Enzyme kinetics of the human norovirus protease control virus polyprotein processing order

•This is the first presentation of the enzyme kinetics of a human norovirus ProPol protease activity.•Viral protease enzymatic efficiencies at NoV ORF1 cleavage sites control its processing order.•Differences in enzyme efficiency were primarily due to differences in enzyme turnover (kcat).•ProPol demonstrated equivalent or superior cleavage efficiency to Pro at all ORF1 cleavage sites.

The human norovirus (NoV) polyprotein is cleaved into mature non-structural proteins by both mature NoV protease (Pro, NS6) and its un-cleaved precursor (ProPol, NS6-7). Processing order is well-established with 'early' and 'late' cleavages, but the governing enzymatic mechanisms are unknown. Enzyme kinetics of a GII Pro and ProPol were analyzed using synthetic peptides representing the five natural polyprotein cleavage sites. The relative efficiency of cleavage of the individual peptides was consistent with established polyprotein processing order, and primarily correlated with enzyme turnover (kcat). Enzymatic efficiencies (kcat/Km) of ProPol at all five sites were equivalent to, or greater than, that of Pro. Binding affinities (Km) for the two least efficiently cleaved sites (p20/VPg, VPg/Pro) were 2-4-fold higher than the other sites. This work further defines the role of ProPol in NoV polyprotein cleavage, and demonstrates that human norovirus polyprotein processing order is primarily an inherent property of enzymatic activity.