Computational protocol: The Hemagglutinin-Neuraminidase (HN) Head Domain and the Fusion (F) Protein Stalk Domain of the Parainfluenza Viruses Affect the Specificity of the HN-F Interaction

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Protocol publication

[…] Subconfluent BHK cells (ca. 3.0 × 106) in one well of six-well culture plate were transfected with 0.2 or 1.0 μg/well of the pcDL-SRα expression vector encoding each HN protein together with 2 μg/well of the pcDL-SRα expression vector encoding each F protein by using X-tremeGENE 9 DNA transfection kit (Roche Diagnostics). After 12 h or 24 h of incubation at 37°C, the cells were fixed with 4% paraformaldehyde in PBS, washed three times with PBS, treated with methanol, and stained with Giemsa’s solution (Sigma). A photomicrograph, which corresponded to an area of 0.75 mm2 (1.0 mm × 0.75 mm) of the BHK cell monolayer, was taken and the areas (number of pixels) occupied by the fused (syncytial) cells were measured with the aid of a graphics software, NIH ImageJ ver.1.45s. The percentage of the syncytial areas to the total area (0.75 mm2 = 3,871,488 pixels) was regarded as the fusion index. Randomly taken 10 photographs in one well (9 cm2) were measured for each sample and the average fusion index (%) and standard deviation were determined. Statistical significance of the data was evaluated with one-way ANOVA (Games-Howell method) using the SPSS Ver. 24 software (IBM). Differences were considered statistically significant if p < 0.05. [...] Molecular modeling of the HN and F proteins was performed on the automated comparative protein modeling server, SWISS-MODEL, by using the crystal structures of the PIV5 (WR) F protein (PDB ID: 4WSG) and the PIV5 (W3A) HN protein (PDB ID: 4JF7) as the templates, respectively. The crystal structure of the PIV5 (W3A) HN head domain (PDB ID: 1Z4Z) was also used as a template. The downloaded PDB files were analyzed with the aid of a graphics software, Waals (Altif Laboratories, Tokyo, Japan). The quality of the model structures was assessed with QMEAN on SWISS-MODEL (Supplementary Figure ). […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Viruses, Human poliovirus 1 Mahoney,
Diseases Paramyxoviridae Infections
Chemicals Amino Acids