Selective membrane permeabilization by the rotavirus VP5* protein is abrogated by mutations in an internal hydrophobic domain.
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.
Dowling
W
W
Department of Medicine, SUNY at Stony Brook, Stony Brook, New York 11794-8173, USA.
Denisova
E
E
LaMonica
R
R
Mackow
E R
ER
D020816:Amino Acid Motifs; D002460:Cell Line; D002463:Cell Membrane Permeability; D003911:Dextrans / Q000737:chemistry; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009598:Nitrophenylgalactosides / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000737:chemistry; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000378:metabolism; D012401:Rotavirus / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D003911:Dextrans; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008081:Liposomes; D010713:Phosphatidylcholines; D016601:RNA-Binding Proteins; D017361:Viral Nonstructural Proteins; C015219:fluorescein isothiocyanate dextran; C104282:nsp1 protein, Rotavirus; C079234:NS35 protein, rotavirus; C055012:2-nitrophenylgalactoside; D009598:Nitrophenylgalactosides; C024098:6-carboxyfluorescein; D016650:Fluorescein-5-isothiocyanate