The mammalian SKI complex is a broad-spectrum antiviral drug target that upregulates cellular cholesterol to inhibit viral replication.

There is a need for the development of broad-spectrum antiviral compounds that can act as first-line therapeutic countermeasures to emerging viral infections. Host-directed approaches present a promising avenue of development and carry the benefit of mitigating risks of viral escape mutants. We have previously found the SKI (super killer) complex to be a broad-spectrum, host-target with our lead compound ("UMB18") showing activity against influenza A virus, coronaviruses, and filoviruses. The SKI complex is a cytosolic RNA helicase, and we previously found that UMB18 inhibited viral RNA production but did not further define the mechanism. Here, we demonstrate that UMB18 directly binds to SKIC8 of the SKI complex, and transcriptomic analysis of UMB18-treated A549 cells revealed an upregulation of genes in the mevalonate pathway, which drives cholesterol synthesis. Further investigation validated the genetic upregulation and confirmed an increase in total cellular cholesterol. This upregulation was dependent on the sterol regulatory element-binding proteins (SREBPs) and their regulator SCAP, the major regulators for cholesterol and fatty acid synthesis. Depletion of the SREBPs or SCAP with siRNA, or extraction of cholesterol with methyl β-cyclodextrin, attenuated UMB18 antiviral activity, emphasizing the role of increased cholesterol synthesis in this mechanism of action. Our findings further define the antiviral mechanism of a developmental host-directed therapeutic approach with broad applicability against emerging viral pathogens. IMPORTANCE: The COVID-19 pandemic has underscored the need for effective countermeasures to emerging pathogens. Our research builds upon our published data on a novel antiviral compound termed UMB18. We have found UMB18 capable of inhibiting replication of influenza A virus, coronaviruses, and the filoviruses Marburg and Ebola virus, but did not fully define a mechanism of action. Here, we demonstrate that UMB18 exerts antiviral activity by modulating cellular cholesterol levels. By targeting the SKI complex, UMB18 triggers an increase in endogenous cellular cholesterol, which disrupts the fine balance that viruses rely on for efficient infection. We demonstrate that this mechanism inhibits replication of SARS-CoV-2, revealing a previously undescribed host-directed strategy for antiviral intervention. These findings highlight UMB18's potential as a broad-spectrum antiviral agent and pave the way for further research into its mechanism and therapeutic applications, offering a promising avenue for development of antiviral countermeasures to current, novel, and emerging pathogens.