Hepatitis C virus (HCV) infection promotes autophagic degradation of viral replicative intermediates for sustaining replication and spread. The excessive activation of autophagy can induce cell death and terminate infection without proper regulation. A prior publication from this laboratory showed that an adaptive cellular response to HCV microbial stress inhibits autophagy through beclin 1 degradation. The mechanisms of how secretory and degradative autophagy are regulated during persistent HCV infection is unknown. This study was performed to understand the mechanisms of viral persistence in the absence of degradative autophagy, which is essential for virus survival. Using HCV infection of a CD63-green fluorescence protein (CD63-GFP), labeled stable transfected Huh-7.5 cell, we found that autophagy induction at the early stage of HCV infection increased the degradation of CD63-GFP that favored virus replication. However, the late-stage of persistent HCV infection showed impaired autophagic degradation, leading to the accumulation of CD63-GFP. We found that impaired autophagic degradation promoted the release of extracellular vesicles and exosomes. The impact of blocking the release of extracellular vesicles (EVs) on virus survival was investigated in persistently infected cells and sub-genomic replicon cells. Our study illustrates that blocking EV and exosome release severely suppresses virus replication without effecting host cell viability. Furthermore, we found that blocking EV release triggers interferon lambda 1 secretion. These findings suggest that the release of EVs is an innate immune escape mechanism that promotes persistent HCV infection. We propose that inhibition of extracellular vesicle release can be explored as a potential antiviral strategy for the treatment of HCV and other emerging RNA viruses.

Hepatitis C virus (HCV) infection compromises the natural defense mechanisms of the liver leading to a progressive end stage disease such as cirrhosis and hepatocellular carcinoma (HCC). The hepatic stress response generated due to viral replication in the endoplasmic reticulum (ER) undergoes a stepwise transition from adaptive to pro-survival signaling to improve host cell survival and liver disease progression. The minute details of hepatic pro-survival unfolded protein response (UPR) signaling that contribute to HCC development in cirrhosis are unknown. This study shows that the UPR sensor, the protein kinase RNA-like ER kinase (PERK), mediates the pro-survival signaling through nuclear factor erythroid 2-related factor 2 (NRF2)-mediated signal transducer and activator of transcription 3 (STAT3) activation in a persistent HCV infection model of Huh-7.5 liver cells. The NRF2-mediated STAT3 activation in persistently infected HCV cell culture model resulted in the decreased expression of hepatocyte nuclear factor 4 alpha (HNF4A), a major liver-specific transcription factor. The stress-induced inhibition of HNF4A expression resulted in a significant reduction of liver-specific microRNA-122 (miR-122) transcription. It was found that the reversal of hepatic adaptive pro-survival signaling and restoration of miR-122 level was more efficient by interferon (IFN)-based antiviral treatment than direct-acting antivirals (DAAs). To test whether miR-122 levels could be utilized as a biomarker of hepatic adaptive stress response in HCV infection, serum miR-122 level was measured among healthy controls, and chronic HCV patients with or without cirrhosis. Our data show that serum miR-122 expression level remained undetectable in most of the patients with cirrhosis (stage IV fibrosis), suggesting that the pro-survival UPR signaling increases the risk of HCC through STAT3-mediated suppression of miR-122. In conclusion, our data indicate that hepatic pro-survival UPR signaling suppresses the liver-specific HNF4A and its downstream target miR-122 in cirrhosis. These results provide an explanation as to why cirrhosis is a risk factor for the development of HCC in chronic HCV infection.

Chronic hepatitis C virus (HCV) infection causes hepatocellular carcinoma (HCC). Although HCV clearance has been improved by the advent of direct-acting antiviral agents (DAA), retrospective studies have shown that the risk of subsequent HCC, while considerably decreased compared with active HCV infection, persists after DAA regimens. However, either the mechanisms of how chronic HCV infection causes HCC or the factors responsible for HCC development after viral eradication in patients with DAA treatments remain elusive. We reported an in vitro model of chronic HCV infection and determined Wnt/β-catenin signaling activation due to the inhibition of GSK-3β activity via serine 9 phosphorylation (p-ser9-GSK-3β) leading to stable non-phosphorylated β-catenin. Immunohistochemical staining demonstrated the upregulation of both β-catenin and p-Ser9-GSK-3β in HCV-induced HCC tissues. Chronic HCV infection increased proliferation and colony-forming ability, but knockdown of β-catenin decreased proliferation and increased apoptosis. Unexpectedly, Wnt/β-catenin signaling remained activated in chronic HCV-infected cells after HCV eradication by DAA, but metformin reversed it through PKA/GSK-3β-mediated β-catenin degradation, inhibited colony-forming ability and proliferation, and increased apoptosis, suggesting that DAA therapy in combination with metformin may be a novel therapy to treat HCV-associated HCC where metformin suppresses Wnt/β-catenin signaling for HCV-infected patients.