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Selective membrane permeabilization by the rotavirus VP5* protein is abrogated by mutations in an internal
W Dowling1, E Denisova, R LaMonica
1Department of Medicine, SUNY at Stony Brook, Stony Brook, New York 11794-8173, USA.
Abstract:
Rotavirus infectivity is dependent on the proteolytic cleavage of the VP4 spike protein into VP8* and VP5* proteins. Proteolytically activated virus, as well as expressed VP5*, permeabilizes membranes, suggesting that cleavage exposes a membrane-interactive domain of VP5* which effects rapid viral entry. The VP5* protein contains a single long hydrophobic domain (VP5*-HD, residues 385 to 404) at an internal site. In order to address the role of the VP5*-HD in permeabilizing cellular membranes, we analyzed the entry of o-nitrophenyl-beta-D-galactopyranoside (ONPG) into cells induced to express VP5* or mutated VP5* polypeptides. Following IPTG (isopropyl-beta-D-thiogalactopyranoside) induction, VP5* and VP5* truncations containing the VP5*-HD permeabilized cells to the entry and cleavage of ONPG, while VP8* and control proteins had no effect on cellular permeability. Expression of VP5* deletions containing residues 265 to 474 or 265 to 404 permeabilized cells; however, C-terminal truncations which remove the conserved GGA (residues 399 to 401) within the HD abolished membrane permeability. Site-directed mutagenesis of the VP5-HD further demonstrated a requirement for residues within the HD for VP5*-induced membrane permeability. Functional analysis of mutant VP5*s indicate that conserved glycines within the HD are required and suggest that a random coiled structure rather than the strictly hydrophobic character of the domain is required for permeability. Expressed VP5* did not alter bacterial growth kinetics or lyse bacteria following induction. Instead, VP5*-mediated size-selective membrane permeability, releasing 376-Da carboxyfluorescein but not 4-kDa fluorescein isothiocyanate-dextran from preloaded liposomes. These findings suggest that the fundamental role for VP5* in the rotavirus entry process may be to expose triple-layered particles to low [Ca](i), which uncoats the virus, rather than to effect the detergent-like lysis of early endosomal membranes.
Insights
Rotavirus VP5* protein, after cleavage from VP4, permeabilizes cell membranes. Specific hydrophobic residues within VP5*-HD are crucial for this function, enabling viral entry.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Rotavirus infectivity relies on VP4 spike protein cleavage into VP8* and VP5*.
- Cleaved VP5* protein is implicated in membrane permeabilization for viral entry.
Purpose of the Study:
- To investigate the role of the VP5* hydrophobic domain (VP5*-HD) in mediating cellular membrane permeabilization.
- To identify specific residues and structural features within VP5*-HD essential for membrane interaction.
Main Methods:
- Expression of wild-type and mutated VP5* proteins in cells.
- Assessing cellular permeability using o-nitrophenyl-beta-D-galactopyranoside (ONPG) uptake.
- Analyzing VP5* truncations and site-directed mutants for membrane permeabilization activity.
- Liposome-based assays to determine size-selective release of molecules.
Main Results:
- VP5* and truncations containing VP5*-HD permeabilized cells to ONPG, unlike VP8*.
- C-terminal truncations removing the GGA motif within VP5*-HD abolished permeability.
- Site-directed mutagenesis confirmed the requirement of specific residues within VP5*-HD, particularly glycines.
- VP5* induced size-selective permeability in liposomes, releasing small molecules but not large dextrans.
Conclusions:
- The VP5*-HD is essential for VP5*-mediated membrane permeabilization.
- Conserved glycines and a potentially random coiled structure within VP5*-HD are critical for function, not just hydrophobicity.
- VP5* likely facilitates rotavirus entry by enabling particle uncoating under specific ionic conditions rather than direct membrane lysis.