Soluble Respiratory Syncytial Virus Fusion Protein in the Fully Cleaved, Pretriggered State Is Triggered by Exposure to Low-Molarity Buffer

Abstract

The paramyxovirus fusion (F) glycoprotein is anchored in the virion membrane in a metastable, pretriggered form. Once triggered, the F protein undergoes a dramatic conformational extension that inserts its hydrophobic fusion peptide into the target cell membrane, then folds back on itself to bring the membranes together and initiate fusion. Unlike most other paramyxoviruses, the respiratory syncytial virus (RSV) F protein alone is sufficient to mediate membrane fusion and virus infection. To study the triggering mechanism of the RSV F protein, we have generated a soluble F (sF) protein by replacing the transmembrane and cytoplasmic tail domains with a 6His tag. The sF protein is secreted efficiently from 293T cells in a fully cleaved form. It is recognized by neutralizing monoclonal antibodies, appears spherical by electron microscopic analysis, and is not aggregated, all consistent with a native, pretriggered trimer. The sF protein was purified on a Ni ؉2 column and eluted with 50 mM phosphate buffer containing 500 mM NaCl and 250 mM imidazole. Dialysis against 10 mM buffer caused the sF protein to trigger, forming "hat pin"-shaped molecules that aggregated as rosettes, characteristic of the posttriggered form. Further dialysis experiments indicated that the efficiency of triggering correlated well with the reduction of buffer molarity. Reduction of buffer molarity by dilution also resulted in exposure of the fusion peptide, as detected by liposome association, confirming sF protein triggering. Mutation of the furin cleavage site adjacent to the fusion peptide prevented liposome association, further confirming that association is via the fusion peptide.

Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis

“…This attachment protein-independent fusion activation has been well characterized in human RSV, bovine RSV, and ovine RSV (14)(15)(16). Recently, it was found that the F proteins of hMPV and aMPV also induce fusion without their attachment G proteins (17)(18)(19)(20), suggesting that the G protein is dispensable for attachment and fusion.…”

“…Another unique characteristic of hMPV entry is that fusion of some hMPV strains requires low pH, whereas fusion of all other paramyxoviruses occurs at neutral pH (17,18,22). In addition, fusion of hMPV in cell culture requires the addition of exogenous protease (17,18), unlike the F protein of RSV but similar to the F proteins of some of the members of the Paramyxovirinae, such as Sendai virus, parainfluenza virus type 1, and avirulent strains of Newcastle disease virus (16). Together these results suggest that hMPV fusion is characterized by a unique combination of features making it distinct from other paramyxoviruses.…”

Attenuation of Respiratory Syncytial Virus–Induced and RIG-I–Dependent Type I IFN Responses in Human Neonates and Very Young Children

“…The differences may be explained by the distinct viral entry mechanisms. Unlike influenza virus, vesicular stomatitis virus, or Newcastle disease virus, RSV fusion does not require endosomal maturation and acidification (41,42), which in turn is a necessary step for endosomal TLR function. Therefore, the ssRNA genome may not be accessible to TLR7 upon RSV entry of human pDCs.…”

The Heptad Repeat C Domain of the Respiratory Syncytial Virus Fusion Protein Plays a Key Role in Membrane Fusion

“…This deposited fragment contains the motavizumab epitope and, according to the description in the Protein Data Bank (PDB), was derived as a by-product of cellular protease cleavage at some time during expression or purification. An alternate 30-kDa by-product corresponding to the remaining C terminus of F 1 (as determined by Western blotting to a C-terminally bound purification tag) has also been observed by other investigators during recombinant F protein expression (17). This product is absent in the cleavage-deficient mutations (E82A, L83A, V90A, and L93A) and reduced in others (Y86A and L96A).…”

“…This transition is irreversible under physiological conditions and renders the fusion protein inactive. Heat, low-molarity buffer, and freeze-thaw cycles have all been shown to cause this unidirectional conformation change (17,19,20). Recently, a number of conformation-specific antibodies have been characterized.…”