Hepatitis C Virus
4. Virus Produktion System
Although HCV replicons are instrumental to study various aspects of the HCV life cycle they do not allow analyses of all aspects. For instance, cell culture-adapted replicons do not support production of infectious virus particles. This limitation has now been overcome by the first establishment of a cell culture system that supports the complete HCV replication cycle in cultured human hepatoma cells ( Wakita et al., 2005; Lindenbach et al., 2005; Zhong et al., 2005). This system is based on a particular HCV isolate that was cloned from a Japanese patient with fulminant hepatitis C, hence the name JFH-1 for this isolate. In contrast to all other HCV replicons available thus far, JFH-1 replicates to very high levels and does not require these replication enhancing mutations. Upon transfection of the complete JFH-1 genome or a chimeric JFH-1 genome into human hepatoma cells, HCV particles were produced that are infectious for naive cells as well as for chimpanzees. Moreover, infection can be neutralized with antibodies directed against the envelope protein E2 or the putative (co)receptor CD81 or by immunoglobulins from patient sera, although neutralization efficiency is variable often low in the latter case. Cell culture grown HCV has a spherical morphology with an outer diameter of about 55nm and an inner structure presumably representing the nucleocapsid with a diameter of about 30nm. Virus particles have a heterogenous density which may be due to association with cellular components.
This virus system has recently been advanced by generation of reporter virus genomes that greatly facilitate measurement of RNA replication and infection studies. They are also of great use for drug screening purposes and for evaluation of antiviral drugs and drug resistance.
Huh-7 cells are transfected with genomic HCV RNA obtained by in vitro transcription from the cloned JFH-1 genome (upper panel) or a mutant that lacks the envelope glycoprotein genes (JFH1ΔE1-E2). Four days later, culture supernatant is harvested and used to inoculate naive Huh-7 cells (schematic in the middle panel). Forty eight hours after inoculation, cells are analyzed for infection by using a NS3-specific immunofluorescence assay (red staining in the IF pictures in the lower panels). Infection was performed directly with supernatant (untreated, left panels) or with supernatant containing a CD81-specific antibody (anti-CD81, right panels) or with supernatant containing an irrelevant isotype-matched antibody (Mab-control, middle panels). Nuclear DNA was stained with DAPI (blue staining). Infection can be neutralized by anti CD81 demonstrating that this cell surface molecule plays an important role in infection by HCV.