Screening for Hepatitis C Virus (HCV) protein interactions by the use of intergenotypic replicon chimeras

Chronic Hepatitis C Virus (HCV) infection is a major cause of liver cirrhosis, hepatocellular carcinoma and liver transplantation. Currently, no vaccine is available and therapeutic options are limited. A hallmark of the HCV life cycle is the formation of a membrane bound replication complex that depends on the concerted interplay of all HCV non-structural proteins (NS). In this study, we investigate protein interactions of the NS proteins that are required for the formation of a functional replication complex. On the background of a subgenomic genotype 1b replicon, we created a panel of chimeras, where sequences of interest were replaced from a genotype 2a replicon. Constructs were analysed in respect to RNA replication, subcellular localization of proteins and polyprotein processing. Furthermore, to localize interacting domains, selected chimeras were analysed for the development of second site changes by sequencing replicon RNA derived from transfected cells. Potential compensatory mutations are reengineered into the parental replicon construct and analysed for replication. Even though intracellular protein localization and polyprotein processing were preserved, some chimeric constructs showed a severe defect in RNA replication, indicating that sequence specific protein interactions within the replication complex were disrupted. Interestingly, even single amino acid exchanges are sufficient to reduce replication dramatically. Sequencing studies revealed a panel of second site changes that might reflect areas of protein interaction. Currently, these mutations are under investigation and results on their impact on replication will be presented. The mapping of protein interactions between HCV NS proteins will contribute to the understanding of replication complex formation and identification of potential antiviral targets.