Multiple enzyme activities of flavivirus proteins

Area of Science:

• Virology
• Molecular Biology
• Drug Discovery

Background:

• Dengue viruses (DENV) possess (+) sense RNA genomes encoding a polyprotein precursor.
• Viral proteins NS2B, NS3, and NS5 are crucial for DENV replication, with NS2B/NS3 forming a serine protease complex.

Purpose of the Study:

• To identify small molecules inhibiting DENV protease activity.
• To elucidate the functions of DENV NS3 and NS5 proteins in viral replication.
• To investigate the role of the conserved 184RKRK motif in DENV-2 NS3.

Main Methods:

• High-throughput screening of a chemical library using a protease assay.
• In vitro assays for RNA-dependent RNA polymerase activity using infected cell lysates or purified NS5.
• Cell-based DENV-2 replicon system with a luciferase reporter.

Main Results:

• Over 200 compounds demonstrated ≥50% inhibition in the protease assay.
• DENV NS3 exhibits RNA-stimulated NTPase, RNA helicase, and 5'-RNA triphosphatase activities.
• NS3 enzyme activities are enhanced by NS5 interaction, and the 184RKRK motif is vital for RNA binding and enzyme modulation.

Conclusions:

• Small molecules capable of inhibiting DENV protease activity were identified.
• The study highlights the enzymatic functions of NS3 and its interaction with NS5.
• The 184RKRK motif is critical for DENV RNA binding and replication, offering potential therapeutic targets.