Secretion of Hepatitis C Virus Replication Intermediates Reduces Activation of Toll-Like Receptor 3 in Hepatocytes.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infections most often result in chronic outcomes, although the virus constantly produces replication intermediates, in particular double-stranded RNA (dsRNA), representing potent inducers of innate immunity. We aimed to characterize the fate of HCV dsRNA in hepatocyte cultures to identify mechanisms contributing to viral persistence in presence of an active innate immune response. METHODS: We analyzed hepatocyte-based culture models for HCV for induction of innate immunity, secretion of virus positive- or negative-strand RNA, and viral replication using different quantification methods and microscopy techniques. Expression of pattern recognition receptors was reconstituted in hepatoma cells by lentiviral transduction. RESULTS: HCV-infected cells secrete substantial amounts of virus positive- and negative-strand RNAs in extracellular vesicles (EVs), toward the apical and basolateral domain of hepatocytes. Secretion of negative-strand RNA was independent from virus production, and viral RNA secreted in EVs contained higher relative amounts of negative-strands, indicating that mostly virus dsRNA is released. A substantial part of viral replication complexes and dsRNA was found in the endosomal compartment and multivesicular bodies, indicating that secretion of HCV replication intermediates is mediated by the exosomal pathway. Block of vesicle release in HCV-positive cells increased intracellular dsRNA levels and increased activation of toll-like receptor 3, inhibiting HCV replication. CONCLUSIONS: Using hepatocyte-based culture models for HCV, we found a portion of HCV dsRNA intermediates to be released from infected cells in EVs, which reduces activation of toll-like receptor 3. This represents a novel mechanism how HCV evades host immune responses, potentially contributing to viral persistence.

Interaction and Mutual Activation of Different Innate Immune Cells Is Necessary to Kill and Clear Hepatitis C Virus-Infected Cells.

Innate immune cells can sense hepatitis C virus (HCV)-infected cells and respond with anti-viral actions including secretion of interferons (IFNs). In previous studies, the response of individual innate immune cells against HCV was analyzed in detail. We hypothesized that interaction of multiple innate immune cells increases the magnitude of the immune response and eventually leads to clearance of HCV-infected cells. To investigate this, we co-cultured Huh-7 HCV subgenomic replicon (SGR) cells with peripheral blood mononuclear cells (PBMCs). We confirm secretion of IFNα by plasmacytoid dendritic cells (pDCs) and IFNγ by natural killer (NK) cells in the co-culture setup. Moreover, we observed that also monocytes contribute to the anti-viral response. Flow cytometry and ImageStream analysis demonstrated that monocytes take up material from HCV SGR cells in co-culture with PBMCs. Preceding the uptake, PBMCs caused apoptosis of HCV SGR cells by tumor necrosis factor-related apoptosis inducing ligand (TRAIL) expression on NK cells. We observed that only the interplay of monocytes, pDCs, and NK cells resulted in efficient clearance of HCV SGR cells, while these cell populations alone did not kill HCV SGR cells. Despite similar TRAIL receptor expression on Huh-7 control cells and HCV SGR cells, HCV activated PBMCs specifically killed HCV SGR cells and did not target Huh-7 control cells. Finally, we showed that HCV replicating cells per se are sensitive toward TRAIL-induced apoptosis. Our results highlight the importance of the interplay of different innate immune cells to initiate an efficient, rapid, and specific response against HCV-infected cells.