Rotavirus capsid protein VP5* permeabilizes membranes

Area of Science:

• Virology
• Molecular Biology
• Cell Biology

Background:

• Rotavirus infectivity and membrane permeabilization depend on the proteolytic cleavage of the VP4 outer capsid spike protein into VP8* and VP5* fragments.
• The specific role of the VP5* fragment in membrane permeabilization has not been fully elucidated.

Purpose of the Study:

• To investigate the function of the VP5* cleavage fragment in permeabilizing biological membranes.
• To identify the regions within VP5* responsible for membrane interaction and permeabilization.

Main Methods:

• Purification of expressed VP5* and truncated VP5* proteins using nickel affinity chromatography.
• Assay of VP5* proteins' ability to permeabilize large unilamellar vesicles (LUVs) loaded with carboxyfluorescein (CF).
• Inhibition studies using VP5*-specific and VP8*-specific neutralizing monoclonal antibodies.

Main Results:

• VP5* and its truncations, but not VP4 or VP8*, effectively permeabilized LUVs, releasing CF.
• VP5*-induced membrane permeabilization was concentration and temperature-dependent, with optimal activity at pH 7.35 and 37°C.
• Permeabilization was independent of divalent cations and cholesterol but was inhibited by VP5*-specific neutralizing antibodies, indicating specific conformational epitopes.
• Truncated VP5* proteins, including residues 265-474, retained membrane-permeabilizing activity, suggesting the fusion region is within these domains.

Conclusions:

• The VP5* fragment is a distinct membrane-permeabilizing protein essential for rotavirus infectivity.
• VP5* can permeabilize membranes independently of other rotavirus proteins, highlighting its direct role in viral entry.
• Specific regions within VP5* are responsible for membrane interaction and permeabilization, providing targets for antiviral strategies.