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

Abstract

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.

N-linked glycosylation is required for optimal proteolytic activation of membrane-bound transcription factor CREB-H

“…Cells were maintained at 37°C in a humidified atmosphere at 5% CO 2 . Transfection of cells was carried out as previously described (Kok et al, 2007;Choy et al, 2008a).…”

CRISPR/Cas9-mediated genome editing of Epstein–Barr virus in human cells

“…Primers for BZLF1 were 59-GCCACCTTTGCTATCTTTG-CT-39 (forward) and 59-TTCCATTAGTAAACGAGGCGTG-39 (reverse). Primers for EBER and the BamHI W region were as described previously (Lan et al, 2007;Choy et al, 2008a). qRT-PCR was performed using a SYBR Premix Ex Taq system (TaKaRa) as described previously (Kew et al, 2013;Siu et al, 2014).…”

MIP-T3 Is a Negative Regulator of Innate Type I IFN Response

“…Primers for amplification of GAPDH and procedures for semiquantitative RT-PCR have been described elsewhere (40,41).…”

“…AGAAAUGUCA GCCUCCAUCt t-39 (antisense for si3C), 59-GUGCA-GAGCA CAAACAGAAt t-39 (sense for si3D), and 59-UUCUGUUUGU GCUCUGCACt t-39 (antisense for si3D). Transfection of small interfering RNA (siRNA) into HEK293 cells was performed as described (40) using Lipofectamine 2000 according to the manufacturer's protocol. Transfection of siRNA into MEF cells was performed using X-treme-Gene siRNA transfection reagents (Roche).…”