Characterization and inhibition of norovirus proteases of genogroups I and II using a fluorescence resonance energy transfer assay

Abstract

Noroviruses are the major cause of food- or water-borne gastroenteritis outbreaks in humans. The norovirus protease that cleaves a large viral polyprotein to nonstructural proteins is essential for virus replication and an attractive target for antiviral drug development. Noroviruses show high genetic diversity with at least five genogroups, GI-GV, of which GI and GII are responsible for the majority of norovirus infections in humans. We cloned and expressed proteases of Norwalk virus (GI) and MD145 virus (GII) and characterized the enzymatic activities with fluorescence resonance energy transfer substrates. We demonstrated that the GI and GII proteases cleaved the substrates derived from the naturally occurring cleavage site in the open reading frame (ORF) 1 of G1 norovirus with similar efficiency, and that enzymatic activity of both proteases was inhibited by commercial protease inhibitors including chymostatin. The interaction of chymostatin to Norwalk virus protease was validated by nuclear magnetic resonance (NMR) spectroscopy.

Fig. 1

Norovirus genome organization and proteolytic map. A. The cleavages at NS2/3 (between NS1–2 and NS3 proteins) and NS3/4 sites in ORF1 occur more efficiently than other cleavage sites in ORF1 of GI and GII noroviruses. *Cleavage dipeptide. B. Cleavage dipeptide and surrounding residues (P7–P7′) at NS2/3 site in ORF1 of GI and GII noroviruses. The designation of substrate residues for P1 and P1′ starts at the scissile bond and counts toward the N- or C-terminus, respectively, as suggested by Schechter and Berger (1967).

Fig. 2

The enzyme kinetics of NVpro and MD145pro with fluorogenic substrates EPDFHLQGPEDLAK (A) and DFHLQGP (B). The reaction velocity as a function of the substrate concentration was plotted for each protease. NVpro and MD145pro are denoted by the solid line with open circles and the dotted line with filled rectangles, respectively.

Fig. 3

Z factor analysis. The scatter plot represents positive control data (filled diamond data points) and negative control data (filled round data point). Solid lines represent mean fluorescence, and dashed lines represent three standard deviations above and below each set of positive control data points. Experiments are repeated more than three times with consistent results.

Fig. 4

DMSO tolerance. Substrate was incubated with NVpro in the presence of 0–6% DMSO (v/v) in assay buffer at 37 °C. Fluorescence signals were measured at 1 h incubation using a plate reader. *Statistically significant (p < 0.05) compared to DMSO 0% by Student's t-test.

Fig. 5

Inhibitor profiles. A range of commercially available protease inhibitors were tested for their activity against NVpro and MD145pro. Each bar represents the mean percentage of substrates (± SEM) cleaved by each protease in the absence (control) or presence of each inhibitor at 50 μM. NVpro and MD145pro are denoted by the dark and light bars, respectively.

Fig. 6

Dose–responsive curves of chymostatin on NVpro and MD145pro. The dot represents percentage of substrates cleaved by NVpro in the presence of chymostatin at different concentrations, compared with control. NVpro and MD145pro are denoted by the solid line with open circles and the dotted line with filled rectangles, respectively.

Fig. 7

Chymostatin specifically interacts with NVpro. A. Weighted chemical shift differences of the ¹H and ¹⁵N resonances for NVpro when titrated with increasing amounts of chymostatin. Largest values (0.15) and asterisks are used to indicate the residues that cannot be assigned after adding 2-fold molar excess of chymostatin due to the peak disappearance or broadness. Weighted chemical shift difference is calculated by an equation, Δd = [1/2(d_H2 + 1/25d_N2)]1/2. B. Overlay of ¹H–¹⁵N HSQC spectra of NVpro in the absence (black) and in the presence of 2-fold excess of chymostatin (blue). C. Comparison of 1H–¹⁵N HSQC spectra of NVpro in the absence and presence of 0.3 mM (2-fold molar excess) PB compound shows that PB titrated as a control causes generally no chemical shift perturbation on the spectra.

Fig. 8

HADDOCK docking model of NVpro complexed with chymostatin. The structure belonging to the lower energy cluster is presented. Chymostatin is shown as stick model (green). Active (red) and passive residues (pink) are labeled. (B) Close up view of chymostatin binding site.

Fig. 9

Purification of NVpro. A. His-tagged NVpro was purified by size exclusion chromatography. B. SDS-PAGE analysis of purified NVpro. The untagged version of the protease has a predicted molecular weight of 19 kDa. The (His) 6-tagged proteases are shifted to a slightly higher molecular weight.