Structural and conformational requirements of membrane fusion mediated by the Parainfluenza virus 5 fusion protein
Fusion of biological membranes is dictated by the interaction between specialized membrane proteins and the lipid bilayer. Parainfluenza virus 5 (PIV5) mediates fusion using two surface glycoproteins: the fusion protein (F) and the attachment protein hemagglutinin-neuraminidase (HN). Activation of membrane fusion of PIV5 typically occurs at neutral pH, and involves binding of HN to sialic acid, interaction of HN with the F protein, insertion of the F fusion peptide into the target membrane, and refolding of the F protein to the final postfusion form that provides the necessary energy for membrane merger.
The F protein contains a hydrophobic fusion peptide, a transmembrane domain (TM), and a cytoplasmic tail (CT). The ectomain and CT of the F protein and their role in fusion have been extensively studied. Different natural isolates of PIV5 have F proteins with CTs of varying length, either a short (20 residues) or long (42 residues). A long tail porcine isolate of PIV5, known as SER, was reported to be an exception to the dogma of paramyxovirus fusion at neutral pH in it requires a low pH step for fusion (S. Seth, A. Vincent, and R.W. Compans, J. Virol. 77: 6520-6527, 2003). However, by using multiple assays we could not find a requirement for low pH triggering of PIV5 SER fusion.
Although the contributions of the ectodomain and CT of PIV5 F to membrane fusion have been well studied, little is known about the role of the PIV5 F TM domain in fusion. Alanine scanning mutageneis determined the TM domain of F is sequence dependent, and hydrophobic string of residues cannot substitute for the TM domain in fusion. Continued substitution revealed residues L486 and I488 play a key role in fusion, where the hydrophobicity of the side chains at these residues affects the interplay of the F protein and the lipid bilayer during membrane merger. Our studies suggest the TM domain is involved in the lipidic steps of fusion, and mutants L486A and I488A are trapped at the lipid stalk intermediate of membrane fusion. Oxidative cross-linking studies of the TM domain indicate the TM regions of the F trimer are in close proximity, and modeling studies suggest the TM domain is α-helical and forms a modified three-helix bundle.