Promoter activity of SARS coronavirus 5' UTR sequence in eukaryotic cells.
OBJECTIVE
To investigate 5'UTR sequence in different SARS-CoV isolates, to identify the secondary structure, and to test the promoter activity of the cDNA sequence corresponding to SARS-CoV 5'UTR in eukaryotic cells.


METHODS
101 SARS-CoV 5'UTR were aligned. One typical sequence containing full 264 nt was then subjected to be predicted its secondary structure. The 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. Then the recombinant plasmids were transfected into HepG2 cells and the luciferase activities were detected. A set of deletion mutant plasmids, of which pGL3-5' UTR-1, pGL3-5' UTR-2, pGL3-5'UTR-3 and pGL3-5'UTR-4 are with 3, 2, 1, and 0 residual stem-loops of 3' termini respectively,were constructed from pGL3-5'UTR and were transfected into HepG2 cells to express reporter gene luc+, with pGL3-5'UTR containing full sequence as control. The luciferase activities expressed by the plasmids were measured. And then the total RNA of the transfected cells was extracted. Subsequently, by 5' Rapid Amplication of cDNA Ends (5'RACE), the PCR product was sequenced. The luciferase expressed by pGL3-5'UTR in various cells, the lung carcinoma cell line A549, hepatoma cell line HepG2, kidney cell Vero E6, cervical cancer cell line HeLa and human umbilical vein endothelial cell line ECV304 were measured and compared with each other.


RESULTS
The full sequence of the SARS-CoV 5' UTR is a 264nt, and 18 deletion mutants were found. Totally, 5 site substitutions were found in 101 5'UTR sequences. The SARS-CoV 5'UTR RNA folded to form a stable secondary structure containing four stem-loop domains. The biggest and most complex one is the stem-loop II appearing a pseudoknot. Comparing with pGL3-a-5'UTR, pGL3-5'UTR expressed luciferase obviously. Both pGL3-5'UTR containing full sequence and pGL3-5'UTR-1 containing three stem-loops of 3' termini expressed the luciferase well. However, when lost stem-loop I and II , the pGL3-5'UTR-2, pGL3-5'UTR-3 and pGL3-5'UTR-4 almost didn't express luciferase. The 56th nucleotide of SARS-CoV 5'UTR was found to be the initiation site for transcription. Transfected with expression luciferase plasmid pGL3-5' UTR in which SARS-CoV 5' UTR acts as the promoter, the luciferase could express in five cell lines in different degrees. Ranked by the luciferase activity from the highest to the lowest, the order is A549, HepG2, ECV304, HeLa and Vero E6.


CONCLUSIONS
A: The 5'UTR sequences of different SARS-CoV isolates are relatively conserved, and a full sequence would form a secondary structure containing four stem-loop domains. B: The cDNA sequence corresponding to SARS-CoV 5'UTR possessed a promoter activity in eukaryotic cells. C: The promoter domain of the SARS-CoV 5'UTR contains both stem-loop I and II. D: The 56th nucleotide and its down stream TRS of SARS-CoV 5'UTR plays a key role in regulating transcription. E: Cells sourced from various tissues can provide efficient accessory factors for SARS-CoV 5'UTR sequence that acts as a promoter, and the lung-sourced cells may be the most suitable.
Zhang
Jian-jun
JJ
Institute for Viral Hepatitis, Chongqing University of Medical Sciences, China.
Huang
Ai-long
AL
Shi
Xiao-ling
XL
Zhang
Xiao-feng
XF
D020121:5' Untranslated Regions / Q000737:chemistry / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics*; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014709:Vero Cells
D020121:5' Untranslated Regions; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D008156:Luciferases