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Effective Inhibition of Mrna Accumulation and Protein Expression of H5N1 Avian Influenza Virus NS1 Gene in Vitro by Small Interfering Rnas

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Effective Inhibition of Mrna Accumulation and Protein Expression of H5N1 Avian Influenza Virus NS1 Gene in Vitro by Small Interfering Rnas Folia Microbiol (2013) 58:335–342 DOI 10.1007/s12223-012-0212-8 Effective inhibition of mRNA accumulation and protein expression of H5N1 avian influenza virus NS1 gene in vitro by small interfering RNAs Hanwei Jiao & Li Du & Yongchang Hao & Ying Cheng & Jing Luo & Wenhua Kuang & Donglin Zhang & Ming Lei & Xiaoxiao Jia & Xiaoru Zhang & Chao Qi & Hongxuan He & Fengyang Wang Received: 18 June 2012 /Accepted: 7 November 2012 /Published online: 29 November 2012 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2012 Abstract Avian influenza has emerged as a devastating protein expressing HEK293 cell lines were established, four disease and may cross species barrier and adapt to a new small interfering RNAs (siRNAs) targeting NS1 gene were host, causing enormous economic loss and great public designed, synthesized, and used to transfect the stable cell health threats, and non-structural protein 1 (NS1) is a mul- lines. Flow cytometry, real-time quantitative polymerase tifunctional non-structural protein of avian influenza virus chain reaction, and Western blot were performed to assess (AIV) that counters cellular antiviral activities and is a the expression level of NS1. The results suggested that virulence factor. RNA interference (RNAi) provides a pow- sequence-dependent specific siRNAs effectively inhibited erful promising approach to inhibit viral infection specifi- mRNA accumulation and protein expression of AIV NS1 cally. To explore the possibility of using RNAi as a strategy in vitro. These findings provide useful information for the against AIV infection, after the fusion protein expression development of RNAi-based prophylaxis and therapy for plasmids pNS1-enhanced green fluorescent protein (EGFP), AIV infection. which contain the EGFP reporter gene and AIV NS1 as silencing target, were constructed and NS1-EGFP fusion Abbreviations AIV Avian influenza viruses HA Hemagglutinin Hanwei Jiao, Li Du, and Yongchang Hao contributed equally to this work. NS1 Non-structural protein 1 : : : : : : : NEP Nuclear export protein H. Jiao L. Du Y. Hao Y. Cheng D. Zhang M. Lei X. Jia RNAi RNA interference F. Wang (*) College of Agriculture, Hainan Key Laboratory of Tropical dsRNA Double-stranded RNA Animal Reproduction & Breeding and Epidemic Disease IFN Interferon Research, Animal Genetic Engineering Key Laboratory of Haikou, CEFs Chicken embryo fibroblasts Hainan University, Haidian Island, EGFP Enhanced green fluorescent protein Haikou 570228, People’s Republic of China e-mail: [email protected] siRNAs Small interfering RNAs : shRNA Short hairpin RNAs J. Luo H. He (*) miRNA microRNAs Key Laboratory of Animal Ecology and Conservation Biology, DMEM Dulbecco’s modified Eagle’s medium National Research Center for Wildlife Born Diseases, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, FBS Fetal bovine serum People’s Republic of China FCM Flow cytometry e-mail: [email protected] RT-PCR Reverse transcription-polymerase chain reaction W. Kuang : X. Zhang : C. Qi College of Life Sciences, Huazhong Normal University, M-MLV Moloney murine leukemia virus Wuhan 430079, People’s Republic of China PBS Phosphate balanced solution 336 Folia Microbiol (2013) 58:335–342 RPLP0 Acidic ribosomal protein P0 As a process of post-transcriptional gene silencing medi- SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel ated by short dsRNA, RNA interference (RNAi) causes electrophoresis degradation of target mRNAs and has been utilized as a IgG Immunoglobulin G therapeutic strategy by knockdown key target of pathogen GAPDH Glyceraldehyde-3-phosphate dehydrogenase or receptor (Hannon 2002). HRP Horseradish peroxidase Small interfering RNAs (siRNAs) specific for matrix mAB monoclonal Ab (M2), nucleocapsid protein (NP), and RNA transcriptase ECM Enhanced chemiluminescent method (PA) genes of H5N1 AIV could effectively inhibit the ex- CCD Charge-coupled device pression of the corresponding viral protein and potently PA RNA transcriptase inhibit AIV production (Ge et al. 2003; Zhou et al. 2007; M2 Matrix Sui et al. 2009; Zhang et al. 2009). However, few studies NP Nucleocapsid protein have been performed to explore the possibility of using MDCK Madin–Darby canine kidney RNAi against NS1 as a strategy for inhibiting AIV H5N1 CPSF30 Cellular cleavage and polyadenylation replication. specificity factor In this study, to explore the possibility of using RNAi targeting NS1 as a strategy against AIV infection, four siRNAs targeting AIV NS1 gene were designed and chem- ically synthesized, and the cell lines which stably express Introduction pNS1-enhanced green fluorescent protein (EGFP) were established and used to evaluate the siRNAs inhibition Avian influenza has emerged as a devastating disease and effects against NS1. Our results indicated that specific may cross species barrier and adapt to a new host, causing siRNAs capable of reducing mRNA accumulation and pro- enormous economic loss and great public health threats. In tein expression of NS1 were identified, which might be of 1997, H5N1 avian influenza virus (AIV) infected 18 hospi- importance as a promising approach for development of talized patients in the Hong Kong Special Administrative anti-AIV therapeutics and knockdown analysis of AIV NS1. Region, China; six of the patients died (Bender et al. 1999). Analysis of the sequences of all eight RNA segments of the influenza A/Goose/Guangdong/1/96 (H5N1) virus, isolated Materials and methods from a sick goose during an outbreak in Guangdong, China, in 1996, revealed that the hemagglutinin (HA) gene of the Virus and gene virus was genetically similar to those of the H5N1 viruses isolated in Hong Kong in 1997, which suggested that the Influenza A virus (A/environment/Qinghai/1/2008(H5N1) H5N1 viruses isolated from the Hong Kong outbreaks de- was isolated from bird feces in Qinghai, People’s Republic rived their HA genes from a virus similar to the A/Goose/ of China. The accession number of NS1 nucleotide sequen- Guangdong/1/96 virus or shared a progenitor with this ces in GeneBank is FJ455824. goose pathogen (Xu et al. 1999). Since 2003, it has been found in many countries in Asia, Europe, and Africa. These Cell line wide distributions and host range, and the ability to transmit interspecies indicate it has pandemic potential. HEK293 cell line (human embryonic kidney cell line) was As the non-structural protein of AIV, non-structural pro- obtained from the Cell Bank of the Chinese Academy of tein 1 (NS1) is a nuclear, dimeric protein that is highly Science (Shanghai, China) and routinely grown at 37 °C, in expressed in infected cells and has double-stranded RNA Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, (dsRNA)-binding activity (Hatada and Fukuda 1992; USA) containing 10 % heat-inactivated fetal bovine serum Nemeroff et al. 1995). It is a multifunctional protein that (FBS; Gibco BRL, USA) supplemented with penicillin counters cellular antiviral activities and is a virulence factor (100 U/mL; Gibco BRL, USA) and streptomycin (100 U/mL; (Li et al. 2006; Min and Krug 2006). Majority of these Gibco BRL,USA). functions are connected with overcoming of host antiviral innate immunity mainly via the inhibition of type I interfer- Expression constructs on (IFN) response (Ehrhardt et al. 2010; Panshin et al. 2010). NS1 gene is critical for the pathogenicity of AIV in In order to establish NS1-EGFP fusion protein expressing chickens, and the amino acid residue Ala149 correlates with stable cell lines, AIV-NS1 cDNA fragment obtained from the ability of these viruses to antagonize IFN induction in plasmid pcDNA3.1-NS1 digested with Xho1 and EcoR1 chicken embryo fibroblasts (CEFs) (Long et al. 2008). was cloned into eukaryotic expression vector pEGFP-N1 Folia Microbiol (2013) 58:335–342 337 (Clontech, Japan) digested with Xho1 and EcoR1 to obtain solution of PepMute™ Transfection Buffer (siRNA final the recombinant fusion protein expressing plasmid, pNS1- concentration is 20 nmol/L). Add 2.0 μLPepMute™ EGFP. All constructs were confirmed by DNA sequencing, reagent, mix by pipetting up and down, and incubate for and EndoFree™ Plasmid Maxi Kit (QIAGEN, Germany) 15 min at reverse transcription (RT). Add the transfection was used to extract the recombinant plasmid for further mix to the cells dropwise, and gently rock the plate back transfection. and forth. Design and synthesis of siRNAs Fluorescence observation and FCM analysis Four siRNAs, NS1-siRNA1, NS1-siRNA2, NS1-siRNA3, Expression of NS1-EGFP and EGFP in the stable cells and NS1-siRNA4, which were targeted NS1, were designed transfected with siRNAs was examined by fluorescent mi- according to the basic principles of siRNA design and croscopy (Olympus X71, Japan) at 24, 48, and 72 h after synthesized by Shanghai GenePharma Co. (Shanghai, transfection. At 48 h after transfection, the cells were China; Table 1). Scramble sequence was synthesized and digested with 0.25 % trypsin, washed with phosphate bal- used as negative control, and effective EGFP-siRNA was anced solution (PBS), and resuspended in PBS for flow also synthesized and used as positive control. cytometry (FCM) assay using a FACS Calibur flow cytom- eter (Becton Dickinson, USA) with filters (emission, Expression plasmids transfection and establishment of NS1- 507 nm; excitation, 488 nm) for EGFP expression. EGFP expressing stable cell lines Samples (106 cells each) were counted and analyzed with CellQuest software (Becton Dickinson, USA). Non- The plasmid DNA was induced into HEK293 cell as de- transfected HEK293 cells were used as control. scribed previously (Liu et al. 2011). Lipofectamine™ 2000 was used to carry pNS1-EGFP and pEGFP-N1 into Real-time quantitative RT-PCR HEK293 cell. After transfection, limiting dilution cloning in 96-well plates was performed as described of Watson Real-time quantitative RT-polymerase chain reaction (PCR) (1979). Cells which stably incorporated the expression plas- was performed as described previously (Liu et al. 2011). mid pNS1-EGFP and pEGFP-N1 were selected by survival Briefly, at 48 h post-transfection, high-quality total RNA in the presence of G418 (500 μg/mL; Gibco BRL, USA).
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