Study On The Promoter Activity In The SARS Coronavirus 5' UTR Sequence

According to WHO, 8096 SARS cases had occured with 774 decedents of them all over the world up to July 8 in 2003. Researchers in 13 nations and regions all over the world collaborated together in the research on the SARS-CoV, and very quickly obtained the important breakthrough. A new coronavirus, SARS-CoV had been isolated from sufferers' specimen and had been confirmed to be the pathogen of the new disease by Koch's Postulate. The SARS-CoV is a new challenge to human being, and many of its characters were infered from the other three known coronavirus groups by some theory models. After all, SARS-CoV is not the other three known coronavirus groups, and a great deal of experimental researchs are still to be done to explicit its source, evolvement, duplication, transcription, pathogenesis, mutation and immunological response. Then the Results from above research would be the foundation to guide the prophylaxis and treatments. These experiments presented below were designed to explore the role of SARS-CoV 5' non-translated region(SARS-CoV 5'UTR) in regulating the replicationg of SARS-CoV, by analyzing the sequence features of SARS-CoV 5'UTR and by studying its promoter activity. PART Ⅰ ANALYSIS OF THE SARS-CoV 5'UTR SEQUENCE FEATURES Objectives: To understand the 5'UTR sequence informations among the different SARS-CoV isolates, and to predict its secondary structure for further studying on its function. Methods: 106 SARS-CoV 5'UTR obtained from GenBank were aligned with software (DNAStar-MegAlign) and by BLAST. One typical RNA sequence containing full 264nts was then subjected to software RNADraw3.0 to be predicted its secondary structure. Results: A. Identities between the SARS-CoV 5'UTR sequence: The full sequence 264nts, and 18 deletion mutants were found. Moreover, all deletion mutants lost their various 5' terminus fragments. Totally, 5 site substitution mutants were found in 101 5'UTR sequences (mutant rate 1.92*10-4). B. Secondary structure of SARS-CoV 5'UTR RNA: Predicted by RNADraw3.0, SARS-CoV 5'UTR RNA folds to form a stable secondary structure containing four stem-loop domains with a lowest free energy of -79.5kcal at 37℃. The first stem-loop is formed by the nucleiotides between nt 7 and nt 31. The biggest and most complex domain is the second stem-loop formed by the nucleiotides between nt 42 and nt 221, which exhibits a pseudoknot. The third stem-loop is formed by the nucleiotides between nt 227 and nt 251, and the fourth is by the nucleiotides between nt 252 and nt 261.Conclusions: The 5'UTR senquences of different SARS-CoV isolates are relatively conserved, and a full sequence would form a secondary structure containing four stem-loop domains, and its may have a promoter-like activity. PART Ⅱ STUDY ON THE PROMOTER ACTIVITY OF THE cDNA CORRESPONDING TO SARS-CoV 5'UTR Objectives: To investigate the promoter activity of the cDNA sequence corresponding to SARS-CoV 5'UTR in eukaryotic cells, consequently to reveal the regulation mechanism for SARS-CoV replication. Methods : Two plasmids, nominated pGL3-5'UTR and pGL3-a-5'UTR, were constructed by substitution of SV40 promoter with SARS-CoV 5'UTR cDNA or its antisense sequence, respectively. In the recombinant plasmids, reporter gene luc+ is driven by SARS-CoV 5'UTR cDNA or its antisense sequence, respectively. Then pGL3-5'UTR and pGL3-a-5'UTR were transfected into HepG2 cells and the firefly luciferase activities were detected by Dual-Luciferase Reporter Assay System (Promega) controlled with a positive plasmid, pGL3-Control, in which luc+ is driven by SV40 promoter and with a negative plasmid, pGL3-Enhancer without any promoter. Results : Compared with pGL3-Enhancer, pGL3-Control andpGL3-5'UTR expressed firefly luciferase obviously (FL/RL were 0.051, 4.066 and 1.429, respectively ).However, pGL3-a-5'UTR did not express firefly luciferase (FL/RL was 0.087). Conclusions:The cDNA sequence corresponding to SARS-CoV 5'UTR possessed a promoter activity in eukaryotic cells.This results may reveals that S...