Abstract
Norovirus is the leading cause of acute gastroenteritis worldwide, infecting ~685 million people each year. No approved vaccines or antivirals are available, creating a large global burden in both economic and societal costs. During the viral life cycle, the norovirus ORF1 is translated into a polyprotein that is cleaved by the norovirus protease (Pro) into the mature version of its non-structural proteins, making Pro a popular target for antiviral research. Pro processes the polyprotein at different cleavage efficiencies, leading to the formation of precursor proteins such as the protease, polymerase precursor (ProPol). It is known that the activity for ProPol is different from Pro. However, it is not known whether Pro-based antivirals will affect ProPol differently from Pro. Additionally, the Km for both Pro and ProPol was required to be established in the laboratory before testing against Pro-based antivirals.
This project investigated whether ProPol should be developed separately from Pro as a protease-based antiviral target. The objective was achieved by first expressing and purifying GII.4 Pro and ProPol using a SUMO-expression system, providing proteins without tags that resemble native proteins produced during infection. Then, the catalytic parameters were compared before testing the efficacy of protease inhibitors against human norovirus Pro and ProPol.
Fluorescent (FRET-based) peptide substrates based on the NS2/3 cleavage site were used to determine both the proteolytic activity and inhibitory profiles of Pro and ProPol. We showed that no differences were observed for the maximum velocity (Vmax) and enzymatic turnover number (kcat) between Pro and ProPol. However, the Km (which is associated with the affinity) for each of the three biological replicates were significantly different, suggesting that ProPol has a 3.4 to a 5-fold reduced affinity for the substrate compared to Pro. Additionally, the catalytic efficiencies (kcat / Km) were statistically significant between Pro and ProPol, with values of 1.5x104 ± 4x103 M-1 min-1 and 6x103 ± 4x103 M-1 min-1, respectively. However, such differences were too small to suggest a difference between the catalytic efficiency of Pro and ProPol.
The antiviral activities of protease inhibitors GC376, MAP-I, and Compound F were tested for their efficacy against Pro and ProPol. We report that both GC376 and MAP-I were inhibitory to the proteolytic activity of Pro and ProPol with comparable half-maximal inhibitory concentration (IC50) values. In contrast Compound F inhibited both Pro and ProPol with IC50 values of 5.11 ± 0.52 μM and 8.24 ± 0.99 μM respectively. Although the IC50 values were statistically significant, such differences were too small to suggest that Compound F inhibited Pro more than ProPol. Although such differences may not justify the need for specialised ProPol inhibitors to be provided, we showed that ProPol could be inhibited by Pro inhibitors when tested against the targeted NS2/3 sequence.