Development of tools to investigate resistance of HCV genotype 3 to NS5A inhibitors

HCV infection leads to liver failure. Genotype 3 (GT3) is known to respond poorly to newly-developed direct-acting antivirals, especially inhibitors of the multifunctional NS5A protein.
This work reports the establishment of efficient transient replication of the S52 GT3 sub-genomic replicon (SGR) by further culture adaptation of S52 in the context of an optimised luciferase reporter. Also documented is the development of hepatoma cells with immune-attenuating modifications and expressing the lipid binding factor hSEC14L2 to support transient replication of S52. In parallel, stable replication of S52 in SGR-harbouring cells was used to investigate the differences between early and established replication.
Differences in sensitivity to the NS5A inhibitor Daclatasvir (DCV) in both transient and stable S52 were observed compared to other genotypes, and between transient and stable replication. In addition, it is shown here that the resistance-associated substitution (RAS) Y93H conferred a significant fitness cost which is not apparent for stable S52 selected with DCV, despite this RAS being detected. This thesis explores the molecular basis of such an observation and highlights a potential mechanism which warrants further research.
The role of RAS in the development of resistance is still unclear though this work reports that the presence of a RAS within a mixed population greatly influenced the development of resistance to DCV in vitro.
Moreover, this work identified that a cellular metabolism-regulating factor, AMP-mediated protein kinase (AMPK), may be differentially regulated during GT3 infection compared to other GTs. This thesis presents the hypothesis that AMPK regulation by HCV may contribute to hepatic steatosis as a direct consequence of viral infection, which is unique to GT3.
More insight into the propensity of GT3 to develop resistance can aid further antiviral design, and an understanding of the molecular basis of steatosis offers a rationale for treating the symptoms of HCV in addition to direct targeting of the virus.