STRUCTURAL STUDIES OF ZIKA VIRUS NS3 PROTEASE AND NS2B MEMBRANE PROTEIN: EXPRESSION, PURIFICATION AND NANODISCS ASSEMBLY

In 2015, Brazil recorded several cases of infections caused by Zika virus (ZIKV). The infection reached epidemic proportions in South America and quickly spread to other regions. ZIKV infection can cause severe neurological damage, such as Congenital Zika Syndrome and Guillain-Barré Syndrome. The NS2B membrane protein is the cofactor of the NS3 protease (NS3pro). Thus, the NS2B-NS3 viral complex is essential for virus replication and represents an attractive drug target. Membrane proteins are unstable in solution; therefore, a membrane mimetic such as nanodiscs is interesting for dynamic and structural investigations. This work aims to study the structure and dynamics of NS3pro and NS2B assembled into nanodiscs by Nuclear Magnetic Resonance (NMR) in solution. Nanodiscs membrane scaffold proteins (MSPs), NS2B, and NS3pro were cloned employing pET-28a vector and transformed into E. coli BL21(DE3) strain. Expression tests were performed in Luria Bertani medium under different parameters to determine the optimal expression conditions for all the proteins. MSPs were expressed and lysed, and the purification protocol is in progress. NS3pro was also expressed in a minimal medium (M9) isotopically labeled with ¹⁵N. NS3pro was purified by nickel affinity chromatography (IMAC) and size-exclusion chromatography (SEC). The sample was subjected to structural studies by NMR on a Bruker 600 MHz spectrometer. NS3pro optimal expression conditions were obtained at 37°C for 3 hours or 18°C for 16 hours. After cell lysis, the NS3pro was extracted from inclusion bodies with SDS 1%. 1D ¹H and 2D [¹H,¹⁵N]-HSQC spectra of NS3pro showed low and line broadening signals. The oligomers were observed by SEC, and the results showed the self-cleavage of NS3pro, requiring the use of SDS for protein stabilization. Activity assays are ongoing, and we are producing different MSP constructions to produce nanodiscs with low diameters to study the structure of NS2B and its interaction with NS3pro. In conclusion, this work will be fundamental for developing and optimizing lead compounds with antiviral activity.