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.