GII.4 Norovirus Protease Shows pH-Sensitive Proteolysis with a Unique Arg-His Pairing in the Catalytic Site

Abstract

Human noroviruses (NoVs) are the main cause of epidemic and sporadic gastroenteritis. Phylogenetically, noroviruses are divided into seven genogroups, with each divided into multiple genotypes. NoVs belonging to genogroup II and genotype 4 (GII.4) are globally most prevalent. Genetic diversity among the NoVs and the periodic emergence of novel strains present a challenge for the development of vaccines and antivirals to treat NoV infection. NoV protease is essential for viral replication and is an attractive target for the development of antivirals. The available structure of GI.1 protease provided a basis for the design of inhibitors targeting the active site of the protease. These inhibitors, although potent against the GI proteases, poorly inhibit the GII proteases, for which structural information is lacking. To elucidate the structural basis for this difference in the inhibitor efficiency, we determined the crystal structure of a GII.4 protease. The structure revealed significant changes in the S2 substratebinding pocket, making it noticeably smaller, and in the active site, with the catalytic triad residues showing conformational changes. Furthermore, a conserved arginine is found inserted into the active site, interacting with the catalytic histidine and restricting substrate/inhibitor access to the S2 pocket. This interaction alters the relationships between the catalytic residues and may allow for a pHdependent regulation of protease activity. The changes we observed in the GII.4 protease structure may explain the reduced potency of the GI-specific inhibitors against the GII protease and therefore must be taken into account when designing broadly cross-reactive antivirals against NoVs. IMPORTANCE Human noroviruses (NoVs) cause sporadic and epidemic gastroenteritis worldwide. They are divided into seven genogroups (GI to GVII), with each genogroup further divided into several genotypes. Human NoVs belonging to genogroup II and genotype 4 (GII.4) are the most prevalent. Currently, there are no vaccines or antiviral drugs available for NoV infection. The protease encoded by NoV is considered a valuable target because of its essential role in replication. NoV protease structures have only been determined for the GI genogroup. We show here that the structure of the GII.4 protease exhibits several significant changes from GI proteases, including a unique pairing of an arginine with the catalytic histidine that makes the proteolytic activity of GII.4 protease pH sensitive. A comparative analysis of NoV protease structures may provide a rational framework for structure-based drug design of broadly cross-reactive inhibitors targeting NoVs.

In crystallo-screening for discovery of human norovirus 3C-like protease inhibitors

“…However, a neighbouring dimer in the crystal structure forms an interface of comparable buried surface area (692.3 Å 2 ) between chains labelled A and D chains and likewise for chains labelled the B and C. This result indicates that higher order oligomers may possibly be formed by SV3CP dimers, such as the putative tetramer shown in Fig. 1 b. Intriguingly, a number of other human GI and GII noroviral protease structures ( Nakamura et al, 2005 , RCSB ID: 1wqs; Muzzarelli et al, 2019 , RCSB ID: 6b6i; Viskovska et al, 2019 , RCSB ID: 6nir) form essentially the same tetrameric assembly in the crystals, as shown in Supplementary Fig. 1 .…”

Molecular Evolution of the Protease Region in Norovirus Genogroup II

“…A previous study reported that the amino acids adjacent to the active sites in the Pro protein of enterovirus 71 play a crucial role in protease activity based on the reduction of activity caused by the mutations of the residues (Wang et al, 2011). Moreover, Viskovska et al (2019) reported that the NoV GII protease showed activity in a pH-dependent manner by interactions between the residues H30 and R112. In this study, we found that these amino acids were conserved in most GII genotypes, except for GII.P25 ( Supplementary Table S3).…”

“…Many studies have been conducted into the development of drug candidates (Hussey et al, 2011;Muhaxhiri et al, 2013;Muzzarelli et al, 2019;Viskovska et al, 2019). A recent report demonstrated different effectiveness of antivirals against GI and GII NoVs, suggesting that the amino acid diversity in Pro may result in different effectiveness of drugs (Viskovska et al, 2019). It is important to understand the evolution of the Pro region of GII NoV, because this virus is the predominant genogroup in patients with NoV infection.…”

Human Norovirus Proteins: Implications in the Replicative Cycle, Pathogenesis, and the Host Immune Response

“…An analysis of the GII.4 protease crystal structure shows that this protein presents changes in the S2 substrate-binding pocket and the active site (catalytic triad residues) compared to those sites in the GI homologous protein. Another characteristic of the active site of the GII.4 protease is that there is a conserved arginine that interacts with the catalytic histidine, which restricts the access of inhibitors or substrates to the S2 pocket and makes the proteolytic activity of this protease sensitive to pH changes (75). Thus, the GI protease inhibitors may not be effective for the GII.4 protease.…”

“…However, the ProPol precursor has two enzymatic functions: protease and polymerase activities. It should be noted that the ProPol complex has a higher enzymatic performance compared to the activity of both proteins separately (75,77,90). Subsequently, the three precursors are cleaved by the action of the ProPol complex, which in turn self-cleaves and generates the six individual non-structural proteins.…”