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. 2014 Oct;88(19):11611-6.
doi: 10.1128/JVI.01726-14. Epub 2014 Jul 16.

Structural insights into the human metapneumovirus glycoprotein ectodomain

Cedric Leyrat  1 , Guido C Paesen  1 , James Charleston  1 , Max Renner  1 , Jonathan M Grimes  2
Affiliations

Structural insights into the human metapneumovirus glycoprotein ectodomain

Cedric Leyrat et al. J Virol. 2014 Oct.

Abstract

Human metapneumovirus is a major cause of respiratory tract infections worldwide. Previous reports have shown that the viral attachment glycoprotein (G) modulates innate and adaptive immune responses, leading to incomplete immunity and promoting reinfection. Using bioinformatics analyses, static light scattering, and small-angle X-ray scattering, we show that the extracellular region of G behaves as a heavily glycosylated, intrinsically disordered polymer. We discuss potential implications of these findings for the modulation of immune responses by G.

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Figures

FIG 1
FIG 1
Computational analysis of HMPV (A and C) and HRSV (B and D) G sequence conservation, order/disorder propensity, and glycosylation sites. (A and B) The predicted disorder propensities and sequence conservation profiles are shown by black and red lines, respectively. Meta-disorder predictions were calculated following procedures described in reference . Sequence conservation was calculated using AL2CO (60) by applying a sliding average on a 20-residue window. (C and D) Location of predicted glycosylation sites along the amino acid sequence, shown as histogram bars, based on GlycoPred server output (61). HMPV sG (strain NL-1-00 [A1]) is predicted to contain 5 N-linked and 59 O-linked glycosylation sites, constituting an average of one site for every three residues.
FIG 2
FIG 2
Biophysical characterization of HMPV sG. (A) Molecular mass determination of HMPV sG using SEC-MALLS-RI. The protein was purified by size exclusion chromatography on an S200 column equilibrated with 20 mM Tris (pH 7.5) and 150 mM NaCl prior to analysis. The black line shows the SEC elution profile as monitored by refractometry. The red line shows the molecular mass calculated from light scattering and refractometry data. (B, C, D, and E) Small-angle X-ray scattering (SAXS) experiments; (B) scattering curves of sG measured at concentrations of 4, 6, and 8 mg/ml are shown in green, red, and black, respectively; (C) Kratky plots showing linear behavior in the high Q range; (D) Guinier plots showing linear behavior in the low Q range; (E) distance distribution functions [P(r)] calculated using GNOM (62).
FIG 3
FIG 3
Analysis of sG flexibility using the ensemble optimization method (EOM). (A and B) Calculated radius of gyration (Rg) and maximal intramolecular distance (Dmax) distributions of the starting (black line) and optimized ensembles (red line). (C) Fitted SAXS profile of sG measured at 8 mg/ml. The experimental data are shown in black and the theoretical scattering from the optimized ensemble in red. (D) Variation of the goodness of fit (χexp) as a function of the optimized ensemble size.
FIG 4
FIG 4
Comparison of the molecular dimensions of the extracellular regions of the HMPV F and G proteins, illustrating the G protein induced steric hindrance potentially hampering host factor-F protein interactions. The extracellular region of the Toll-like receptor 4–myeloid differentiation factor 2 (TLR-4/MD-2) complex (PDB identifier 3FXI) is represented as an example of such host factor. Homology models of the F protein trimers in the pre- and postfusion states form protrusions of 13 and 17 nm, respectively. Models were generated in HOMER (63) and are represented as red and blue surfaces. The homodimeric TLR-4 is shown in cyan and MD-2 in magenta, and three superimposed low-resolution models of sG are shown in orange, wheat color, and yellow. The anti-HMPV DS7 Fab bound to the prefusion model of F is shown in green based on PDB identifier 4DAG.
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