RNA binding by human Norovirus 3C-like proteases inhibits proteaseactivity

A highly active, fluorescence-based, in vitro assay for human Norovirus protease from genogroup I and II viruses was optimized utilizing as little as 0.25 μM enzyme, pH 7.6, and substrate:enzyme of 50–100. Activity in Tris–HCl or sodium phosphate buffers was 2-fold less than HEPES, and 2-fold lower for buffer concentrations over 10 mM. Protease activity at pH 7.6 was 73% (GI) or 63% (GII) of activity at the optimal pH 9.0. Sodium inhibited activity 2–3 fold, while potassium, calcium, magnesium, and manganese inhibited 5–10 fold. Differences in efficiency due to pH, buffer, and cations were due to changes in kcat and not Km. Norovirus protease bound short RNAs representing the 3′ or 5′ ends of the virus, inhibiting protease activity (IC50 3–5 μM) in a non-competitive manner. Previous reports indicated participation of the protease in the Norovirus replicase complex. The current studies provide initial support for a defined role for the viral protease in Norovirus replication.

► A systematic characterization of component requirements for NoV protease is presented.
► Cations, pH, and buffers affect catalytic activity (Kcat), not binding affinity (Km).
► Enzymatic efficiency of proteases from GI and GII viruses was identical.
► The first demonstration of RNA binding by a human Norovirus protease is presented.
► RNA binding inhibits Norovirus protease activity in a non-competitive manner.