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Glycogen Synthase Kinase 3β Enhances Hepatitis C Virus Replication by Supporting miR-122.

ABSTRACT: Hepatitis C virus (HCV) infection is associated with alterations in host lipid and insulin signaling cascades, which are partially explained by a dependence of the HCV life cycle on key molecules in these metabolic pathways. Yet, little is known on the role in the HCV life cycle of glycogen synthase kinase 3 (GSK3), one of the most important kinases in cellular metabolism. Therefore, the impact of GSK3 on the HCV life cycle was assessed in human hepatoma cell lines harboring subgenomic genotype 1b and 2a replicons or producing cell culture-derived HCV genotype 2a by exposure to synthetic GSK3 inhibitors, GSK3 gene silencing, overexpression of GSK3 constructs and immunofluorescence analyses. In addition, the role of GSK3 in hepatitis E virus (HEV) replication was investigated to assess virus specificity of the observed findings. We found that both inhibition of GSK3 function by synthetic inhibitors as well as silencing of GSK3β gene expression resulted in a decrease of HCV replication and infectious particle production, whereas silencing of the GSK3α isoform had no relevant effect on the HCV life cycle. Conversely, overexpression of GSK3β resulted in enhanced HCV replication. In contrast, GSK3β had no effect on replication of subgenomic HEV replicon. The pro-viral effect of GSK3β on HCV replication was mediated by supporting expression of microRNA-122 (miR-122), a micro-RNA which is mandatory for wild-type HCV replication, as GSK3 inhibitors suppressed miR-122 levels and as inhibitors of GSK3 had no antiviral effect on a miR-122-independent HCV mutant. In conclusion, we have identified GSK3β is a novel host factor supporting HCV replication by maintaining high levels of hepatic miR-122 expression.

SUBMITTER: Saleh M

PROVIDER: S-EPMC6278592 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The small GTPase Rab27a has been shown to control membrane trafficking and microvesicle transport pathways, in particular the secretion of exosomes. In the liver, high expression of Rab27a correlates with the development of hepatocellular carcinoma. We discovered that low abundance of Rab27a resulted in decreased hepatitis C virus (HCV) RNA and protein abundances in virus-infected cells. Curiously, both cell-associated and extracellular virus yield decreased in Rab27a depleted cells, suggesting that reduced exosome secretion did not cause the observed effect. Instead, Rab27a enhanced viral RNA replication by a mechanism that involves the liver-specific microRNA miR-122. Rab27a surrounded lipid droplets and was enriched in membrane fractions that harbor viral replication proteins, suggesting a supporting role for Rab27a in viral gene expression. Curiously, Rab27a depletion decreased the abundance of miR-122, whereas overexpression of miR-122 in Rab27a-depleted cells rescued HCV RNA abundance. Because intracellular HCV RNA abundance is enhanced by the binding of two miR-122 molecules to the extreme 5' end of the HCV RNA genome, the diminished amounts of miR-122 in Rab27a-depleted cells could have caused destabilization of HCV RNA. However, the abundance of HCV RNA carrying mutations on both miR-122-binding sites and whose stability was supported by ectopically expressed miR-122 mimetics with compensatory mutations also decreased in Rab27a-depleted cells. This result indicates that the effect of Rab27a depletion on HCV RNA abundance does not depend on the formation of 5' terminal HCV/miR-122 RNA complexes, but that miR-122 has a Rab27a-dependent function in the HCV lifecycle, likely the downregulation of a cellular inhibitor of HCV gene expression. These findings suggest that the absence of miR-122 results in a vulnerability not only to exoribonucleases that attack the viral genome, but also to upregulation of one more cellular factor that inhibit viral gene expression.

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Glycogen Synthase Kinase 3β Enhances Hepatitis C Virus Replication by Supporting miR-122.

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