Novel perspectives for hepatitis A virus therapy revealed by comparative analysis of hepatitis C virus and hepatitis A virus RNA replication

Abstract

Hepatitis A virus (HAV) and hepatitis C virus (HCV) are two positive-strand RNA viruses sharing a similar biology, but causing opposing infection outcomes, with HAV always being cleared and HCV establishing persistence in the majority of infections. To gain deeper insight into determinants of replication, persistence, and treatment, we established a homogenous cell-culture model allowing a thorough comparison of RNA replication of both viruses. By screening different human liver-derived cell lines with subgenomic reporter replicons of HAV as well as of different HCV genotypes, we found that Huh7-Lunet cells supported HAV-and HCV-RNA replication with similar efficiency and limited interference between both replicases. HAV and HCV replicons were similarly sensitive to interferon (IFN), but differed in their ability to establish persistent replication in cell culture. In contrast to HCV, HAV replicated independently from microRNA-122 and phosphatidylinositol 4-kinase IIIa and b (PI4KIII). Both viruses were efficiently inhibited by cyclosporin A and NIM811, a nonimmunosuppressive analog thereof, suggesting an overlapping dependency on cyclophilins for replication. However, analysis of a broader set of inhibitors revealed that, in contrast to HCV, HAV does not depend on cyclophilin A, but rather on adenosine-triphosphate-binding cassette transporters and FK506-binding proteins. Finally, silibinin, but not its modified intravenous formulation, efficiently inhibited HAV genome replication in vitro, suggesting oral silibinin as a potential therapeutic option for HAV infections. Conclusion: We established a cell-culture model enabling comparative studies on RNA replication of HAV and HCV in a homogenous cellular background with comparable replication efficiency. We thereby identified new host cell targets and potential treatment options for HAV and set the ground for future studies to unravel determinants of clearance and persistence. (HEPATOLOGY 2015;62:397-408) H epatitis C virus (HCV) and hepatitis A virus (HAV) are two hepatotropic positive-strand RNA viruses sharing a similar biology, but causing very different infection outcomes. Worldwide, approximately 160 million people are infected with HCV, a member of the Flaviviridae family. HCV establishes persistence in up to 70%-80% of cases, which often results in severe liver damage. This high rate of persistence is quite unusual for a positive-strand RNA virus, and the underlying reasons are only poorly understood. 1 In contrast, HAV, a picornavirus causing acute self-limited hepatitis, never leads to chronic infection. Counterintuitive to infection outcomes, HCV usually induces a high, long-lasting interferon

DDX60L Is an Interferon-Stimulated Gene Product Restricting Hepatitis C Virus Replication in Cell Culture

“…Interestingly, DDX60L had no impact on HAV replication, although HAV and HCV share similar biologies, the same tropism, and an identical sensitivity to IFNs in Huh-7 cells (34). Two recent large-scale screens of ISGs identified other genes that act specifically on HCV but not on either of the picornaviruses poliovirus or coxsackie B virus (15,17), stressing the need for a specific yet complementary set of genes required for effective defense against different viruses.…”

“…We furthermore assessed inhibition of HCV replication by IFN-␥ in naive Huh6 and Huh-7-Lunet using genotype 2a firefly luciferase (FLuc) reporter replicons (LucJFH1 [40]), since only JFH1-based replicons replicated robustly enough in Huh6 cells to allow transient-replication assays (34). Indeed, HCV replication was completely abolished in Huh-7 cells (IC 90 ϭ 0.18 ng/ml) ( Fig.…”

“…HCV and HAV reporter replicons used for transient-transfection assays were described before: pFKI389Luc-EI/NS3-3=_ET (LucCon1ET) (32), pFKI389Luc-EI/NS3-3=_JFH (LucJFH1) (33), or pT7-18f-Luc (LucHAV) (34,35). The plasmid used for generation of reporter virus JcR2A was described before (36), as was the monocistronic HCV reporter replicon pFK-I389Luc-ubi/NS3-3=/JFH1 (33).…”

The cyclophilin-inhibitor alisporivir stimulates antigen presentation thereby promoting antigen-specific CD8+ T cell activation

“…Alisporivir (Novartis, Basel, Switzerland) was dissolved to 20 mg/ml in DMSO. Detailed information on other inhibitors can be found in [13] and Supplementary materials and methods.…”

“…Cyp-inhibitors cover a broad range of cellular targets, including adenosine triphosphate binding cassette (ABC)-transporters, Na + -taurocholate cotransporting polypeptide (SLC10A1, NTCP) or calcineurin [24,25]. Therefore, we tested a panel of compounds with different inhibition profiles [13] for their effect on MHC-I surface expression, using non-cytotoxic concentrations ( Supplementary Fig. 5C).…”