Structural Basis of Substrate Specificity and Protease Inhibition in Norwalk Virus

Abstract

cNorwalk virus (NV), the prototype human calicivirus, is the leading cause of nonbacterial acute gastroenteritis. The NV protease cleaves the polyprotein encoded by open reading frame 1 of the viral genome at five nonhomologous sites, releasing six nonstructural proteins that are essential for viral replication. The structural details of how NV protease recognizes multiple substrates are unclear. In our X-ray structure of an NV protease construct, we observed that the C-terminal tail, representing the native substrate positions P5 to P1, is inserted into the active site cleft of the neighboring protease molecule, providing atomic details of how NV protease recognizes a substrate. The crystallographic structure of NV protease with the C-terminal tail redesigned to mimic P4 to P1 of another substrate site provided further structural details on how the active site accommodates sequence variations in the substrates. Based on these structural analyses, substrate-based aldehyde inhibitors were synthesized and screened for inhibition potency. Crystallographic structures of the protease in complex with each of the three most potent inhibitors were determined. These structures showed concerted conformational changes in the S4 and S2 pockets of the protease to accommodate variations in the P4 and P2 residues of the substrate/inhibitor, which could be a mechanism for how the NV protease recognizes multiple sites in the polyprotein with differential affinities during virus replication. These structures further indicate that the mechanism of inhibition by these inhibitors involves covalent bond formation with the side chain of the conserved cysteine in the active site by nucleophilic addition, and such substrate-based aldehydes could be effective protease inhibitors.

Structure determination of Murine Norovirus NS6 proteases with C-terminal extensions designed to probe protease-substrate interactions

“…distinct from that observed in crystals we previously obtained with the mature NS6 protein 94! (Leen, Baeza & Curry, 2012;Muhaxhiri et al, 2013). Unfortunately, in no case did the cleaved by MNV NS6.…”

“…analogues bound to human norovirus NS6 structures (Hussey et al, 2011;Kim et al, 2012; 70! Muhaxhiri et al, 2013) revealed details of the specific contacts made by the P5-P1 residues 71! of peptidyl products of the protease.…”

“…(Chiba virus, Murine norovirus, Norwalk virus, Southampton norovirus) (Nakamura et al,53! 2005; Zeitler, Estes & Venkataram Prasad, 2006;Hussey et al, 2011;Kim et al, 2012;Leen, 54! Baeza & Curry, 2012;Muhaxhiri et al, 2013). The solution structure and dynamics of 55!…”

“…An almost identical packing arrangement was obtained in a 67! different space-group with crystals of Norwalk virus NS6 protease (Muhaxhiri et al, 2013).…”

“…Whereas ORF2, ORF3 and ORF4 each encode single proteins, ORF1 codes for a single large 44! polyprotein (~ 190 kDa) that is processed by the virally-encoded protease at five specific sites 45! to release the six non-structural proteins (NS1/2 -NS7) -and an array of functional 46! precursors -that are required for virus replication (Belliot et al, 2003;Sosnovtsev et al, 47! 2006; Muhaxhiri et al, 2013). The viral NS6 protease present within the C-terminal half of 48!…”