Studies Of RNA Interference On SARS-associated Coronavirus Spike Glycoprotein

The severe acute respiratory syndrome (SARS), also named infectious atypical pneumonia, is a newly described and highly contagious respiratory infection that firstly occurred in late 2002 in Guangdong Province, China, and then spread to more than 30 countries in the following days. The main symptoms were high fever, dry cough and difficult respiratory, and acute respiratory failure was the main cause of the patients' death. Severe harm of human health and social economy progress was caused to happen for the absence of effective prevention and cure methods and immunity of the crowd to this type of new virus. A novel coronavirus, named as severe acute respiratory syndrome-associated coronavirus (SARS-CoV), has been identified as the main causative agent of SARS, which differs from all previously known human coronaviruses and forms a unique group based on the analysis of nucleotides and amino acid sequences and especially the homology comparison of the most conservative region of pol gene. SARS-CoV, belonging to Nidovirales, Coronaviridae, Coronavirus, is an enveloped single-stranded plus-sense RNA virus, approximately 30kb. The genomic organization has the characteristic gene order [5'-replicase (rep), spike (S), envelope (E), membrane (M), and nucleocapsid (N)-3'] and short untranslated regions at both termini. The S protein, with the size of 1,255 amino acids, is pro-glycoprotein located at the viral surface and belongs to type I trans-membrane glycoprotein. It is thought that the binding of the amino terminal of S protein to its receptors on the surface of host cells induces conformational changes in the S protein, leading to the fusion between viral envelope and host cell membrane mediated by the carbonous terminal. Additionally, S protein is one of the most important candidate antigens, which can induce the generation of neutralizing antibodies and has a direct impact on viral pathogenicity once mutated. At present, there is no specific treatment or vaccine for this disease although the studies of antiviral drugs or vaccines have made great progress. RNA interference (RNAi) technology, paid close attention to for its great application potential in diseases' therapy in recent years, may possibly provide a new strategy for the study of anti-SARS drugs.RNAi, originally discovered by Fire in 1998, is induced by double-strand RNA(dsRNA). In the process, dsRNA is cleaved into 21-23 nucleotide dsRNA molecules, known as small interfering RNA or siRNA, by an RNase Ill-like enzyme known as Dicer. These siRNA molecules, with the terminal of 5'-phosphate and 3'-hydroxy with two or three protruded ir-pairing bases, associate with a multiprotein complex known as the RNA-induced silencing complex (RISC) and targets homologous mRNA for degradation to silence the target gene, belongs to post-transcriptional gene silencing. Therefore, RNAi has been employed in the study of gene function and complex signal transduction pathways, and in the gene therapy of human diseases including cancers, neurogenerative diseases and infectious diseases. RNAi, as a new technology of silencing gene expression, has been successfully employed in inhibiting viral duplication in cultured cells such as HIV, HBV/HCV. Therefore, SARS-CoV, single-stranded plus-sense RNA virus, can become the target of RNAi and resist the viral infection by the degradation of viral RNA. In this study, we cloned and expressed SARS-CoV spike gene, which encodes N-terminal amino acid residues 1-690 and contains the identified receptor-binding domain (RBD). Then we designed two siRNAs specific to amino terminal of S protein, EGFP-siRNA and scramble siRNA as positive and negative control respectively, and synthesized them with 17 RiboMAX? expression system in vitro. Our results demonstrated that the codon-optimized spike gene obviously improved S glycoprotein expression in cultured cells. The specific siRNAs corresponding to SARS-CoV spike gene specifically degraded S mRNA, and significantly inhibited S glycoprotein expression.1. Expression of SARS-CoV spike glycoprotein in HEK 293T cellsUsing HEK 293T cells as host cells, we transfected the cells with the wild plasmid pEGFP-S, which encodes 1-2070 nt of SARS-CoV S gene, and pEGFP-optS with partial optimized codon respectively. After 48 hours, quantity and fluorescence intensity in the transfected cells were detected by fluorescence microscope, the expression of S-EGFP fusion protein was detected using Western blot. The results demonstrated that very mild fluorescence was detected in the pEGFP-S transfected HEK 293T cells, while strong fluorescence was observed in the pEGFP-optS transfected cells, and the green fluorescence intensity in the two cells was different. Western blot analysis of the cell lysate also showed that the S-EGFP fusion proteinwas expressed at high level in pEGFP-optS transfected cells but at low level in pEGFP-S transfected cells using anti-GFP monoclonal antibody. However, the molecular weight of expressed S-EGFP is larger than the predicted one (about 82kDa) based on amino acid composition. This may represent glycosylated protein resulted from posttranslational modification by the eukaryotic expression system. After all, this transient expression system, transfected with plasmid pEGFP-optS, effectively expresses SARS-CoV S glycoprotein and is suitable for the use in silencing of SARS-CoV S gene expression by siRNA transcribed in vitro.2. Silencing of SARS-CoV S gene by siRNA in cultured cellsWe adopted transcription in vitro with T7 RNA polymerase to acquire siRNAs. Firstly, the sense and antisense of four siRNA (S-siRNAl, S-siRNA2, scramble siRNA and EGFP-siRNA) templates and T7 promoter primer template were synthesized. The sense and antisense of siRNA were separately transcribed in vitro with T7 RNA polymerase and annealed to form double-strand siRNA. The 5' overhanging leader sequence of the generated dsRNA and DNA template remainders were digested with single-strand specific nuclease (SI nuclease) and RNase-free DNase I, respectively. The double stranded siRNAs of 21 nt in length were obtained. Then the four siRNAs and pEGFP-optS were co-transfected into HEK 293T cells. After 48 hours, green fluorescence was observed using fluorescence microscope; Samples were counted and analyzed with CellQuest software, using non-transfected HEK 293T cells as control. The values were calculated as the percentage of the cell population that exceeded the fluorescence intensity of the control cells and the mean fluorescence intensity of this population; Total RNA from the transfected or non-transfected cells was extracted and semi-quantitative RT-PCR and Real-time PCR were performed using SARS-CoV S and GAPDH gene specific primers after being reverse transcribed into cDNA. The expression of S-EGFP fusion protein was detected by Western blot The results demonstrated that compared with the cells transfected with pEGFP-optS alone (the percentage of fluorescence cells is 30.56% and mean fluorescence intensity is 238.89), the cells co-transfected scramble siRNA gave no significant reduction of EGFP expression, while the EGFP-siRNA gave an about 3.7- and 3.8-fold reduction in percentage of fluorescence cell population and...