Modulation of lipid metabolism by thapsigargin 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. AUTHOR’S SUMMARY Thapsigargin (Tg) is emerging as a potent broad-spectrum host-targeted antiviral, but the specific mechanisms by which it inhibits unrelated viral infections are unclear and its antiviral activity against hepatitis C virus has not been reported. We determined that Tg causes significant changes in host lipid metabolism, resulting in enlarged lipid droplets (LDs) that persist during HCV infection. We demonstrate that Tg-induced enlarged LDs in part contribute to the restriction of HCV RNA replication. However, the most compelling antiviral effect is on the release of infectious virions. We report no differences in intracellular titer, but a significant reduction in extracellular titer, concomitant with a decrease in apolipoprotein (Apo) B and ApoE secretion, which are required for efficient egress of HCV. Together, our data support modulation of lipid metabolism and cellular secretory function by Tg as a previously unidentified antiviral mechanism for Tg and highlight its potential use against other viruses.