Expression and inhibitors screening of St. Louis encephalitis virus NS2B-NS3 protease

Abstract:

[Objective] St. Louis encephalitis virus (SLEV) is a single-stranded and positive-sense RNA virus, belonging to the Flaviviridae family. Non-structural protein 3 (NS3) encoded by flaviviruses plays an important role in virus replication and polyprotein precursor modification. NS2B is an essential element that acts as a co-factor to enhance the activity of NS3. Therefore, the NS2B-NS3 protease is considered as a crucial target for anti-flavivirus drug development. In the current study, we constructed recombinant plasmid of SLEV NS2B-NS3 protease, and expressed and purified the protein to screen inhibitors. [Methods] We obtained the NS2B-NS3 recombinant gene by PCR and subcloned the prokaryotic recombinant expression plasmid. The NS2B-NS3 protease was expressed in soluble form induced by isopropyl β-D-1-thiogalactopyranoside in Escherichia coli BL21(DE3). The fusion protein was purified with Ni⁺-NTA affinity column. Then the serine protease activity was determined by a fluorescence resonance energy transfer assay and the screening platform for inhibitors was constructed. [Results] The purity NS2B-NS3 protease was up to 95%. After screening for 700 old drugs, we found that procyanidine efficiently inhibited the enzymatic activity of the NS2B-NS3 protease. [Conclusion] Our current study provided a convenient and high-throughput method for screening of SLEV NS2B-NS3 protease inhibitors. Porcyanidine has been found to inhibit the activity of SLEV NS2B-NS3 protease for the first time, and might become a potential drug to treat SLEV infection.