Studies On Hepatitis G Virus As A Replicable Vector For Gene Therapy And Inhibition Of Gene Expression And Viral Replication By Small Interfering RNA

Hepatitis G virus (HGV) possesses a positive-sense, single-stranded RNA genome approximately 9.4Kb in length that encodes a single, long open reading frame (ORF). The ORF is franked at the 5' end by a nontranslated region (NTR) that functions as an internal ribosome entry site (IRES) and at the 3' end by a highly conserved sequence essential for viral replication. Studies supported that there exist two HGV variants with different tissue tropism as lymphotropism and hepatotropism. We once constructed the full length HGV cDNA clone pHGVqz in 1998. It has been confirmed that this clone was replicable in macaques, transgenic mice or cultured cells. Our former studies demonstrated the HGVqz was a hepatotropic variant. Sequence analysis of the viral genome revealed that HGV was a member of the Flaviviridae family and the genome organization was found to be organized similarly to that of HCV. HCV subgenomic RNA replicons, which the structural genes were replaced by a selectable marker gene (Neo) and IRES could self-replicate in human hepatoma cell line, Huh7 efficiently. We constructed the HGV subgenomic RNA replicons by replacing the structural genes with the enhanced green fluorescent protein (EGFP) or TNF-a and encephalomyocarditis virus (ECMV) IRES and neomycin phosphotransferase (neo) genes to seek its possibility as a hepatotropic viral vector for gene therapy.1. Establishment of Huh7 cells expressing HGV structural proteinsThe aim of this study is to characterize the structural proteins of HGV and provide the structural proteins for the package of the HGV-like particle. The structural genes of HGV were amplified with PCR and cloned into pVAX 1.0 vector. The hygromycin resistance gene was inserted downstream from the HGV structural genes. The recombinant eukaryotic expressing vector pVAX.EH was used to transfect human hepatoma cell Huh7 by liposome-mediated gene transfer method. The resistant cell clones were obtained after 400u,g/ml hygromycin selection for 20 days. The expression of HGV structural proteins at mRNA and protein levels was found by RT-PCR and Western blot. A 45kDa expected protein band of HGV E2 was identified with HGV E2 MAb and serum of HGV infected patient. One protein band, which was greater than 66kDa was identified with serum of HGV, infected patient. Structural proteins of HGV were expressed in Huh7 cells and could be identified with HGV E2 MAb and serum of HGV infected patient. There exists the precursor protein before the cleavage of the structural proteins of HGV.2. Replication and expression of HGV subgenomic replicons in human hepatoma cell HuhThe transcription plasmids of HGV subgenomic replicons RNAs were constructed with four steps. First, the sequence of HGV 5'NTR fused to 12 amino acids of the capsid coding region and the sequence of EGFP or TNF were fused with SOEing PCR. Second, this fragment (HGV STsTTR-EGFP or HGV 5*NTR-TNF) was ligated to the sequences of IRES-neo with T4 DNA ligase. Third, the sequences of HGV dNS2-NS3-NS4-NS5-3'NTR were amplified with SOEing PCR and T4 DNA ligase and fused to the sequence of HDV antigenomic ribozyme. Finally, the sequences of STCTR-EGPF-IRES-neo or STSTTR-TNF-IRES-neo and the sequences of dNS2-NS3-NS4-NS5-3'NTR-Ribo were ligated with T4 DNA ligase and inserted into the plasmid pGEM-3zf (+). Then the transcription plasmids of pHGVrepEGFP and pHGVrepTNF were constructed. HGVrepEGFP and HGVrepTNF RNAs were transcribed with T7 RNA polymerase in vitro and transfected into Huh cells by liposome-mediated gene transfer method. The G418 resistant cell clones were picked after 600 ug/ml G418 selection for 35 days. The formation of G418-resistant colonies demonstrated that HGVrepEGFP and HGVrepTNF could express the neomycin phosphotransferase. These G418 resistant cells expressed HGV non-structural proteins detectable by Western blot method with serum of HGV infected patient. Production of HGV subgenomic RNA transcripts and presence of replicative negative strand of HGVrepEGFP or HGVrepTNF was confirmed by RT-PCR assay and Northern blot indic...