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 VPS*, permeabilizes membranes, suggesting that cleavage exposes a membrane-interactive domain of VPS* which effects rapid vital entry. The VPS* protein contains a single long hydrophobic domain (VPS*-HD, residues 385 to 404) at an internal site. In order to address the role of the VPS*-HD in permeabilizing cellular membranes, we analyzed the entry of o-nitrophenyl-β-D-galactopyranoside (ONPG) into cells induced to express VPS* or mutated VPS* 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 VPS* 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 VPS-HD further demonstrated a requirement for residues within the HD for VPS*-induced membrane permeability. Functional analysis of mutant VPS*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 VPS* 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.