Inhibition Of SARS-CoV Replication By Small Interfering RNAs

Severe acute respiratory syndrome (SARS) is a dramatic example of an emerging infectious disease in humans at the beginning of 21s' century. Our country is one of the mostly epidemic areas. A novel coronavirus is the cause of SARS and called SARS Coronavirus (SARS-CoV) by WHO. Currently, no effective antiviral strategy exists for the treatment of infection with SARS—CoV. This situation has spurred urgently the interest in search for new method against SARS.RNAi is a method of sequence-specific mRNA knockdowns and has been a focus of attention due to its effectivity, specific and long duration of effect. Small interfering RNAs (siRNAs) is a silencing agent in process of RNAi, which has been used in the experimental treatments for several important viruses and has shown a promising prospect as an antiviral strategy. Therefore RNAi maybe provide a new method for prevention of SARS.The object of our study is to observe the inhibition of SARS-CoV replication by siRNA and to explore the feasibility of RNAi as an antiviral strategy against SARS infection. There are two parts in this article.1. Construction of siRNAs and observation of inhibition of replicase gene expression by siRNAsThe replicase polyprotein induces the transcription of SARS-CoV genome and spike protein is important for binding to cellular receptor and for mediating the fusion of viral and host membranes, both of these processes being critical for virus entry into host cells. Then nine siRNA expression plasmids (pS01~09) , which direct the synthesis of siRNAs derived from the replicase gene and the spike gene of SARS-CoV, were constructed. pS01~07were targeting replicase gene and pS08~pS09 were targeting spike gene. We also constructed recombinant plasmid (pEGFP-REP), which can express GFP-replicase fusion protein in mammalian cells. Then we cotransfected the siRNA expression plasmid which targeted replicase gene and pEGFP-REP into 293T cells and found that replicase gene can be effectively silenced by siRNA which targeted 3504nt-3524nt of SARS-CoV through fluorescent microscopy and flow cytometry.2. Interference of SARS - CoV replication in transformed Vero cells by siRNAsVero cells were stably transformed with siRNA expression plasmids. Clonal cell lines were selected by growth in hygromycin B-containing medium and were characterized further by PCR analysis to determine the organization of plasmid DNA in the clonal cell lines. Cell lines, in which the plasmid DNA was correctly integrated into the genome, were challenged with SARS-CoV BJ01 strain .At 4th day, virus titers were determined. The postchallenged cells were fixed in the slip and immunofluorescence assays (IFA) were performed with SARS patient sera and mouse anti-human antibodies. Two of the clonal cell lines, V/pS05 and V/pS08, were resistant to SARS-CoV challenge, with more than 95% of the cells showing no SARS-CoV antigens accumulation and virus titers decreased by 103 to 101 folds. In addition, the level of replicase gene reduced to 103 folds in clonal cells(V/pS05) by real-time RT-PCR analysis, which suggested that the degradation of RNA was the real antiviral mechanism of RNAi. These results were consistent with RNAi as the mechanism of resistance to SARS-CoV in transformed Vero cells.In conclusion, we obtained two siRNAs that can inhibit SARS replication and successfully demonstrated that siRNA- mediate interference can act as a viral defense mechanism for SARS-CoV in mammalian cells. The two targeting sites are 6743nt-6761nt and...