Dengue virus (DENV) is a Flavivirus that is primarily transmitted by Aedes aegypti mosquitoes and infects an estimated 390 million people worldwide, causing approximately 25,000 deaths per year. While most infections are asymptomatic or cause mild fever, some individuals develop severe dengue; a life-threatening disease characterised by vascular leakage due to endothelial dysfunction.
The DENV protein NS4B is a highly hydrophobic, non-structural transmembrane protein that is essential for viral RNA replication and immune evasion. NS4B is located in the virus-modified endoplasmic reticulum and has been shown to physically interact with other viral non-structural proteins such as NS1 and NS3, with these interactions being essential for DENV replication.
A recent study has described a highly potent DENV inhibitor (JNJ-A07) that blocks the interaction between NS3 and NS4B. While this inhibitor has a great therapeutic potential, it is unknown whether it affects interactions other than the disruption of the NS3:NS4B complex. It is important to determine the exact mechanism(s) of action of this drug and any unanticipated ‘off-target’ effects.
Here, we have investigated the efficacy of JNJ-A07 against DENV2 in Huh 7.5 hepatoma cells in time-of-addition studies using live cell imaging and a unique reporter virus that encodes a fluorescent protein insertion within NS1. These studies have revealed the dynamics of the antiviral effects of JNJ-A07 at high temporal resolution and have confirmed that its antiviral activity is greatest when it is applied during the early stages of infection. Furthermore, the inhibitor decreased the colocalization between NS4B and NS1, dsRNA and the viral Envelope protein. These effects on viral protein colocalization are currently being explored using a non-structural viral protein expression system that accurately reflects normal viral protein localization and membrane rearrangements. Proteomics studies will then seek to identify whether JNJ-A07 disrupts other NS4B interactions with viral and host cell factors. It is hoped that an improved understanding of the mechanism of action of JNJ-A07 will inform mechanisms of antiviral drug resistance and facilitate the development of analogous and complementary antiviral therapies that build upon the great promise of this potent, clinically relevant, and unique class of DENV inhibitors.