Structures of the compact helical core domains of feline calicivirus and murine norovirus VPg proteins

Abstract

We 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.

Fig 1

¹H-¹⁵N HSQC and hetNOE data for FCV and MNV VPg proteins. (A) ¹H-¹⁵N HSQC spectrum for FCV VPg. (B) ¹H-¹⁵N hetNOE measurements for full-length FCV VPg, plotted against residue number. The gap in the data corresponds to amides from residues 26 to 38 inclusive, which were not able to be assigned from data collected at 298 K. (C) ¹H-¹⁵N hetNOE measurements for MNV VPg full length (1 to 124) (filled diamonds) and MNV VPg 11-85 (open squares). The backbone amides of residues 1 to 10 and 86 to 124 were not assigned.

Fig 2

Solution structure of FCV VPg 9-79. (A) Backbone trace of the 20 lowest-energy conformers of FCV VPg 9-79, calculated using ARIA (62) and CNS (60), including a final water refinement. The nucleotide-accepting tyrosine (Y24) side chain of one of the models is shown. (B) Representative conformer of FCV VPg 9-79, with selected (mainly hydrophobic) side chains shown as sticks and colored by atom type (tan, carbon; red, oxygen; blue, nitrogen). The N and C termini are indicated. (C) FCV VPg 9-79 rotated by 180° compared to panel B and with key charged and polar residues involved in electrostatic interactions shown as sticks. Polar interactions are indicated by dashed black lines.

Fig 3

NMR analysis of the MNV VPg benefitted from knowledge of the structure of FCV VPg. (A) Amino acid sequence alignment of FCV and MNV VPg proteins, performed using ClustalW. Positions of the helices detected through structural analysis are indicated as shaded boxes above and below the sequences (green for FCV and brown for MNV). Dashed lines indicate disordered portions of the polypeptide backbone. The nucleotidylated Tyr in each sequence is shown in boldface. Within the alignment, the asterisk, colon, and period characters denote identical, very similar, and similar amino acids, respectively. (B) Overlay of ¹H-¹⁵N HQSC spectra of MNV VPg constructs used to probe the extent of the structure core of the protein: blue, VPg 11-85; black, VPg 1-124; red, VPg 11-62.

Fig 4

Solution structure of MNV VPg 11-85. (A) Backbone trace of the 20 lowest-energy conformers of MNV VPg 11-85, calculated by ARIA (62) and CNS (60). The side chain of the nucleotide-accepting tyrosine from one of the conformers is indicated. (B) Representative conformer of MNV VPg 11-85. The N and C termini are indicated. (C) Overlay of the structured cores of FCV VPg (tan) and MNV VPg (green); the backbone RMSD between the two structures is ∼2 Å. (D) Representative conformer of MNV VPg 11-85, with selected (mainly hydrophobic) side chains shown as sticks and colored by atom type. (E) MNV VPg 11-85 rotated by 110° compared to panel D and with key charged and polar residues involved in electrostatic interactions shown as sticks. Polar interactions are indicated by dashed black lines.

Fig 5

Effects of pH, NaCl, and temperature on the ¹H-¹⁵N HSQC spectrum of MNV VPg 1-124. (A to C) Spectra recorded for MNV VPg 1-124 at pH 5 (A), pH 6 (B), or pH 7 (C). The spectra were recorded at 303 K with 100 μM protein dissolved in 50 mM malic acid, 2-(N-morpholino)ethanesulfonic acid (MES), and Tris-HCl at a molar ratio of 1:2:2 (MMT), 300 mM NaCl, and 1 mM DTT, which was titrated to the required pH with HCl or NaOH as appropriate. (D to F) ¹H-¹⁵N HSQC spectra recorded at 303 K with 40 μM protein in 50 mM sodium phosphate (pH 6.5) and 1 mM DTT supplemented with 50 mM NaCl (D), 150 mM NaCl (E), or 300 mM NaCl (F). (G to I) ¹H-¹⁵N HSQC spectra recorded with 590 μM protein in 50 mM sodium phosphate (pH 6.5), 300 mM NaCl, and 1 mM DTT at 278 K (G), 293 K (H), or 303 K (I).

Fig 6

Effects of site-directed mutations on the stability of the core structures of MNV and FCV VPg. The amide region of the 1D ¹H NMR spectra of MNV VPg 1-124 (A) and FCV VPg 1-111 (B). In each case, the region of the spectrum shown is the structured amide region of a 1D protein spectrum, which is not overlapped by aromatic signals. The peaks at 10.0 to 10.3 ppm derive from the NH^ε of Trp side chains.

Fig 7

Effects of mutations on MNV VPg on RNA synthesis by MNV NS7^pol. The bar graph shows results from the cell-based reporter assay detecting NS7^pol products by the RIG-I innate immune receptor. Each bar contains the results from 3 independent transfected HEK293T cells, and the results are expressed as a ratio of the firefly (FF) luciferase driven by the IFN-β promoter to a constitutive Renilla luciferase (Ren Luc) that was coexpressed within the same cells. Within the bar graph, the solid line represents reporter levels from the NS7^pol in the absence of coexpressed VPg. The dashed line denotes the background reporter levels in the absence of RNA synthesis by the NS7^pol. Bars identified with asterisks denote results that are statistically different (P < 0.05) from those of the polymerase alone. Under the graph is a schematic representation of key features within the MNV VPg molecule. The thick lines represent the two α-helices in the core, and the bold “Nuc” denotes the location of the nucleotidylation tyrosine. The tyrosines that form a hydrophobic region within the core are identified by black diamonds. The salt bridge between residues R32 and D48 is indicated by a dashed line. The gel images above the graph are from Western blots of immunoprecipitated VPg in cultures tested in parallel to the reporter assay. The identities of VPg and the fractions of the VPg molecules used by the NS7^pol for RNA synthesis are shown to the left of the images. All samples were from one experiment but cropped to allow alignment of the Western blot results to those of the reporter assay.

Fig 8

How does MNV VPg interact with its polymerase? Comparison of the structures of polymerases and VPg proteins from MNV and FMDV. (A) Crystal structure of MNV NS7^pol (67) and solution structure of MNV VPg 11-85 (this work). The active site of the polymerase, where nucleotidylation takes place, is colored blue. It is not yet clear what conformational changes are required for MNV VPg to be accommodated within the active site. (B) Cocrystal structure of FMDV 3D^pol and VPg (75), with VPg also shown extracted from the structure on the right (for ease of comparison with panel A). Only residues 1 to 15 of FMDV VPg were visible in the crystal structure.