Soluble respiratory syncytial virus fusion protein in the fully cleaved, pretriggered state is triggered by exposure...

Area of Science:

• Virology
• Structural Biology
• Molecular Biology

Background:

• Paramyxovirus fusion (F) glycoproteins are metastable and require triggering to mediate membrane fusion.
• Respiratory syncytial virus (RSV) F protein alone mediates fusion, unlike other paramyxoviruses.
• Understanding RSV F protein triggering is crucial for developing antiviral strategies.

Purpose of the Study:

• To investigate the triggering mechanism of the RSV F protein.
• To characterize a soluble form of the RSV F protein (sF).
• To determine the role of buffer conditions in F protein conformational changes.

Main Methods:

• Generation of a soluble RSV F protein (sF) with a 6His tag.
• Purification of sF protein using Ni(+2) affinity chromatography.
• Induction of sF protein triggering via dialysis and buffer molarity reduction.
• Detection of fusion peptide exposure using liposome association assays.
• Analysis of sF protein conformation by electron microscopy.

Main Results:

• Soluble RSV F protein (sF) was successfully produced, purified, and characterized as a pre-triggered trimer.
• Dialysis against low molarity buffer triggered sF protein, inducing conformational changes to a post-triggered form.
• Triggering efficiency correlated with reduced buffer molarity.
• Fusion peptide exposure, indicated by liposome association, was confirmed upon sF protein triggering.
• Mutating the furin cleavage site abolished liposome association, confirming fusion peptide-mediated binding.

Conclusions:

• Buffer molarity is a critical factor in triggering the RSV F protein.
• The soluble F protein serves as a valuable tool for studying RSV fusion mechanisms.
• The findings provide insights into the conformational changes governing paramyxovirus-host cell membrane fusion.