Modulation of lipid metabolism by thapsigarin inhibits hepatitis C virus infection by multiple mechanisms

Abstract

Thapsigargin (Tg), an inducer of endoplasmic reticulum stress and the unfolded protein response (UPR), has broad-spectrum antiviral activity, although the underlying mechanisms remain unclear. Here, we characterized its antiviral mechanism(s) using hepatitis C virus (HCV) as a model. Pre-treatment with Tg partially inhibited HCV RNA replication, but more strongly reduced extracellular viral titer and RNA, suggesting a block to later stages of infection. Its antiviral effects against HCV infection are likely UPR-independent, since treatment with tunicamycin, which activates the UPR by a different mechanism, did not exert the same antiviral effect. Given the importance of lipid droplets (LDs) and lipid metabolism in mediating HCV assembly and egress, we examined Tg-mediated effects on lipid homeostasis. Tg treatment upregulated the expression of lipid synthesis genes, including FASN and DGAT1/2, and led to the accumulation of enlarged LDs. Tg also induced expression of CIDE-C, a mediator of LD fusion. Silencing CIDEC expression impaired Tg-induced LD enlargement and rescued viral RNA replication, but not extracellular titer, from Tg inhibition, demonstrating that Tg-mediated LD remodeling contributes to replication defects without significantly affecting assembly or egress. Intracellular viral titers were unchanged in Tg-treated cells, indicating intact virion assembly but a defect in secretion. Consistently, Tg treatment reduced both apolipoprotein B and E secretion, but not that of Gaussia luciferase, suggesting that specific disruption of the very low-density lipoprotein secretion pathway by Tg could impair efficient HCV egress. Together, our findings reveal that modulation of lipid homeostasis by Tg inhibits HCV RNA replication and egress by distinct mechanisms. This work has antiviral implications for other viruses that rely on lipid metabolism during infection.