RESUMO
Apoptosis of infected cells represents a key host defense mechanism against viral infections. The impact of apoptosis on the elimination of hepatitis C virus (HCV)-infected cells is poorly understood. The TRAIL has been implicated in the death of liver cells in hepatitis-infected but not in normal liver cells. To determine the impact of TRAIL on apoptosis of virus-infected host cells, we studied TRAIL-induced apoptosis in a tissue culture model system for HCV infection. We demonstrated that HCV infection sensitizes primary human hepatocytes and Huh7.5 hepatoma cells to TRAIL induced apoptosis in a dose- and time-dependent manner. Mapping studies identified the HCV nonstructural proteins as key mediators of sensitization to TRAIL. Using a panel of inhibitors targeting different apoptosis pathways, we demonstrate that sensitization to TRAIL is caspase-9 dependent and mediated in part via the mitochondrial pathway. Sensitization of hepatocytes to TRAIL-induced apoptosis by HCV infection represents a novel antiviral host defense mechanism that may have important implications for the pathogenesis of HCV infection and may contribute to the elimination of virus-infected hepatocytes.
Assuntos
Apoptose/imunologia , Caspase 9/fisiologia , Hepacivirus/imunologia , Hepatócitos/patologia , Hepatócitos/virologia , Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Comunicação Autócrina/genética , Comunicação Autócrina/imunologia , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Células Cultivadas , Deleção de Genes , Hepacivirus/genética , Hepatócitos/enzimologia , Hepatócitos/imunologia , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Mitocôndrias/enzimologia , Mitocôndrias/genética , Proteínas do Core Viral/biossíntese , Proteínas do Core Viral/genética , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/biossíntese , Proteínas não Estruturais Virais/genética , Proteínas Estruturais Virais/biossíntese , Proteínas Estruturais Virais/genéticaRESUMO
Hepatitis C virus (HCV) is a major cause of liver disease, but therapeutic options are limited and there are no prevention strategies. Viral entry is the first step of infection and requires the cooperative interaction of several host cell factors. Using a functional RNAi kinase screen, we identified epidermal growth factor receptor and ephrin receptor A2 as host cofactors for HCV entry. Blocking receptor kinase activity by approved inhibitors broadly impaired infection by all major HCV genotypes and viral escape variants in cell culture and in a human liver chimeric mouse model in vivo. The identified receptor tyrosine kinases (RTKs) mediate HCV entry by regulating CD81-claudin-1 co-receptor associations and viral glycoprotein-dependent membrane fusion. These results identify RTKs as previously unknown HCV entry cofactors and show that tyrosine kinase inhibitors have substantial antiviral activity. Inhibition of RTK function may constitute a new approach for prevention and treatment of HCV infection.
Assuntos
Receptores ErbB/fisiologia , Hepacivirus/fisiologia , Hepatite C/fisiopatologia , Hepatite C/virologia , Receptor EphA2/fisiologia , Internalização do Vírus , Animais , Antígenos CD/fisiologia , Antivirais/farmacologia , Sequência de Bases , Linhagem Celular , Claudina-1 , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Cloridrato de Erlotinib , Hepacivirus/efeitos dos fármacos , Hepatite C/prevenção & controle , Hepatite C/terapia , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Ligantes , Proteínas de Membrana/fisiologia , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , Receptor EphA2/antagonistas & inibidores , Receptor EphA2/genética , Tetraspanina 28 , Internalização do Vírus/efeitos dos fármacosRESUMO
Donor T cell alloreactivity can be efficiently controlled by retrovirus-mediated ex vivo transfer of a "suicide" gene encoding the wild-type herpes simplex virus thymidine kinase (wtHSV-tk) gene, allowing gene-modified cells (GMCs) to be sensitive to ganciclovir (GCV). A limitation to this approach was related to the presence of an inactive form of the wtHSV-tk gene, resulting from alternative splicing. A corrected HSV-tk (cHSV-tk) gene was developed in order to circumvent this problem and was fused to a truncated splice variant of the human CD34 molecule (tCD34) suitable for the selection of retrovirally transduced GMCs. We demonstrate now that, despite this correction, CD34-positive, but GCV-resistant, HUT and primary GMCs can still be generated after transduction with a retroviral vector encoding a tCD34/cHSV-tk fusion protein (FuProtein). Deletions in the HSV-tk part of the transgene account in part for this resistance. However, an additional mechanism involving proteolytic-dependent "breakage" of the FuProtein has been observed: the CD34 part of the FuProtein can be detected by Western blot, separated from its HSV-tk part. Although the HSV-tk protein alone is not detectable in GCV-resistant tCD34/cHSV-tk-transduced HUT cells, it can be detected in 293T cells transduced with another tCD34/HSVTK fusion vector, demonstrating that a posttranslational effect leads to the breakage of the FuProtein. This is to our knowledge the first example of a loss of function of a FuProtein, of which one part is still expressed while the other one, suffering a selection pressure, is no longer detectable.