Cell culture systems for hepatitis C virus: Useful for computational modeling of the viral life cycle?

Poster number: 0

Ralf Bartenschlager

  1. Department of Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 345, Heidelberg, Germany

About 15 years ago, the genome of the virus responsible for the majority of post-transfusion hepatitis, HCV, was cloned. It was grouped as a novel member of the family Flaviviridae, which have in common an enveloped particle that harbors an RNA genome of positive polarity. The HCV genome has a length of about 9,600 nucleotides and it carries a single long open reading frame that is flanked by short non-translated regions required for RNA translation and replication. By analogy to other positive strand RNA viruses, upon infection the viral genome is liberated into the cytoplasm and translated at the rough endoplasmic reticulum where viral proteins together with viral RNA and cellular proteins form a replication factory designated the ‘membranous web’. Positive strand RNA is amplified via negative strand intermediates and ultimately packed into viral capsids that acquire their envelope at intracellular membranes. Virus is probably released via the constitutive secretory pathway.

Studies of the viral replication cycle have only recently become possible with the development of the HCV replicon system. Initially, it was based on subgenomic, G418-selectable viral RNAs (replicons) that self-amplify in human hepatoma cells (Huh-7) to very high levels. Subsequent studies led to the identification of cell culture adaptive mutations and highly permissive cells, enabling the development of transient HCV replication assays and selectable full length genomes. Very recently, we developed a system that supports the complete viral life cycle in Huh-7 cells. Upon transfection of these cells with a chimeric genome, virus particles are released that spread within the culture with rather fast kinetics. More importantly, virus can be stably propagated in cell culture by serial passage in Huh-7 cells without reduction of titre. These cell culture systems are instrumental to gain insight into the mechanisms of the HCV replication cycle. Moreover, the systems may be useful to quantitatively assess and model parts of the viral life cycle.