RESUMO
Short hairpin RNAs (shRNAs) efficiently inhibit gene expression by RNA interference. Here, we report the efficient inhibition by DNA-based vector-derived shRNAs of core protein expression in Huh-7 cells. The shRNAs were designed to target the core region of the hepatitis C virus (HCV) genome. The core region is the most conserved region in the HCV genome, making it an ideal target for shRNAs. We identified an effective site on the core region for suppression of the HCV core protein. The HCV core protein in core protein-expressing Huh-7 cells was downregulated by core protein-shRNA expression vectors (core-shRNA-452, 479, and 503). Our results support the feasibility of using shRNA-based gene therapy to inhibit HCV core protein production.
Assuntos
Vetores Genéticos/farmacologia , RNA Interferente Pequeno/farmacologia , Proteínas do Core Viral/antagonistas & inibidores , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Hepacivirus/efeitos dos fármacos , Hepacivirus/fisiologia , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND/AIMS: The development of an efficient in vitro infection system for HCV is important in order to develop new anti-HCV strategy. Only Huh7 hepatocyte cell lines were shown to be infected with JFH-1 fulminant HCV-2a strain and its chimeras. Here we aimed to establish a primary hepatocyte cell line that could be infected by HCV particles from patients' sera. METHODS: We transduced primary human hepatocytes with human telomerase reverse transcriptase together with human papilloma virus 18/E6E7 (HPV18/E6E7) genes or simian virus large T gene (SV40 T) to immortalize cells. We also established the HPV18/E6E7-immortalized hepatocytes in which interferon regulatory factor-7 was inactivated. Finally we analyzed HCV infectivity in these cells. RESULTS: Even after prolonged culture HPV18/E6E7-immortalized hepatocytes exhibited hepatocyte functions and marker expression and were more prone to HCV infection than SV40 T-immortalized hepatocytes. The susceptibility of HPV18/E6E7-immortalized hepatocytes to HCV infection was further improved, in particular, by impairing signaling through interferon regulatory factor-7. CONCLUSIONS: HPV18/E6E7-immortalized hepatocytes are useful for the analysis of HCV infection, anti-HCV innate immune response, and screening of antiviral agents with a variety of HCV strains.
Assuntos
Hepacivirus/fisiologia , Hepatócitos/virologia , Fator Regulador 7 de Interferon/antagonistas & inibidores , Antígenos CD/genética , Linhagem Celular , Células Cultivadas , Primers do DNA/genética , Proteínas de Ligação a DNA/genética , Genótipo , Células HeLa , Hepacivirus/genética , Hepacivirus/isolamento & purificação , Hepacivirus/patogenicidade , Hepatite C/virologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/fisiologia , Papillomavirus Humano 18/genética , Humanos , Fator Regulador 7 de Interferon/genética , Interferon Tipo I/farmacologia , Proteínas Oncogênicas Virais/genética , RNA Interferente Pequeno/genética , Proteínas Recombinantes , Telomerase/genética , Tetraspanina 28 , TransfecçãoRESUMO
The baculovirus has recently emerged as a promising vector for in vivo gene therapy. To investigate its potential as a delivery vector for an anti-virus ribozyme targeting HIV-1, we constructed recombinant baculovirus vectors bearing a ribozyme-synthesizing cassette driven by the tRNA(i)(Met) promoter with enhanced transduction efficiency by displaying vesicular stomatitis virus glycoprotein (VSV-G) on the viral envelope. Transduction of HeLa CD4(+) cells with a recombinant baculovirus delivering the HIV-1 U5 gene-specific ribozyme dramatically suppressed HIV-1 expression in this cell line. The VSV-G pseudotyped baculovirus vector-transduced ribozyme potently inhibited HIV-1 replication compared to a recombinant baculovirus vector-transduced ribozyme lacking VSV-G. The use of a baculovirus vector might be beneficial for application in gene therapy.
Assuntos
Baculoviridae/genética , Marcação de Genes/métodos , HIV-1/fisiologia , Glicoproteínas de Membrana/genética , RNA Catalítico/genética , Transfecção/métodos , Proteínas do Envelope Viral/genética , Replicação Viral/genética , Inativação Gênica , Vetores Genéticos/genética , Células HeLa , HumanosRESUMO
We describe a HCV core-short-hairpin RNA (shRNA) expressed from a human U6 Pol II promoter, which synergistically enhanced the inhibition of HCV replication in HCV replicon. We also describe the construction of recombinant baculovirus expressing shRNAs of the HCV core gene under the control of the U6 promoter. The anti-HCV shRNAs may provide a practical basis for applying shRNA-based gene therapy to the treatment of HCV.