Structural and inhibitor studies of norovirus 3C-like proteases.
Noroviruses have a single-stranded, positive sense 7-8kb RNA genome, which encodes a polyprotein precursor processed by a virus-encoded 3C-like cysteine protease (3CLpro) to generate mature non-structural proteins. Because processing of the polyprotein is essential for virus replication, norovirus 3CLpro has been targeted for the discovery of anti-norovirus small molecule therapeutics. Thus, we performed functional, structural and inhibition studies of norovirus 3CLpro with fluorescence resonance energy transfer (FRET) assay, X-ray crystallography, and NMR spectroscopy with a synthetic protease inhibitor. Three 3CLpro from Norwalk virus (NV, genogroup I), MD145 (genogroup II) and murine norovirus-1 (MNV-1, genogroup V) were optimized for a FRET assay, and compared for the inhibitory activities of a synthetic protease inhibitor (GC376). The apo 3D structures of NV 3CLpro determined with X-ray crystallography and NMR spectroscopy were further analyzed. In addition, the binding mode of NV 3CLpro-GC376 was compared with X-ray crystallography and NMR spectroscopy. The results of this report provide insight into the interaction of NV 3CLpro with substrate/inhibitor for better understanding of the enzyme and antiviral drug development.
Department of Biochemistry, Kansas State University, 141 Chalmers Hall, Manhattan, KS 66506, USA.
Daisuke
D
Takahashi
Yunjeong
Y
Kim
Scott
S
Lovell
Om
O
Prakash
William C
WC
Groutas
Kyeong-Ok
KO
Chang
D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D029322:Norovirus / Q000201:enzymology*; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D016415:Sequence Alignment; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry*
D011480:Protease Inhibitors; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases
3C-like protease; 3D structures; Inhibitors; Norovirus