Phenotypic analysis of Hepatitis C virus evolution in the post-transplant setting: Understanding mechanisms of rapid fitness adaptation to a new environment

• Applicant: Professor Dr. Volker Lohmann
• Subject Area: Virology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 519777725

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. HCV is among the most variable viruses, creating a highly diverse quasispecies in every patient, but so far very little is known about the impact of the viral isolate on infection outcome or disease progression. Following the virus evolution in a liver transplant (LTX) patient, we identified a dramatically reduced virus diversity after LTX, along with a strong increase in RNA replication fitness. 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 found a clear correlation between the appearance of highly replicating RFDR mutants after LTX in patients developing a fibrosing cholestatic hepatitis (FCH) due to a fast and severe disease progression, pointing to substantial contribution of HCV replication fitness to direct viral pathogenesis, at least in absence of adaptive immune responses. This project now aims at understanding the general clinical significance of RFDR variants, the definition of sequence patterns governing RNA replication fitness and the mechanism how the RFDR regulates RNA replication, focusing on the hypothesis of a regulation of polymerase activity, which is suggested by literature. Aim 1 will study a wider range of post LTX HCV variants stratified according to disease progression to obtain further evidence for a role of replication fitness in pathogenesis and address the impact of entry and assembly competence to viral fitness. Aim 2 will address the evolution of the RFDR in pre- and post-LTX isolates to study whether high replicator RFDRs are primarily selected upon LTX or already prevalent prior LTX. We will further follow hints in literature on the contribution of high replicator RFDRs to development of hepatocellular carcinoma, direct-acting-antiviral treatment failure and generally high viral titers. In addition, we will assess the possible significance of RFDR regulatory activity beyond HCV genotype 1b. Aim 3 will identify and validate distinct sequence determinants governing replication efficiency, to allow sequence-based predictions and to support a mechanistic understanding. Aim 4 will clarify the mechanism underlying the regulation of RNA replication by the RFDR, supported by structural analyses. In a first step, in vitro assays building on purified polymerase and NS5A variants will be used to find different regulatory activities of RFDR variants, correlating with RNA replication fitness. Cell based studies will complement the in vitro analysis with more unbiased approaches.

• DFG Programme: Research Grants