Small interfering RNA-mediated inhibition of hepatitis G virus gene expression in human hepatoma cell Huh-7.
The RNA interference (RNAi) phenomenon is a recently observed process in which the introduction of a double-stranded, small interfering RNA (siRNA) into a cell causes the specific degradation of a homologous single-stranded RNA. It represents an exciting new technology that could have therapeutic applications for the treatment of viral infections. Since hepatitis G virus (HGV) genome is a positive-sense single-stranded RNA, the replication of HGV does not lead to an integrated DNA genome, suggesting a particularly attractive target for RNAi study that could eliminate viral RNA from infected cells. The eukaryotic expression vector pVAX.EH containing the cDNA sequences of the entire HGV structural genes and hygromycin resistance gene downstream from the encephalomyocarditis virus (ECMV) internal ribosome entry site (IRES) was constructed and transfected into human hepatoma cell Huh-7. The modified cleavage products of the structural proteins of HGV expressed in hygromycin-resistant cell line Huh-7-EH were confirmed by RT-PCR and Western blot methods. Two specific HGV E2 siRNAs (1-E2 siRNA, 2-E2 siRNA) synthesized with T7 RNA polymerase by transcription in vitro were transfected into the Huh-7-EH cells. With the analyses of Western blot and the formation of hygromycin-resistant colonies, the inhibitions of expression of HGV structural protein by two HGV E2 siRNAs were detected and found lasting at least one week. The inhibition of 2-E2 siRNA was stronger and only 1% of the cells treated with 2-E2 siRNA formed hygromycin-resistant colonies. These results support that specific HGV 2-E2 siRNAs mediate the degradation of mRNA spanning from HGV structural gene cDNA to hygromycin resistance gene in a majority of cells. In conclusion, the Huh-7-EH cells expressing HGV structural proteins stably can be used as a cell model for studying the replication of HGV and RNAi and the enlargement of RNAi may exist, in mammalian cells.
Cao
Mingmei
M
Department of Microbiology, Second Military Medical University, Shanghai 200433, China.
Ren
Hao
H
Zhao
Ping
P
Pan
Wei
W
Zhao
Lanjuan
L
Qi
Zhongtian
Z
D000998:Antiviral Agents / Q000494:pharmacology; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D045744:Cell Line, Tumor; D002934:Cinnamates / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004351:Drug Resistance; D019008:Drug Resistance, Neoplasm; D004686:Encephalomyocarditis virus / Q000378:metabolism; D029407:GB virus C / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D006801:Humans; D006921:Hygromycin B / Q000031:analogs & derivatives* / Q000494:pharmacology; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000235:genetics; D014162:Transfection
D000998:Antiviral Agents; D002934:Cinnamates; D018076:DNA, Complementary; D016376:Oligonucleotides, Antisense; D034741:RNA, Small Interfering; D006921:Hygromycin B; C026273:hygromycin A