Author: Thomas Burton
Burton, Thomas, 2025 Molecular Dissection of the Dengue Virus NS1 Protein, Flinders University, College of Medicine and Public Health
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Dengue is the most common mosquito-borne viral disease and is responsible for a major public health burden in tropical and subtropical regions around the world. The dengue virus (DENV) non-structural protein 1 (NS1) has been shown to play roles in pathogenicity, immune system evasion, viral RNA replication and virus production. To date, no antiviral therapeutic for dengue-related illness has been approved for use. Current research on therapeutic candidates that target of NS1 is limited to antibodies, peptides and a heparan sulfate mimetic, with no small molecule inhibitors described. We performed two in vitro high-throughput screening approaches to identify antiviral compounds that target NS1: a protein complementation assay to identify inhibitors of NS1 dimerisation/hexamerisation and; a Nanoluciferase-based thermal shift assay to identify NS1-binding compounds. We also performed an in silico high-throughput drug-like compound screen to identify potential ligands of NS1, focussing on ligands which may bind at the dimerisation interface of NS1.
While we are yet to unambiguously identified direct inhibitors of NS1 function(s), the thermal shift assay screen revealed a group of structurally related analogues with antiviral properties. A Nanoluciferase-based thermal shift assay in conjunction with a Nanoluciferase-tagged dengue reporter virus (DENV2-NS1-NLuc) was used in a high-throughput compound screen of 3,378 drug-like compounds. Validation studies revealed a collection of structurally related compounds which inhibit DENV infection in a hepatoma cell culture model. Following testing of 37 structurally related analogues, we identified a lead compound (PubChem CID: 50839998) which had minimal impact on viral RNA replication and cell viability but inhibited infectious particle production at low micromolar concentrations. Examination of the impact of this compound on viral protein localization by confocal microscopy revealed dose-dependent reductions in the detection of mature Envelope (E) protein, consistent with the observed inhibition of infectious virus production. Further investigation into the mechanism of action of this compound is warranted to determine its exact molecular target(s), while testing of a wider range of structural analogues may enable identification of related compounds with greater efficacy and lower cytotoxicity.
To identify inhibitors of NS1 dimerisation, the NanoBiT® Protein:Protein Interaction System was used. This NanoBiT or ‘split Nanoluciferase’ system involves the NanoLuc subunits SmBiT and LgBiT. Each subunit was fused individually to NS1 in mammalian expression vectors, and in a high-throughput screen, 3,378 drug-like compounds were screened, with luminescence as a readout for NS1 dimerisation levels. While the screen identified three hits, subsequent experiments showed no clear antiviral effects, and the reduction of NS1 dimerisation upon treatment with these compounds could not be verified.
An in silico high-throughput drug-like compound screen revealed two binding pockets at the dimerisation interface of NS1. Approximately 7.8 million compounds from the ZINC20 chemical database were tested computationally for binding at these sites and 24 of the top 60 hits were analysed for antiviral efficacy and cytotoxicity via live cell imaging using an mScarlet-tagged dengue reporter virus. The top hit, Compound V2.3, was further analysed. This compound showed high nanomolar inhibition of infectious particle production, with minimal impact on viral RNA replication and low cytotoxicity. Despite being predicted to bind at the dimerisation interface of NS1, Compound V2.3 treatment did not appear to affect NS1 dimerisation, and the binding of Compound V2.3 to NS1 has not yet been confirmed.
Previous studies have identified several key residues of NS1 that are required for its roles in RNA replication and virus production, through analysis of specific hydrophobic regions, conserved residues across multiple orthoflavivirus species and cysteine residues suspected to enable disulphide bonding. We created a DENV mutant library featuring mutants across the envelope (E), precursor Membrane (prM) and NS1 genes. We applied specific selective pressures to the library, including passaging through Huh7.5 cells (human cell line) and C6/36 cells (mosquito cell lines), and preparations are being made for the passaging of the library through Aedes mosquitoes, both infected and uninfected with the intracellular bacterium Wolbachia. This will allow for identification of DENV mutants which confer resistance to Wolbachia-based suppression of DENV, while also identifying E, prM and NS1 residues that are critical to various DENV lifecycle stages and tropism factors. Additionally, we have prepared a library of DENV mutants featuring mutants across NS1 only, as well as a library of mutants covering NS1 in the DENV subgenomic replicon, which will allow for demarcation of residues required for viral RNA replication from those required for infectious particle production.
Keywords: Virology, Dengue, Virus, Antivirals, Mutagenesis, Wolbachia
Subject: Medicine thesis
Thesis type: Doctor of Philosophy
Completed: 2025
School: College of Medicine and Public Health
Supervisor: Dr Nicholas Eyre