[Effect of structural domain II of HCV 5' noncoding region on its translation initiation activity].
OBJECTIVE
To examine the role of domain II of hepatitis C virus (HCV) 5' noncoding region (5' NCR) in its translation initiation activity.


METHODS
The fragment of HCV 5' NCR with deletions of 5'-proximal 118 nucleotides were amplified with PCR, then was used to substitute for the full length HCV 5' NCR of plasmid pCMVNCRluc, a firefly luciferase (Fluc) eukaryotic expression plasmid regulated by HCV 5' NCR, to generate recombinant plasmid pCNl-d3. pCMVNCRluc, pCNl-d2 (modified pCMVNCRluc with deletions of HCV 5' NCR ntl-43) and pCNl-d3 were transfected into HepG2 cells with liposome transfection protocol, respectively, and the relative luciferase activity was measured to analyze the regulatory effect of the truncated 5'NCR on Flue gene expression. Meanwhile the Flue mRNA levels were detected by RT-PCR.


RESULTS
The recombinant plasmid was successfully constructed. The Flue mRNA levels of the 3 plasmids were not significantly different (P > 0.05), and there were also no significant difference between the relative luciferase activity of plasmids pCNl-d2 and pCMVNCRluc (P > 0.05). However, that of pCNl-d3 was decreased significantly (P < 0.01, compared with pCNl-d2 or pCMVNCRluc).


CONCLUSIONS
Structural domain II of HCV 5' NCR plays an important role in its translation initiation activity.
Liu
Shui-Ping
SP
Department of Microbiology, Xiangya School of Medicine, Central South University, Changsha 410078, China. spliu@mail.csu.edu.cn
Zhao
Jun-Qin
JQ
Li
Hong-Tao
HT
Yang
Tao
T
D020121:5' Untranslated Regions; D056945:Hep G2 Cells; D016174:Hepacivirus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006526:Hepatitis C; D006801:Humans; D009690:Nucleic Acid Conformation; D010442:Peptide Chain Initiation, Translational; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics
D020121:5' Untranslated Regions; D012367:RNA, Viral