| Parainfluenza virus 5 (PIV5) enters host cells by fusing the viral envelope with host cellular membrane. This process is carried out by two viral envelope glycoproteins: the hemagglutinin-neuraminidase (HN) protein, and the fusion (F) protein. Upon binding the cellular receptor, HN activates the F protein to undergo irreversible conformational changes that convert it from its metastable prefusion conformation to a more stable postfusion conformation, and cause membrane fusion.;The C terminus of the F protein consists of a membrane proximal external region (MPER), a transmembrane (TM) domain and a cytoplasmic tail (CT). The role of this region in fusion is not well understood. To study the sequence requirements of the C-terminus, chimeras were constructed of PIV5 F ectodomain, and either the MPER, TM domain or CT of other paramyxovirus F proteins. Chimeric PIV5 F proteins with all the three regions replaced with those of other paramyxoviruses were nonfunctional in fusion. Fusion could be restored by adding a destabilizing hyperfusogenic mutant, indicating an increased energy of activation for the chimeras as compared to wild type (wt) F protein. Replacement of PIV5 F sequences back to the C-terminus chimeras restored fusion to some extent, but this restoration in fusion was not additive, indicating that the C-terminus facilitates fusion in a sequence-specific concerted fashion, such that the activity of the native C-terminus is greater than that of the sum of individual regions. In addition, we made alanine point mutants of the residues of the MPER, and found both hypofusogenic and hyperfusogenic mutants.;We also expressed a double mutant of PIV5 F protein with two residues in the MPER replaced with cysteines. The resultant protein was expressed on the cell surface; it formed disulfide bonded F trimers and was inactive in fusion. Reduction of the disulfide bond restored fusion activity. The data indicate that in addition to dissociation of the three-helix bundle stalk domain of prefusion F, the MPER region also needs to separate for F to be able to refold and cause fusion. |
