INTERACTION STUDIES OF NS2B-NS3 PROTEASE COMPLEX OF ZIKA VIRUS USING NMR: SEARCHING FOR NEW ANTIVIRAL COMPOUNDS

Flaviviruses are pathogens with a single-stranded RNA as their nucleic acid. They are transmitted by arthropods, specifically mosquitoes and ticks. Therefore, they can be defined as an arboviruses and are responsible for diseases relevant to global health, such as yellow fever (YFV), dengue (DENV), and zika (ZIKV). The molecular target of this work is the ZIKV NS2B-NS3 complex, composed of two non-structural proteins that together act in the cleavage of the immature viral polyprotein, essential in the viral replication cycle. In this process, NS2B acts as a cofactor for the NS3 protease, activating it and enabling its catalytic activity. Consequently, this mechanism is crucial for the viral life cycle and presents a promising target for the development of novel antiviral drugs. This research aims to study the molecular basis of these interactions to enhance the screening and selection of fragments by Nuclear Magnetic Resonance (NMR). For this, the complex was cloned into a pETDuet plasmid and expressed in E. coli Rosetta cells. The optimal expression conditions were achieved with 1mM IPTG at 37ºC for 16 hours. Following expression, the complex was purified using a combination of nickel affinity, ion exchange, and size exclusion chromatography (SEC). These steps were followed and analyzed using 15% SDS-PAGE. In SEC, the complex was purified and observed in oligomeric states based on the elution volume. The 15N-TROSY-HSQC NMR spectrum of the complex was performed on a Bruker 900 mHz spectrometer at 298K and exhibited good chemical shift dispersion. However, the spectrum showed a limited number of peaks and broad signals, suggesting a higher molecular weight, corroborating the SEC results. In addition, an enzymatic activity assay was performed using fluorimetry to verify the activity of the complex, which was found to be active and functional even in its oligomeric state. It is concluded that the stabilization of the NS2B-NS3 complex will be essential for fragment-based screening studies by NMR and for the development of antiviral drugs.