title: RNA binding by human Norovirus 3C-like proteases inhibits protease activity.
abstract: A highly active, fluorescence-based, in vitro assay for human Norovirus protease from genogroup I and II viruses was optimized utilizing as little as 0.25μM 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 10mM. 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 3-5μM) 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.
authors: Georgetown University Medical Center, Department of Microbiology and Immunology, 3900 Reservoir Rd., N.W., Med-Dent Building, RM SW319, Washington, DC 20057, USA.
authors: Prasanth
authors: P
authors: Viswanathan
authors: Jared
authors: J
authors: May
authors: Sunghae
authors: S
authors: Uhm
authors: Changsuek
authors: C
authors: Yon
authors: Brent
authors: B
authors: Korba
mesh_terms: D000086042:3C Viral Proteases; D002021:Buffers; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D020536:Enzyme Activators / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000378:metabolism; D029322:Norovirus / Q000201:enzymology*; D011485:Protein Binding; D012367:RNA, Viral / Q000378:metabolism*; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*
chemical_list: D002021:Buffers; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D008670:Metals; D012367:RNA, Viral; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases