Description
As globalization and climate change continues to facilitate the spread of viruses across the planet, the need for new and effective antivirals is appropriately evident. Mosquito-borne arboviruses such as Zika, dengue, and chikungunya (CHIKV) viruses are becoming alarmingly widespread as invasive mosquito species are facilitating increased viral infections across the world. A member of the Togaviridae viral family, CHIKV is an RNA virus that causes high fever, rash, and most notably, debilitating muscle/joint pain. At this time, there is no current vaccine or commercially available antiviral against CHIKV. In 2020, over 60 countries worldwide have reported local transmission of CHIKV by Aedes genus mosquitoes. Furthermore, multiple strains of the virus have appeared with enhanced transmission capabilities. This fact only furthers the need for antiviral research and drug discovery as the threat of a CHIKV pandemic is significant due to the rising global temperature trend that will allow Aedes genus mosquitoes, serving as viral hosts, to spread to new latitudes and environments. This thesis presents a cell-based assay utilizing a previously developed Gal4 transcription factor reporter assay that monitors the protease-activity of the CHIKV Capsid (CHIKV CP) protein through the expression of Green Fluorescent Protein (GFP). This viral protein is a prime target for antiviral development as its enzymatic activity and structural roles are critical for viral replication. This report analyzes a set of engineered CHIKV CP/Gal4 assay retroviral DNA constructs to observe the wild-type protease and understand the importance of specific amino acids on CHIKV CP protease activity, and to serve as controls for future antiviral drug screens. This CHIKV CP/Gal4 reporter assay was developed for potential high-throughput screens with robust, clonal mammalian cell-lines expressing the assay. By using flow-cytometry, GFP expression can be used to assess protease inactivity, thus generating a cell-based platform for drug discovery.
