key: cord-0033668-swk9o0nn authors: Wei-xian, Chen; Juan, Chen; Zhen-zhen, Zhang; Ai-long, Huang title: P19 of tomato bushy stunt virus suppresses RNA silencing induced by short hairpin RNA in mammal cells date: 2008-01-13 journal: Virol Sin DOI: 10.1007/s12250-007-0022-3 sha: 09168fb23c9fc8d8ab0b7dd7b4ff4cf613585916 doc_id: 33668 cord_uid: swk9o0nn To counteract the immune system in parasitic hosts, some viruses encode proteins to suppress the RNA interference (RNAi) effect. In this report, we established two RNAi systems to be easily observed with strong and obvious effect. The function of the P19 of tomato bushy stunt virus, which suppresses RNAi in mammal cells, was then studied using these two systems. Short hairpin RNAs targeting green fluorescence protein (pshRNA-GFP) and firefly luciferase (pshRNA-luc) were designed and inserted into a eukaryotic transcriptional vector pTZU6+1, respectively. The shRNA expressing vectors were co-transfected with plasmids containing the target gene with or without P19. The GFP expression level was assayed by fluorescence microscopy, Western blotting and RT-PCR. The luciferase expression level was analyzed by the dual-luciferase assay system. pshRNA designed in this study down-regulated the target gene specifically and efficiently, with a decrease of expression of both genes of about 70%, respectively. When P19 was introduced into the RNAi systems, the expression of both GFP and the luciferase were mostly recovered compared with the control groups. The RNAi systems of GFP and luciferase were constructed successfully, demonstrating that P19 of tomato bushy stunt virus has the ability to counteract the RNAi effect induced by shRNA in mammal cells. RNA silencing is a unique RNA-guided gene regulatory mechanism that operates in a wide range of eukaryotic organisms from plants to mammals. It Consistent with the notion that RNAi is a natural antiviral mechanism, miRNAs related to certain viruses in cells and siRNA derived from viruses in infection process were recently identified. Furthermore, some viruses were found to be able to encode proteins to suppress RNA silencing (9) . Thus, viruses can antagonize the cell immune response at the gene level and enhance their ability to survive. In this article, we designed short hairpin RNA (shRNA) targeted to the enhanced green fluorescence protein (EGFP) and luciferase genes expressed by vector in mammal cells and determined their abilities to down-regulate the target genes. Efficient screening systems of RNAi suppressor were established. Using this system, we further demonstrated the function of P19 of tomato bushy stunt virus to antagonist RNAi induced by shRNA in mammal cells. Oligonucleotides were synthesized by Shanghai Bioasia Corporation. Sequences corresponding to the siRNA hairpin targets were as follows: shRNA-EGFP (5'-TCGAGGCTGACCCTGAAGTTCATCGAGTAC TGGATGAACTTCAGGGTCAGCTTTTT-3')targeting the EGFP gene at sites 526nt-546nt, shRNA-Luc (5'-T CGAGAAGTGTTGTTCCATTCCATTTCAAGAGA ATGGAATGGAACAACACTTTTTTTTT-3')targeting the luciferase gene at sites 485nt-426nt, and the corresponding reverse sequences were also synthesized. The genome DNA of hepG2 was used as a control. To study the effect of P19 on the GFP RNAi system, Total RNA was extracted from cultured cells posttransfection with the RNAeasy kit (Qiagen) and then the RNA was digested with DNase to exclude DNA contamination. To quantify the RNA from EGFP, the hGAPDH was amplified at the same time as a control. Primers for EGFP in the tests were as follows: forward The reporter values represented averages 1 SD from at least three independent transfections. To construct a RNA interference model in mammal cells, we established the cell line stably expressing GFP (the cell line was named HepG2-GFP) by G418 selection culture. We obtained a cell line with strong green fluorescence observed by fluorescence microscopy after one-month culture. The insertion of EGFP into genome DNA was then confirmed by PCR. We detected the fragment of EGFP with the genome DNA as the template (Fig.1. lane 1) whereas the same fragment did not appear in the control test (Fig.1 ). The tomato bushy stunt virus is a type of plant virus. To confirm its expression in mammal cells, we detected at the mRNA and protein levels by different methods. The mRNA of P19 were detected in cells of HepG2-GFP and HepG2 transfected with the Psg-5mp19 or pcDNAp19-his respectively by RT-PCR. We furthermore detected the P19-his protein by an antibody targeting the his tag using the immunofluenscent method and P19-his proteins were observed to be primarily located in the plasma. Scientists attached much importance to therapy exploitation of RNAi in viral infection diseases and cancer diseases when synthetic siRNAs were found to be able to down-regulate the homologous gene expression by activating the RNAi mechanism in mammal cells (6) . Today, much improvement has been achieved in this field (16) . But the discovery of RNAi suppressors encoded by viruses will bring some new questions to the application of siRNA drugs. Benasser pointed out that HIV could resist the persistent vector-derived shRNAs since its Tas was a RNAi suppressor (1) . It may also be the case that some mammal viruses such as HCV, HBV and SARS-coV may also encode such RNAi suppressors and these factors could influence the therapy strategy of RNAi. There are still many unknown factors to be studied in this field. The knowledge of RNAi suppressor will not only enrich our understanding of RNAi phenomenon and interaction between virus and host but also help Evidence that HIV-1 encodes an siRNA and a suppressor of RNA silencing Role of microRNAs in plant and animal development A cellular microRNA mediates antiviral defense in human cells A viral protein suppresses RNA silencing and binds silencinggenerated, 21-to 25-nucleotide double-stranded RNAs Breakthrough of the Year Duplexes of 21-nucleotide RNAs mediate RNA interference in culture mammalian cells RNA interference P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA function Induction and suppression of RNA silencing by an animal virus Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing A pancreatic islet-specific microRNA regulates insulin secretion A viral suppressor of RNA silencing differentially regulates the accumulation of short interfering RNAs and micro-RNAs in tobacco Probing the microRNA and small interfering RNA pathways with virus-encoded suppressors of RNA silencing RNA viruses as inducers, suppressors and targets of posttranscriptional gene silencing Plant viral suppressors of RNA silencing Therapeutic Potential of RNA Interference RNA silencing in plants---defense and counterdefense How to silence silencing Double-stranded RNA-binding proteins could suppress RNA interferencemediated antiviral defences