Selective membrane permeabilization by the rotavirus VP5* protein is abrogated by mutations in an internal hydrophobic domain
| Autor: | Erich R. Mackow, William Dowling, Rachel LaMonica, Evgeniya A. Denisova |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Rotavirus
Lysis Cell Membrane Permeability Membrane permeability Endosome viruses Immunology Mutant Amino Acid Motifs lac operon Biology Viral Nonstructural Proteins Cleavage (embryo) Microbiology Cell Line Viral entry Virology Fluorescent Dyes RNA-Binding Proteins Dextrans Fluoresceins Nitrophenylgalactosides Cell biology Protein Structure Tertiary Virus-Cell Interactions Membrane Biochemistry Insect Science Liposomes Mutagenesis Site-Directed Phosphatidylcholines Fluorescein-5-isothiocyanate |
| Zdroj: | Journal of virology. 74(14) |
| ISSN: | 0022-538X |
| Popis: | 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-β- d -galactopyranoside (ONPG) into cells induced to express VP5* or mutated VP5* polypeptides. Following IPTG (isopropyl-β- 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. |
| Databáze: | OpenAIRE |
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