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Beeinflussung der Lipidzusammensetzung membranöser viraler Replikationsorganelle durch das Hepatitis C Virus

Fachliche Zuordnung Virologie
Förderung Förderung von 2015 bis 2022
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 278191845
 
Erstellungsjahr 2023

Zusammenfassung der Projektergebnisse

Hepatitis C virus (HCV) is a positive strand RNA virus belonging to the family of Flaviviridae and an important human pathogen causing severe liver disease. During the first funding period we clarified the mechanism of adaptation of HCV to replication in cell culture, limiting an overproduction of Phosphatidylinositol (PI) 4-phoshate PI4P, which is deleterious for most HCV isolates. Thereby we could establish an inhibitor regimen, termed PCi treatment, allowing efficient replication of HCV wt isolates in cell culture. Using this technology, we have recently identified a new HCV genotype 1b wt isolate, termed GLT1, replicating to very high levels in cell culture. Replication of HCV GLT1 can be stimulated by up to 4 orders of magnitude either by PCi treatment or by expression of Sec14L2, a lipid transporter protein expressed in hepatocytes but not in Huh7, arguing again for a critical role of the lipid composition of the viral replication organelles. Sec14L2 was recently identified by others to stimulate replication of HCV wt isolates, but the mechanism is still poorly defined and the effect was very moderate for all isolates tested so far. The dramatic stimulation of GLT1 replication by Sec14L2 or PCi treatment now provides the unique opportunity to understand the requirements of HCV replication in cell culture at a molecular level and to define the impact of Sec14L2 on the lipid and protein composition, as well as the morphology of authentic HCV replication organelles. Therefore the renewal proposal followed three major aims: 1. Understanding the mechanisms underlying efficient GLT1 replication by generating chimeras with a related gt1b isolate. We further wanted to adapt the GLT1 isolate to efficient virion production to obtain the first full-replication cycle culture model for gt1b. 2. We aimed to pursue lipidomic and proteomic comparisons of purified replication organelles in presence and absence of Sec14L2 using a replicase expression model and experimentally validate respective candidate lipids and proteins. 3. We aimed for an ultrastructural comparison of viral replication organelles in presence and absence of Sec14L2 to identify structures favorable or unfavorable for viral replication. Regarding aim 1, we identified a highly variable region in HCV nonstructural protein (NS)5A, which we now term RFDR (replication fitness determining region), substantially increasing RNA replication efficiency by accumulation of mutations. So far, the phenotype appears rather related to the number than to the nature of mutations, as previously found in the context of interferon therapy. We further were successful in adapting the GLT1 isolate to efficient virus production in cell culture, retaining its ability to replicate in vivo, in human liver chimeric mice, thereby generating a highly efficient novel cell culture system for genotype 1b. We could further show that high replicating HCV variants based on RFDR mutations appear after liver transplantation, resulting in severe pathogenesis, suggesting a selective advantage for high replicating variants under immunosuppression. Regarding the mode of action of SEC14L2, using GLT1 as a novel tool to understand the mode of action and determinants of HCV replication (aims 2 and 3), all our unbiased lipidomic and proteomic approaches and ultrastructural analyses did not reveal substantial differences. We therefore conclude that modulation of HCV replication by the RFDR is mediated by intra-replicase interactions, which will be addressed in future studies. For SEC14L2, we tested several hypotheses for its mode action. Since Tocopherol could not rescue GLT1 replication in absence of SEC14L2, it appears unlikely that its mechanism is based on preventing lipid peroxidation, as initially thought. We further found no evidence that SEC14L2 might deplete PI from the HCV replication organelle, thereby limiting PI4P production. Based on a recent publication, we have first evidence that SEC14L2 might directly remove PI4P from the viral replication site, probably in exchange to PI3P, thereby acting similarly as cell culture adaptive mutations and PCi treatment.

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