Viral determinants of hepatitis C virus resistance to interferon and their relevance for broad resistance to HCV specific antivirals.
Final Report Abstract
Infection with hepatitis C virus (HCV) induces chronicity in the majority of infected individuals. Due to its high genetic variability, HCV replicates as a swarm of closely related viral variants within a single infected individual, referred to as quasispecies. The continuous selection of the fittest variants is one of the key determinants of immune evasion and persistence of HCV. Cell culture adaptation of HCV by long-term passaging in the highly permissive human liver cell line Huh-7.5 resulted in the selection of a virus population (p100pop), exhibiting increased replicative fitness. In addition, partial resistance to interferon treatment and HCV-specific antivirals compared to its parental virus Jc1 was also observed. These phenotypic changes correlate with the fixation of numerous coding and noncoding mutations in the p100pop consensus sequence. In this study, we aimed to investigate viral mechanisms conferring cell culture adaptation of p100pop in order to identify principles by which HCV optimizes its replicative fitness. Infection experiments in different liver cancer cell lines revealed that cell culture adaptation of HCV resulted in the expansion of the limited cell tropism of HCV in cell culture. We could show that this is partially conferred by a change in viral dependence on the critical host factor miR-122. In contrast, adaptation to liver cell lines resulted in a modest viral attenuation in primary human hepatocytes, correlating with a greater induction of the innate immune response by p100pop compared to the parental virus. Infection experiments with molecular p100 clones confirmed that adaptive coding mutations are the main driver of enhanced replicative fitness, whereas noncoding mutations contribute only to a minor extent. Replication kinetics of p100/Jc1 chimeras and subgenomic replicons indicate that cell culture adaptation resulted in optimized virus assembly and release processes. Furthermore, our results suggest that adaptive mutations in the structural proteins facilitate virus entry. Our results highlight that the presence of cell culture adapted mutations correlates with changes in cyclophilin A dependence, presenting a possible mechanism by which HCV further optimizes its replicative fitness. In addition, the release of infectious p100pop particles was reduced upon inhibition of protein kinase R autophosphorylation, suggesting another principle of optimized virus-host interaction. Finally, transcriptomic analyses of the host response in Huh-7 cells revealed that p100pop confers dysregulation of pathways involved in endoplasmic reticulum stress signaling and unfolded protein response. Both pathways were previously reported to be hijacked by HCV, suggesting that modulation of the host response may also contribute to the observed enhanced replicative fitness. Taken together, this study highlights different principles by which viral and host determinants can synergistically contribute to optimized fitness of HCV in cell culture. This knowledge will contribute to the understanding of evolved HCV persistence mechanisms.
Publications
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Hepatitis C virus cell culture adaptive mutations enhance cell culture propagation by multiple mechanisms but boost antiviral responses in primary human hepatocytes. Cold Spring Harbor Laboratory.
Frericks, Nicola; Brown, Richard J.P.; Reinecke, Birthe M.; Herrmann, Maike; Brüggemann, Yannick; Todt, Daniel; Miskey, Csaba; Vondran, Florian W. R.; Steinmann, Eike; Pietschmann, Thomas & Sheldon, Julie
