown that transfection of a Zta expression vector in latently infected cells does not activate endogenous Zp. Using chromatin immunoprecipitation (ChIP) assays, we show here that Zta binds to endogenous Zp following induction of the lytic cycle by anti-Ig and TGF-beta1 and that binding occurs early enough to play a role in the activation of Zp. We have also generated a dominant-negative Zta and shown that it inhibits activation of endogenous Zp. These data support a two-step model for Zp activation during reactivation involving initial activation by cellular factors followed by an autoactivation step.

The complete DNA sequence (2734 nucleotides) of the monopartite genome of tomato yellow leaf curl China virus (TYLCCNV), a begomovirus transmitted by the whitefly Bemisia tabaci, was determined. The circular genomic DNA contains six open reading frames (ORFs) encoding proteins of molecular weights >10 kDa, of which two (V1 and V2) are located on the virion-sense strand and four (C1, C2, C3 and C4) on the complementary-sense strand. The ORFs are comparable to those of other whitefly-transmitted begomoviruses with a monopartite genome and to those encoded by DNA-A of bipartite begomoviruses. Sequence comparisons with other geminiviruses showed that TYLCCNV belongs to Begomovirus from the Old World. No putative DNA-B genome was found. Nicotiana species and tomato plants agroinoculated with the TYLCCNV monopartite genome developed typical yellowing and leaf-curling symptoms. The cloned molecule carried all the information needed for virus replication and systemic infection of plants.

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., Author Summary Theileria is the only intracellular eukaryotic parasite known to transform its host cell into a cancer-like state. Infection by the T. annulata parasite causes tropical theileriosis, killing large numbers of cattle in North Africa and Asia, and the related T. parva parasite causes East Coast Fever. We investigated whether transformation of host bovine leukocytes was associated with deregulation of small, non-coding RNAs. We discovered that transformation by Theileria leads to upregulation of an oncogenic small RNA called miR-155 which is contained within the BIC gene. Parasite induction of the microRNA involves activation of the transcription factor c-Jun which controls the BIC gene promoter. We identified a new target for the miR-155; the DET1 protein which is responsible for degradation of the c-Jun factor. This leads to a regulatory feedback loop that is critical for the transformed phenotype of the infected cells. We show that miR-155 expression inhibits DET1 protein translation, leading to accumulation of c-Jun protein and activation of the BIC gene containing miR-155. This is the first study to report regulation of oncogenic non-coding RNAs by Theileria and the novel feedback loop underlying the parasite-induced transformation.

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.

The 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.