Abstract
Flavivirus infection of cells induces massive rearrangements of the endoplasmic reticulum (ER) membrane to form viral replication organelles (ROs) which segregates viral RNA replication intermediates from the cytoplasmic RNA sensors. Among other viral nonstructural (NS) proteins, available evidence suggests for a prominent role of NS4B, an ER membrane protein with multiple transmembrane domains, in the formation of ROs and the evasion of the innate immune response. We previously reported a benzodiazepine compound, BDAA, which specifically inhibited yellow fever virus (YFV) replication in cultured cells and in vivo in hamsters, with resistant mutation mapped to P219 of NS4B protein. In the following mechanistic studies, we found that BDAA specifically enhances YFV induced inflammatory cytokine response in association with the induction of dramatic structural alteration of ROs and exposure of double-stranded RNA (dsRNA) in virus-infected cells. Interestingly, the BDAA-enhanced cytokine response in YFV-infected cells is attenuated in RIG-I or MAD5 knockout cells and completely abolished in MAVS knockout cells. However, BDAA inhibited YFV replication at a similar extent in the parent cells and cells deficient of RIG-I, MDA5 or MAVS. These results thus provided multiple lines of biological evidence to support a model that BDAA interaction with NS4B may impair the integrity of YFV ROs, which not only inhibits viral RNA replication, but also promotes the release of viral RNA from ROs, which consequentially activates RIG-I and MDA5. Although the innate immune enhancement activity of BDAA is not required for its antiviral activity in cultured cells, its dual antiviral mechanism is unique among all the reported antiviral agents thus far and warrants further investigation in animal models in future.
Highlights
Yellow fever (YF), a disease caused by infection of the yellow fever virus (YFV), was once considered as the most dangerous infectious disease with high fatality rate in the beginning of the 19th century
The work reported further demonstrates that BDAA interaction with the YFV nonstructural 4B (NS4B) protein may impair the integrity of viral RNA replication organelles, which inhibits viral RNA replication, and results in the leakage of viral RNA into the cytoplasm to activate RIG-I-like RNA receptors and enhances the innate antiviral immune response
293/IFNβLuc is a HEK293-derived reporter cell line that expresses a firefly luciferase under the control of a human IFN-β promoter. Infection of this cell line with RNA viruses, including dengue virus, YFV, Sendai virus (SeV) and encephalomyocarditis virus (EMCV), activates the reporter gene expression that quantitatively correlates with the levels of virus replication and progeny virus production, and can be inhibited in a dose-dependent manner by known antiviral compounds [16]
Summary
Yellow fever (YF), a disease caused by infection of the yellow fever virus (YFV), was once considered as the most dangerous infectious disease with high fatality rate in the beginning of the 19th century. In a study performed during the 2018 outbreak of YF in Brazil, a 36% fatality rate was observed, and the high viral load was found to be a key determinant of disease severity, suggesting that an effective antiviral drug against YFV can be anticipated to significantly improve the clinical outcome of YF [5]. Antiviral therapy may help control the outbreak by administration to potentially exposed individuals as a prophylaxis since the protection from vaccine will take weeks to become effective [6,7,8]. This approach is important when YF vaccine is in short supply during unexpected outbreaks. The YF antiviral therapeutic candidates are currently limited to repurposed nucleoside analogs originally
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