15th International HIV Drug Resistance Workshop 13-17 June 2006, Sitges, Spain

NEW ACHIEVEMENTS IN HEPATITIS C VIRUS RESEARCH AND THEIR IMPLICATIONS FOR ANTIVIRAL THERAPY

Antivir Ther. 2006; 11:P3 (abstract no. P1)

R Bartenschlager
Department for Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 345, Heidelberg, Germany

Hepatitis C viruses (HCV) comprise a group of positive- strand RNA viruses that together with the flaviviruses and the pestiviruses belong to the Flaviviridae family. As a major cause of acute and chronic liver disease that currently affects 170 million people worldwide and for which no selective therapy exists HCV has received much attention. The viral genome was first molecularly cloned in 1988 and its organization was delineated shortly thereafter. To most viral proteins distinct functions could be ascribed and the 3D crystal structures have been solved for several viral enzymes that are prime targets for antiviral therapy. However, a major hurdle in unravelling the viral life cycle has been the inherent difficulty to propagate HCV in cell culture. A first step to overcome this block was the establishment of selectable, subgenomic replicons that self-amplify to very high levels in the human hepatoma cell line Huh-7. Subsequent studies led to the discovery of more efficient HCV replicons derived from multiple HCV isolates and genotypes, and cell lines other than Huh-7 that also support stable HCV RNA replication. Moreover, replicons that stably express marker genes have been developed that are extensively used both for high throughput screening and for optimizing antiviral compounds. In addition, HCV replicons have been instrumental to select for antiviral drug resistance and to study the mechanisms underlying resistance development.

In spite of this remarkable progress, the replicon system is limited because only the intracellular steps of the HCV life cycle can be studied. This limitation has recently been overcome with the molecular cloning of a novel HCV isolate that was found in the serum of a Japanese patient with fulminant hepatitis. This isolate, designated JFH-1, replicates to exceptionally high levels in cell culture without requirement for replication enhancing mutations. Most importantly, upon transfection of Huh-7 cells with the JFH-1 genome virus particles are released from the cells that are infectious for naïve Huh-7 cells. Infectivity of these particles can be neutralized by antibodies directed against CD81 as well as immunoglobulins from patient serum demonstrating specificity of the infection process. Cell culture-grown HCV is also infectious in vivo and JFH-1 derived particles generated in vivo are infectious for Huh-7 cells demonstrating that cell culture grown HCV is authentic.

To expand the scope of this virus culture system, JFH-1 genomes with reporter genes such as luciferase or green fluorescent protein have been developed. These genomes also support production of infectious HCV particles and have the advantage that infectivity can be quantitated by using simple and reliable reporter gene assays. Furthermore, chimeric genomes carrying the region from core up to NS2 from various HCV isolates fused to the JFH-1 replicase have been generated. These genomes also support production of HCV particles that infect Huh-7 cells in a CD81-dependent manner. Finally, distinct Huh7 cell clones that are highly permissive for HCV infection could be identified allowing the continuous propagation of infectious HCV in cell culture. These novel cell culture systems will allow the development of new antiviral concepts targeting the early and late steps of the HCV life cycle and should facilitate studies on HCV antiviral drug resistance.

2006-06-13
P1

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