Cellular Localization Of SARS-CoV N Protein And Suppression Of It's Expression By RNA Interference

Objective : To study the cellular localization of Severe Acute Respiratory Syndrome Associated Coronavirus (SARS-CoV) nucleocapsid protein, a series of fusion expression plasmids , containing EGFP (enhanced green fluorescent protein)genes and different length SARS-CoV nucleocapsid gene ,were constructed. To observe the effect of RNA interference on SARS-CoV structural gene expression, the following plasmids were constructed respectively: 1. the plasmids containing the structural genes of SARS-CoV and EGFP gene; 2. the shRNA plasmids targeting the structural genes of SARS-CoV. Methods:The sequence of SARS-CoV nucleocapsid protein was analyzed by Prosite and PredictNLS server (prediction and analysis of nuclear localization signals,Columbia University Bioinformatics Center) to identify nuclear localization signals(NLS). Different length fragments of SARS-CoV nucleocapsid gene were obtained by PCR or chemical synthesis. These fragments were cloned into pEGFP-C1 vector to form plasmids pEGFP-C1-N,pEGFP-C1-N1 and pEGFP-C1-N2 respectively. 293 cells were examined by fluorescence microscope and laser confocal microscope after being transfected with pEGFP-C1-N,pEGFP-C1-N1 and pEGFP-C1-N2 plasmid. At the same time, the cellular localization of SARS-CoV nucleocapsid protein was investigated by cell immuno-fluorescence, using FITC-labelled second antibody. The EGFP fusion expression plasmids were constructed by cloning synthesized SARS-CoV structural gene fragments into pEGFP-C1. The designed and synthesized shRNA sequences,containing a 19bp reverse repeated motif of target sequence with 4bp spacer ,were ligated into vector pTZU6+1 to generate shRNA plasmids. The shRNA plasmids were cotransfected with the EGFP fusion expression plasmids encoding the corresponding SARS-CoV proteins at a 5:1 mass ratio. Fluorescence was observed 48h after transfection. The expression levels of GFP and nucleocapsid protein were determined by Western blot respectively. The transcription of N gene was detected by RT-PCR. Results:By using PROSITE, we identified a nuclear localization signal sequence from amino acids 373 to 389 or from amino acids 374 to 390 . However, the analysis of PredictNLS server revealed that the nuclear localization signal region was located between amino acids 36 and 44. Agarose gel electrophoresis of PCR product showed that SARS-CoV Ngene was amplified. Identification of pEGFP-C1-N,pEGFP-C1-N1 and pEGFP-C1-N2 plasmid by restriction enzyme digest showed that different length fragments of SARS-CoV nucleocapsid gene had been cloned into pEGFP-C1. In addition, sequencing of the recombinant plasmids indicated that the inserted sequences were correct. Both the GFP fusion expression strategy and the immune fluorescence showed the following findings: N1 protein(1-187aa) was located in both cytoplasm and cell nucleus;N protein(1-422aa) and N2 protein(161-422aa) were located in cytoplasms . Restriction enzyme digest and DNA sequencing showed that the EGFP fusion expression plasmids(pEGFP-C1-N1 , pEGFP-C1-E ,pEGFP-C1-M,and pEGFP-C1-S) and the shRNA plasmids (pshRNA-N1, pshRNA-N2,pshRNA-E,pshRNA-M and pshRNA-S)were constructed successfully. Among a total of 5 shRNA plasmids,we obtained one shRNA plasmid which could sequence-specifically reduce target gene expression. The introduction of pshRNA-N1 plasmid efficiently and specifically inhibited the synthesis of protein N by western blot analysis of GFP fusion protein. RT-PCR showed that RNAs of N gene were clearly reduced when pEGFP-C1-N1 was cotransfected with pshRNA-N1, whereas the control vector did not exhibit inhibitory effect on N gene transcription. Conclusion : Successfully constructed the recombinant plasmidscontaining different structural genes of SARS-CoV. N1 protein was located in both cytoplasm and nucleus;N protein and N2 protein were located in cytoplasms. shRNAs targeting different SARS-CoV sequences have significantly various inhibitory effects on SARS-CoV gene expression. The introduction of pshRNA-N1 was shown to efficiently and...