A Norovirus Protease Structure Provides Insights into Active and Substrate Binding Site Integrity

Sapoviruses are one of the major agents of acute gastroenteritis in childhood. They form a tight genetic cluster (genus) in the Caliciviridae family that regroups both animal and human pathogenic strains. No permissive tissue culture has been developed for human sapovirus, limiting its characterization to surrogate systems. We report here on the first extensive characterization of the key enzyme of replication, the RNAdependent RNA polymerase (RdRp) pol synthesizes a double-stranded RNA or labels the template 3 terminus by terminal transferase activity. Initiation of RNA synthesis occurs de novo on heteropolymeric templates or in a primerdependent manner on polyadenylated templates. Strikingly, this mode of initiation of RNA synthesis was also described for norovirus, but not for lagovirus, suggesting structural and functional homologies in the RNAdependent RNA polymerase of human pathogenic caliciviruses. This first experimental evidence makes sapovirus 3D pol an attractive target for developing drugs to control calicivirus infection in humans.

Section: Discussionmentioning

confidence: 99%

Structural and Functional Characterization of Sapovirus RNA-Dependent RNA Polymerase

Fullerton

Blaschke²,

Coutard

et al. 2007

“…In addition, the active-site residues of the 3C-like proteases of the NoV Chiba virus strain and the RHDV FRG strain have been identified by site-directed mutagenesis (5,14,36,46). Recently, the X-ray crystal structures of the two 3C-like proteases of NoV (Chiba and Norwalk strains) were determined (27,46). In contrast, although site-directed mutagenesis of the 3C-like proteases of the SaV Mc10 and FCV Urbana strains showed that C in the GDCG motif is crucial for the proteolytic processing activity (28)(29)(30), the remaining amino acids that are important for the activity have not been identified.…”

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confidence: 99%

Highly Conserved Configuration of Catalytic Amino Acid Residues among Calicivirus-Encoded Proteases

Oka

Yamamoto

Yokoyama

et al. 2007

Self Cite

, and H 137 in FCV. A computer-assisted structural analysis showed that despite high levels of diversity in the primary structures of the protease domains in the family Caliciviridae, the configurations of the H, E, C, and H residues are highly conserved, with these residues positioned closely along the inner surface of the potential binding cleft for the substrate. These results strongly suggest that the H, E, C, and H residues are involved in the formation of a conserved catalytic surface of the SaV and FCV 3C-like proteases.

“…The mature proteins include an RNA-dependent RNA polymerase (RdRp) (NS7 pol ), an ATPase (NS3), and proteins that disrupt cellular trafficking (NS1-2 and NS4) (18)(19)(20)(21)(22)(23).…”

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confidence: 99%

Structures of the Compact Helical Core Domains of Feline Calicivirus and Murine Norovirus VPg Proteins

Leen

Kwok

Birtley

et al. 2013

fWe report the solution structures of the VPg proteins from feline calicivirus (FCV) and murine norovirus (MNV), which have been determined by nuclear magnetic resonance spectroscopy. In both cases, the core of the protein adopts a compact helical structure flanked by flexible N and C termini. Remarkably, while the core of FCV VPg contains a well-defined three-helix bundle, the MNV VPg core has just the first two of these secondary structure elements. In both cases, the VPg cores are stabilized by networks of hydrophobic and salt bridge interactions. The Tyr residue in VPg that is nucleotidylated by the viral NS7 polymerase (Y24 in FCV, Y26 in MNV) occurs in a conserved position within the first helix of the core. Intriguingly, given its structure, VPg would appear to be unable to bind to the viral polymerase so as to place this Tyr in the active site without a major conformation change to VPg or the polymerase. However, mutations that destabilized the VPg core either had no effect on or reduced both the ability of the protein to be nucleotidylated and virus infectivity and did not reveal a clear structure-activity relationship. The precise role of the calicivirus VPg core in virus replication remains to be determined, but knowledge of its structure will facilitate future investigations.