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

Abstract

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.

Norovirus Translation Requires an Interaction between the C Terminus of the Genome-linked Viral Protein VPg and Eukaryotic Translation Initiation Factor 4G

“…We have recently determined the structure of the MNV VPg protein (35) and demonstrated that although the central domain consists of two α helices, the N and C termini are largely disordered. To identify the regions within the norovirus VPg protein that are required for the interaction with initiation factors, we targeted several conserved amino acids for alanine mutagenesis.…”

“…The pETM11 plasmid encoding MNV VPg 1–124 is as described elsewhere (35). QuikChange site-directed mutagenesis (Stratagene) was used to produce the MNV VPg 1–124 F123A mutant.…”

“…Expression was induced during mid-log phase by the addition of 1 m m final isopropyl 1-thio-β- d -galactopyranoside in the case of GST for 4 h at 37 °C and for 3.5 h at 30 °C for GST-eIF4GI 4GM. Wild type and F123A MNV VPg were expressed as described previously (35). …”

Comprehensive Review of Human Sapoviruses

“…2) (37,49,68,69). VPg is linked to the 5= end of the viral RNA and is critical for calicivirus genome replication, transcription, and translation (37,70).…”

Sequence specificity for uridylylation of the viral peptide linked to the genome (VPg) of enteroviruses

“…As Table 1 shows, the charges of the sequences surrounding the reactive Tyrosine of the FCV and MNV VPgs are “polar opposites”. While the positive charges of PV-VPg are essential, mutation of negatively charged residues near the uridylylated Tyr in MNV VPgs prevents formation of the VPg-RNA covalent complex and virus replication (Leen et al, 2013). These residues could stabilize the structure through salt bridges (formed perhaps to residues not included in the NMR structure).…”