On the stability of parainfluenza virus 5 F proteins

Abstract

The crystal structure of the F protein (prefusion form) of the paramyxovirus parainfluenza virus 5 (PIV5) WR isolate was determined. We investigated the basis by which point mutations affect fusion in PIV5 isolates W3A and WR, which differ by two residues in the F ectodomain. The P22 stabilizing site acts through a local conformational change and a hydrophobic pocket interaction, whereas the S443 destabilizing site appears sensitive to both conformational effects and amino acid charge/polarity changes.

FIG 1

The atomic model of the F protein of the WR isolate of parainfluenza virus 5. (A) Schematic diagram of the domain organization of PIV5 F-GCNt. Domains I, II, and III are colored in yellow, red, and magenta, respectively. The hydrophobic fusion peptide (FP) is colored in light pink. The locations of P22 and S443, which are mutated to a leucine and a proline, respectively, in the WR F-GCNt construct, are highlighted. (B) Western blot (WB) and a silver-stained (SS) SDS-PAGE gel showing the protein purity post-affinity column purification. (C and D) Electron micrographs of negatively stained WR F-GCNt and W3A F-GCNt, respectively, showing comparable percentages of prefusion trimers in each sample. (E) A cartoon representation of the WR F-GCNt atomic structure colored as in panel A. (F and G) Zoomed-in regions of panel E showing the 2F_o − F_c electron density map at 1 σ around residues 22 and 443, respectively. The W3A F-GCNt model, in cyan, was aligned to the WR F-GCNt model, in gray, in PyMOL to highlight the changes between the two structures.

FIG 2

Mutagenesis analysis for residues P22 and S443. (A) Prefusion F protein expressed on the surface of transfected 293T cells was detected using the prefusion specific monoclonal F1a antibody and flow cytometry. Mean fluorescence intensity (MFI) values were calculated and normalized against wild-type W3A F. The experiments were done in triplicate, and the error bars represent ±1 standard deviation. (B) F protein fusion activity quantified using a luciferase reporter system and normalized against wild-type W3A F plus HN fusion activity. P22 mutations are ordered according to increasing side-chain hydrophobicity, and S443 mutations are ordered by side-chain molecular weight. PIV5 HN was transfected in equal amounts with each construct, with the exception of the multiple-cloning site (MCS) vector negative control. The experiments were done in triplicate, and the error bars represent ± 1 standard deviation. (C) Monitoring F protein activity through syncytium formation of transfected BHK-21 cells. Cells were fixed and stained 18 h posttransfection. “MCS,” “HN only,” and “F only” are negative controls, while “F+HN” is the W3A wild-type F and HN positive control.