+ Site Statistics
+ Search Articles
+ PDF Full Text Service
How our service works
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ Translate
+ Recently Requested

Promoter activity of SARS coronavirus 5' UTR sequence in eukaryotic cells



Promoter activity of SARS coronavirus 5' UTR sequence in eukaryotic cells



Sichuan Da Xue Xue Bao. Yi Xue Ban 37(1): 5-9



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. 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. 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. 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.

Please choose payment method:






(PDF emailed within 1 workday: $29.90)

Accession: 050043876

Download citation: RISBibTeXText

PMID: 16468630


Related references

Differential sensitivities of severe acute respiratory syndrome (SARS) coronavirus spike polypeptide enzyme-linked immunosorbent assay (ELISA) and SARS coronavirus nucleocapsid protein ELISA for serodiagnosis of SARS coronavirus pneumonia. Journal of Clinical Microbiology 43(7): 3054-3058, 2005

Cloning of ACE-2 gene encoding the functional receptor for the SARS coronavirus and its expression in eukaryotic cells. Zhonghua Shi Yan He Lin Chuang Bing du Xue Za Zhi 19(3): 260-263, 2005

Construction of SARS coronavirus partial S gene segments from eukaryotic expression vectors and its expression in Hela cells. Applied Immunohistochemistry & Molecular Morphology 11(4): 296, 2003

Susceptibility of different eukaryotic cell lines to SARS-coronavirus. Archives of Virology 150(5): 1023-1031, 2005

Alisporivir inhibits MERS- and SARS-coronavirus replication in cell culture, but not SARS-coronavirus infection in a mouse model. Virus Research 228: 7, 2017

Mutagenesis of the transmembrane domain of the SARS coronavirus spike glycoprotein: refinement of the requirements for SARS coronavirus cell entry. Virology Journal 6: 230, 2009

Evidence of the recombinant origin of a bat severe acute respiratory syndrome (SARS)-like coronavirus and its implications on the direct ancestor of SARS coronavirus. Journal of Virology 82(4): 1819-1826, 2008

Development of a quantitative assay for SARS coronavirus and correlation of GAPDH mRNA with SARS coronavirus in clinical specimens. Journal of Clinical Pathology 58(3): 276-280, 2005

Difference in receptor usage between severe acute respiratory syndrome (SARS) coronavirus and SARS-like coronavirus of bat origin. Journal of Virology 82(4): 1899-1907, 2008

Mouse studies of SARS coronavirus-specific immune responses to recombinant replication-defective adenovirus expressing SARS coronavirus N protein. Hong Kong Medical Journal 15(Suppl. 2): 33-36, 2009

Detection of specific antibodies to severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein for serodiagnosis of SARS coronavirus pneumonia. Journal of Clinical Microbiology 42(5): 2306-2309, 2004

Severe Acute Respiratory Syndrome (SARS) Coronavirus ORF8 Protein Is Acquired from SARS-Related Coronavirus from Greater Horseshoe Bats through Recombination. Journal of Virology 89(20): 10532-10547, 2015

Longitudinal profile of immunoglobulin G (IgG), IgM, and IgA antibodies against the severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein in patients with pneumonia due to the SARS coronavirus. Clinical and Diagnostic Laboratory Immunology 11(4): 665-668, 2004

Anti-SARS virus antibody responses against human SARS-associated coronavirus and animal SARS-associated coronavirus-like virus. Chinese Medical Journal 117(11): 1723-1725, 2004