Inhibitor RNA blocks the protein translation mediated by hepatitis C virus internal ribosome entry site in vivo.
OBJECTIVE
To investigate the inhibitory effect of hepatitis C virus internal ribosome entry site (HCV IRES) specific inhibitor RNA (IRNA) on gene expression mediated by HCV IRES in vivo.


METHODS
By using G418 screening system, hepatoma cells constitutively expressing IRNA or mutant IRNA (mIRNA) were established and characterized, and HCV replicons containing the 5' untranslated region (5'UTR) were constructed by using the same method. Cotransfection of pCMVNCRluc containing HCV 5'UTR-luc fusion genes and eukaryotic vector of IRNA into human hepatic carcinoma cells (HepG2) was performed and the eukaryotic expression plasmid of IRNA was transfected transiently into HCV replicons. pCMVNCRluc or pCDNA-luc was cotransfected with pSV40-beta Gal into IRNA expressing hepatoma cells by using lipofectamine 2000 in vitro. Then the reporting gene expression level was examined at 48 h after transfection by using a luminometer and the expressing level of HCV C antigen was analysed with a confocal microscope.


RESULTS
Transient expression of IRES specific IRNA could significantly inhibit the expression of reporter gene and viral antigen mediated by HCV IRES by 50% to 90% in vivo, but mIRNA lost its inhibitory activity completely. The luciferase gene expression mediated by HCV IRES was blocked in the HHCC constitutively expressing IRNA. At 48 h after transfection, the expression level of reporter gene decreased by 20%, but cap-dependent luciferase gene expression was not affected. IRNA could inhibit the HCV replicon expression 24 h after transfection and the highest inhibitory activity was 80% by 72 h, and the inhibitory activity was not increased until 7d after transfection.


CONCLUSIONS
IRNA can inhibit HCV IRES mediated gene expression in vivo.
Liang
Xue-Song
XS
Department of Infectious Diseases, Changhai Hospital, Second Military Medical University, Shanghai, China. liangxuesong2000@163.com
Lian
Jian-Qi
JQ
Zhou
Yong-Xing
YX
Wan
Mo-Bin
MB
D006528:Carcinoma, Hepatocellular; D015967:Gene Expression Regulation, Viral; D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D008113:Liver Neoplasms; D014176:Protein Biosynthesis; D012367:RNA, Viral; D012093:Replicon / Q000235:genetics; D012270:Ribosomes / Q000235:genetics* / Q000821:virology*; D014162:Transfection
D012367:RNA, Viral