Shaping the lipid landscape of the membraneous viral replication organelles by Hepatitis C virus

• Applicant: Professor Dr. Volker Lohmann
• Subject Area: Virology
• Term: from 2015 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 278191845

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. We have recently identified the lipid kinase phosphatidylinositol 4-phosphate kinase IIIα (PI4KIIIa, PI4KA) as an essential host factor of HCV replication. PI4KA converts phosphatidylinositol to phosphatidylinositol 4-phosphate (PI4P). The enzymatic activity of PI4KA is activated by HCV, resulting in strongly elevated intracellular PI4P levels. During the recent funding period we found that activation of PI4KA in Huh7 hepatoma cells is deleterious for most HCV isolates due to an excess of PI4P, explaining the inefficient replication of patient derived viruses in cell culture. Surprisingly, we could demonstrate that replication enhancing adaptive mutations in fact rely on a loss of function mechanism, abrogating activation of PI4KA to compensate for high PI4KA expression levels in hepatoma cells compared to primary hepatocytes. Based on this finding we could establish a regimen based on PI4KA/Casein Kinase Iα (CKIα) treatment, allowing efficient replication of HCV wt isolates in cell culture. Using this technology we 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 PI4KA/CKIα treatment or by expression of Sec14L2, a lipid transporter protein expressed in hepatocytes but not in Huh7, arguing 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 PI4KA/CKIα inhibition 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 this renewal proposal follows three major aims: 1. Understanding the mechanisms underlying efficient GLT1 replication by generating chimeras with a related gt1b isolate. We will also try to adapt the GLT1 isolate to efficient virion production to obtain the first full-replication cycle culture model for gt1b.2. We will 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. An ultrastructural comparison of viral replication organelles in presence and absence of Sec14L2 will identify structures favorable or unfavorable for viral replication. The distribution of candidate lipids relative to viral replication vesicles will be analyzed by super-resolution microscopy and live cell imaging.

• DFG Programme: Research Grants