Description
Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus responsible for outbreaks in Africa, Asia, Europe, and Caribbean islands, and has now become a major emerging threat on a global scale. The virus is transmitted to humans by Aedes aegypti and Aedes albopictus mosquitoes in urban areas. Symptoms of CHIKV infection include severe joint pain, photophobia, high fever, and petechial rash. CHIKV contains an error-prone RNA-dependent RNA polymerase, which leads to frequent mutations that can result in immune escape and resistance. Despite several CHIKV outbreaks, currently there are no approved drugs or vaccines available to counter this virus, which emphasize the need of novel tools for drug discovery initiatives. CHIKV depends on proteolytic processing carried out by viral and host proteases (PRs) at various stages of its life cycle. Unlike in most viruses, the CHIKV capsid (CP) is a serine PR, and thus is a potential antiviral target. The main objective of this thesis project is to engineer constructs expressing CP to be used in the development of a cell-based assay to serve as a high-throughput screening platform to identify CHIKV inhibitors. Our group has previously published an assay in cells that monitors the activity of the HIV-1 PR. We are now adapting the assay to monitor the activity of the CHIKV CP. The assay relies on three components: a Gal4/PR fusion, a reverse tetracycline transactivator (rtTA) off/on system for inducible activation in the presence of doxycycline (Dox), and the green fluorescent protein (GFP) under the Gal4 promoter. In this system, an active CP can cleave and disrupt the Gal4 fusion, which results in loss of GFP expression. On the contrary, when an active CP is inhibited or mutated, the Gal4 fusion remains intact, leading to GFP expression. To date, we have obtained retroviral plasmids with both wild type and mutants (I227K and S213A) CHIKV CP sequences. Following transfection into mammalian cells, we have confirmed the constructs’ expression by western blotting and assessed their localization by confocal microscopy. We expect to utilize the CP constructs to generate stable cell lines for the development of an assay to search for molecules against CHIKV.
