Identification of Chikungunya virus nucleocapsid core assembly modulators

Abstract

The alphavirus Chikungunya virus is transmitted to humans via infected mosquitos. Most infected humans experience symptoms which can range from short-term fatigue and fever to debilitating arthritis that can last for months or years. Some patients relapse and experience symptoms months or years after the initial bout of disease. The capsid protein of Chikungunya virus forms a shell around the viral RNA genome; this structure is called the nucleocapsid core.The core protects the genome during virus transmission and with the correct environmental trigger, this proteinaceous shell dissociates and releases the viral genome to initiate infection. We hypothesized that targeting compounds to interfere with the nucleocapsid core's function would constrain virus spread either by inhibiting the release of viral genomes during entry or by reducing the number of infectious virus particles assembled. We implemented a high throughput, in vitro, FRET-based assay to monitor nucleic acid packaging by purified Chikungunya capsid protein as a proxy for nucleocapsid core assembly and disassembly. We screened 10,000 compounds and found 45 that substantially modulated the assembly of core-like particles. A subset of compounds was selected to study their effects in virus-infected vertebrate cells. Our results show that four compounds inhibit infectious virus production by at least 90% in a dose-dependent manner. The most promising inhibitor was tested and found to reduce the amount of nucleocapsid cores inside the cell during Chikungunya virus infection. These compounds could be the foundation for anti-viral therapeutics.Keywords (6 max): Chikungunya virus; antiviral compounds; nucleocapsid core; fluorescence quenching-based high throughput screen

Specific Recognition of a Stem-Loop RNA Structure by the Alphavirus Capsid Protein

“…The SFV and RRV Cp were expressed in Rosetta2 bacteria as previously described [ 20 ], and purified by affinity chromatography using Strep-Tactin sepharose (IBA Lifesciences, Goettingen, Germany). The CHIKV Cp was expressed by inducing Rosetta2 cells at an OD 600 ~0.5 for 4 h at 37 °C [ 23 ], and purified as for the RRV and SFV Cps except using twice the starting material. 1 µg of each protein was subjected to SDS-PAGE followed by Coomassie staining to assess protein integrity and purity.…”