RESUMEN
The hepatitis C virus (HCV) NS5A protein is highly phosphorylated by cellular protein kinases. To study how NS5A might be integrated in cellular kinase signalling, we isolated phosphoproteins from HuH-7 hepatoma cells that specifically interacted with recombinant NS5A protein. Subsequent mass spectrometry identified the adaptor protein amphiphysin II as a novel interaction partner of NS5A. Mutational analysis revealed that complex formation is primarily mediated by a proline-rich region in the C-terminal part of NS5A, which interacts with the amphiphysin II Src homology 3 domain. Importantly, we could further demonstrate specific co-precipitation and cellular co-localization of endogenous amphiphysin II with NS5A in HuH-7 cells carrying a persistently replicating subgenomic HCV replicon. Although the NS5A-amphiphysin II interaction appeared to be dispensable for replication of these HCV RNAs in cell culture, our results indicate that NS5A-amphiphysin II complex formation might be of physiological relevance for the HCV life cycle.
Asunto(s)
Hepacivirus/fisiología , Proteínas del Tejido Nervioso/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas no Estructurales Virales/metabolismo , Citoplasma/química , Técnica del Anticuerpo Fluorescente Indirecta , Hepacivirus/química , Hepacivirus/genética , Humanos , Immunoblotting , Leucina Zippers , Quinasas Quinasa Quinasa PAM , Mutación , Proteínas del Tejido Nervioso/química , Prolina , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , Proteínas Recombinantes/metabolismo , Replicón , Células Tumorales Cultivadas , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Replicación Viral , Proteina Quinasa Quinasa Quinasa 11 Activada por MitógenoRESUMEN
The development of a subgenomic replicon derived from the hepatitis C virus (HCV) strain Con1 enabled the study of viral RNA replication in Huh-7 cells. The level of replication of replicons, as well as full-length Con1 genomes, increased significantly by a combination of two adaptive mutations in NS3 (E1202G and T1280I) and a single mutation in NS5A (S2197P). However, these cell culture-adaptive mutations influenced in vivo infectivity. After intrahepatic transfection of chimpanzees, the wild-type Con1 genome was infectious and produced viral titers similar to those produced by other infectious HCV clones. Repeated independent transfections with RNA transcripts of a Con1 genome containing the three adaptive mutations failed to achieve active HCV infection. Furthermore, although a chimpanzee transfected with RNA transcripts of a Con1 genome with only the NS5A mutation became infected, this mutation was detected only in virus genomes recovered from serum at day 4; viruses recovered at day 7 had a reversion back to the original Con1 sequence. Our study demonstrates that mutations that are adaptive for replication of HCV in cell culture may be highly attenuating in vivo.
Asunto(s)
Adaptación Fisiológica/genética , Hepacivirus/genética , Proteínas no Estructurales Virales/genética , Replicación Viral/genética , Adaptación Fisiológica/fisiología , Animales , Genoma Viral , Hepacivirus/fisiología , Humanos , Mutagénesis , Pan troglodytes , ARN Viral/biosíntesis , Replicón , Células Tumorales Cultivadas , Proteínas no Estructurales Virales/fisiología , Replicación Viral/fisiologíaRESUMEN
Persistent infection with hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. All treatments known so far rely on the antiviral activity of interferon alfa (IFN-alpha) that is given alone or in combination with ribavirin. Unfortunately, only a fraction of the patients clear the virus during therapy and for those who do not respond there is currently no alternative treatment. Selectable subgenomic HCV RNAs (replicons) have been recently used to investigate the effect of IFN-alpha on HCV replication. However, it has not yet been analyzed whether other cytokines also play a role in the innate immune response against HCV. Here we show that IFN-gamma inhibits protein synthesis and RNA replication of subgenomic and genomic HCV replicons. We further show that the inhibitory action of IFN-gamma does not rely on the production of nitric oxide or the depletion of tryptophan. In conclusion, our results suggest that cytotoxic T cells and natural killer cells may contribute to HCV clearance not only by cell killing but also by producing IFN-gamma, thereby enhancing the intracellular inhibition of viral replication.
Asunto(s)
Hepacivirus/efectos de los fármacos , Interferón gamma/farmacología , ARN Viral/biosíntesis , Replicación Viral/efectos de los fármacos , Hepacivirus/genética , Humanos , Interferón Tipo I/biosíntesis , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/fisiología , Óxido Nítrico Sintasa de Tipo II , Replicón/efectos de los fármacos , Triptófano/metabolismo , Células Tumorales Cultivadas , Proteínas no Estructurales Virales/metabolismoRESUMEN
The recently developed subgenomic hepatitis C virus (HCV) replicons were limited by the fact that the sequence encoding the structural proteins was missing. Therefore, important information about a possible influence of these proteins on replication and pathogenesis and about the mechanism of virus formation could not be obtained. Taking advantage of three cell culture-adaptive mutations that enhance RNA replication synergistically, we generated selectable full-length HCV genomes that amplify to high levels in the human hepatoma cell line Huh-7 and can be stably propagated for more than 6 months. The structural proteins are efficiently expressed, with the viral glycoproteins E1 and E2 forming heterodimers which are stable under nondenaturing conditions. No disulfide-linked glycoprotein aggregates were observed, suggesting that the envelope proteins fold productively. Electron microscopy studies indicate that cell lines harboring these full-length HCV RNAs contain lipid droplets. The majority of the core protein was found on the surfaces of these structures, whereas the glycoproteins appear to localize to the endoplasmic reticulum and cis-Golgi compartments. In agreement with this distribution, no endoglycosidase H-resistant forms of these proteins were detectable. In a search for the production of viral particles, we noticed that these cells release substantial amounts of nuclease-resistant HCV RNA-containing structures with a buoyant density of 1.04 to 1.1 g/ml in iodixanol gradients. The same observation was made in transient-replication assays using an authentic highly adapted full-length HCV genome that lacks heterologous sequences. However, the fact that comparable amounts of such RNA-containing structures were found in the supernatant of cells carrying subgenomic replicons demonstrates a nonspecific release independent of the presence of the structural proteins. These results suggest that Huh-7 cells lack host cell factors that are important for virus particle assembly and/or release.