Cell-to-cell spreading of RNA interference in mammalian cells

Presentation no. 2593 ; Poster Board no. B603published_or_final_versio

Identification of a Novel Marine Fish Virus, Singapore Grouper Iridovirus-Encoded MicroRNAs Expressed in Grouper Cells by Solexa Sequencing

BACKGROUND: MicroRNAs (miRNAs) are ubiquitous non-coding RNAs that regulate gene expression at the post-transcriptional level. An increasing number of studies has revealed that viruses can also encode miRNAs, which are proposed to be involved in viral replication and persistence, cell-mediated antiviral immune response, angiogenesis, and cell cycle regulation. Singapore grouper iridovirus (SGIV) is a pathogenic iridovirus that has severely affected grouper aquaculture in China and Southeast Asia. Comprehensive knowledge about the related miRNAs during SGIV infection is helpful for understanding the infection and the pathogenic mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: To determine whether SGIV encoded miRNAs during infection, a small RNA library derived from SGIV-infected grouper (GP) cells was constructed and sequenced by Illumina/Solexa deep-sequencing technology. We recovered 6,802,977 usable reads, of which 34,400 represented small RNA sequences encoded by SGIV. Sixteen novel SGIV-encoded miRNAs were identified by a computational pipeline, including a miRNA that shared a similar sequence to herpesvirus miRNA HSV2-miR-H4-5p, which suggests miRNAs are conserved in far related viruses. Generally, these 16 miRNAs are dispersed throughout the SGIV genome, whereas three are located within the ORF057L region. Some SGIV-encoded miRNAs showed marked sequence and length heterogeneity at their 3' and/or 5' end that could modulate their functions. Expression levels and potential biological activities of these viral miRNAs were examined by stem-loop quantitative RT-PCR and luciferase reporter assay, respectively, and 11 of these viral miRNAs were present and functional in SGIV-infected GP cells. CONCLUSIONS: Our study provided a genome-wide view of miRNA production for iridoviruses and identified 16 novel viral miRNAs. To the best of our knowledge, this is the first experimental demonstration of miRNAs encoded by aquatic animal viruses. The results provide a useful resource for further in-depth studies on SGIV infection and iridovirus pathogenesis

Utility of Epstein-Barr virus-encoded small RNA promoters for driving the expression of fusion transcripts harboring short hairpin RNAs

To induce RNA interference (RNAi), either small interfering RNAs (siRNAs) are directly introduced into the cell or short hairpin RNAs (shRNAs) are expressed from a DNA vector. At present, shRNAs are commonly synthesized by RNA polymerase III (Pol III) promoters of the H1 and U6 RNAs. In this study, we designed and characterized a new set of plasmid vectors driven by promoters of the Epstein-Barr virus (EBV)-encoded small RNAs (EBERs). The EBERs are the most abundant transcript in infected cells and they are transcribed by Pol III. We showed that the EBER promoters were able to drive the expression of shRNA fusion transcripts. siRNAs processed from these fusion transcripts specifically and effectively inhibited the expression of homologous reporter or endogenous genes in various types of cells. The partial EBER sequences in the fusion transcripts did not activate double-stranded RNA-dependent protein kinase or suppress RNAi. In nasopharyngeal carcinoma cells, the EBER2 promoter was stronger than the H1 and U6 promoters in shRNA synthesis, leading to more effective knockdown of the target genes. Taken together, our findings suggest that the EBER promoters fundamentally different from those of H1 and U6 can be used to drive the intracellular expression of shRNAs for effective silencing of target genes in mammalian cells and particularly, in EBV-infected cells.link_to_subscribed_fulltex

miR-BART5 is an anti-apoptotic miRNA encoded by EBV and inhibits PUMA to promote host cell survival

Conference Theme: Frontier in Life Science

Epstein-Barr virus-encoded microRNA targets PUMA to promote tumor cell survival

INTRODUCTION. Epstein-Barr virus (EBV) is a γ-herpesvirus that is constantly associated with nasopharyngeal carcinoma (NPC). EBV is the first human virus found to express microRNAs (miRNAs), but the functions of these viral miRNAs are largely unknown. Here, we identified and characterized a cellular target of an EBV miRNA known as miR-BART5, which is conserved with rhesus lymphocryptovirus miR-rL1-8 and abundantly expressed in NPC cells. METHOD. miRanda and RNAhybrid were used to predict the potential targets of miR-BART5. Luciferase reporter assay was performed to verify the target site. Gain and loss of miR-BART5 function were interrogated for influence on the expression of target gene. Target expression in EBVassociated NPC tissue samples was also examined. Apoptosis assays were conducted to determine the biological function of miR-BART5 and its target gene in EBV-positive tumor cells. RESULTS. By in silico analysis and screening with luciferase reporter assays, we showed that miR-BART5 targets the 3′-untranslated region of a cellular mRNA transcript encoding p53 upregulated modulator of apoptosis (PUMA). The target site of miR-BART5 was identified and characterized. Regulation of the endogenous PUMA expression was verified by overexpressing miRBART5. In addition, an anti-miR-BART5 oligonucleotide inhibitor was used to confirm that reduction of endogenous miR-BART5 activity in NPC cells led to alteration in PUMA expression. Consistent with a role in the development of NPC, PUMA was found to be significantly underexpressed in about 60% of human NPC tissues constitutively harboring EBV. More importantly, miRBART5-expressing NPC cells were less sensitive to proapoptotic agents adriamycin and etoposide. In addition, apoptosis can be induced in these cells by inhibiting miR-BART5 activity. DISCUSSION. In this work we sought to investigate the function of an EBV miRNA in pathogenesis, particularly in establishing latency in epithelial cells. PUMA was characterized to be a target of miR-BART5. PUMA is a proapoptotic protein belonging to the “Bcl-2 homology 3 domain (BH3)-only” group of the Bcl-2 family. Although it is an immediate downstream target of p53, PUMA can also induce p53-independent apoptosis in response to a large variety of stimuli, including serum starvation, cytokine withdrawal, kinase inhibitors, phorbol esters, glucocorticoids, ischemia-reperfusion, endoplasmic reticulum stress, proteosome inhibition, HIV envelope glycoprotein complex, p73 and FOXO3a. It is not surprising that PUMA is implicated in our work as a critical factor in the prevention of apoptosis in EBV-positive NPC cells. Our findings suggest a model for viral promotion of host cell survival in which EBV encodes an miRNA to repress the expression of PUMA and consequently facilitate the establishment of stable latent infection. ACKNOWLEDGMENT. This work was supported in part by Fogarty International Center of National Institutes of Health (R01 TW06186-01). REFERENCES 1. Pfeffer, S., Zavolan, M., Grasser, F.A., Chien, M., Russo, J.J., Ju, J., John, B., Enright, A.J., Marks, D., Sander, C., and Tuschl, T. (2004) Science 304, 734-736 2. Cai, X., Schafer, A., Lu, S., Bilello, J.P., Desrosiers, R.C., Edwards, R., Raab-Traub, N., and Cullen, B.R. (2006) PLoS Pathog. 2, e23 3. Nakano, K., and Vousden, K.H. (2001) Mol. Cell 7, 683-694 4. Yu, J., Zhang, L., Hwang, P.M., Kinzler, K.W., and Vogelstein, B. (2001) Mol. Cell 7, 673-68

miR-BART5 is an anti-apoptotic miRNA encoded by EBV and inhibits PUMA to promote host cell survival

Epstein-Barr virus (EBV) is a herpesvirus associated with nasopharyngeal carcinoma (NPC) and other types of epithelial and lymphoid malignancies. EBV is the first human virus found to express microRNAs (miRNAs), but the functions of these viral miRNAs are largely unknown. Here, we identified and characterized a cellular target of an EBV miRNA known as miR-BART5, which is conserved with rhesus lymphocryptovirus miR-rL1-8 and abundantly expressed in NPC cells. By in silico analysis and functional screening with luciferase reporter assays, we showed that miR-BART5 targets the 3-untranslated region of a cellular mRNA transcript encoding p53 upregulated modulator of apoptosis (PUMA), a mediator of p53-dependent and –independent apoptosis. The target site of miR-BART5 was identified and characterized. Regulation of the endogenous PUMA expression was verified by overexpressing miR-BART5. In addition, a synthetic anti-miR-BART5 oligonucleotide inhibitor was used to confirm that reduction of endogenous miR-BART5 in NPC cells led to alteration in PUMA expression. Consistent with a role in the development of NPC, PUMA was found to be significantly underexpressed in about 60% of human NPC tissues constitutively harboring EBV. More importantly, miR-BART5-expressing NPC cells were less sensitive to proapoptotic agents adriamycin and etoposide. In addition, apoptosis can be induced in these cells by inhibiting miR-BART5 activity. Taken together, our findings suggest a model for viral promotion of tumor cell survival in which EBV encodes an miRNA to repress the expression of PUNA and consequently facilitate the establishment of stable latent infection and the development of epithelial carcinoma