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

• Published in: Gene therapy Vol. 15; no. 3; pp. 191 - 202
• Main Authors: Choy, E Y-W, Kok, K-H, Tsao, S W, Jin, D-Y
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.02.2008; Nature Publishing Group
• Subjects: Anesthesia. Intensive care medicine. Transfusions. Cell therapy and gene therapy; Applied cell therapy and gene therapy; Biological and medical sciences; Biomedical and Life Sciences; Biomedicine; Biotechnology; Cell Biology; Cell Line, Tumor; Cellular biology; DNA-directed RNA polymerase; Double-stranded RNA; Epstein-Barr virus; Feasibility Studies; Fundamental and applied biological sciences. Psychology; Gene Expression; Gene Silencing; Gene Therapy; Genetic Engineering; Genetic Therapy - methods; Genetic Vectors - pharmacology; Health. Pharmaceutical industry; Herpesvirus 4, Human - genetics; Human Genetics; Humans; Industrial applications and implications. Economical aspects; Kinases; Mammalian cells; Medical sciences; Nanotechnology; Nasopharyngeal carcinoma; Nasopharyngeal Neoplasms - metabolism; Nasopharyngeal Neoplasms - therapy; original-article; Promoter Regions, Genetic; Promoters; Protein kinase; Recombinant Fusion Proteins - genetics; Ribonucleic acid; RNA; RNA Interference; RNA polymerase; RNA Polymerase III - genetics; RNA viruses; RNA, Messenger - analysis; RNA, Small Interfering; RNA-mediated interference; siRNA; Transcription; Transfusions. Complications. Transfusion reactions. Cell and gene therapy; Viruses; RNA polymerase III-dependent promoter; small interfering RNA (siRNA); RNA interference; Epstein–Barr virus-encoded small RNA (EBER); Epstein–Barr virus (EBV); Gammaherpesvirinae; Enzyme; Herpesviridae; Transferases; siRNA; Epstein Barr virus; Gene expression; DNA-directed RNA polymerase; Virus; Gene silencing; Nucleotidyltransferases; Epstein-Barr virus-encoded small RNA (EBER); Short hairpin RNA; Gene therapy; Epstein-Barr virus (EBV)
• ISSN: 0969-7128; 1476-5462
• DOI: 10.1038/sj.gt.3303055

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.