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. 2004 Feb;78(4):2037-44.
doi: 10.1128/jvi.78.4.2037-2044.2004.

Discrete domains within the rotavirus VP5* direct peripheral membrane association and membrane permeability

Nina E Golantsova  1 , Elena E Gorbunova, Erich R Mackow
Affiliations

Discrete domains within the rotavirus VP5* direct peripheral membrane association and membrane permeability

Nina E Golantsova et al. J Virol. 2004 Feb.

Abstract

Cleavage of the rotavirus spike protein, VP4, is required for rotavirus-induced membrane permeability and viral entry into cells. The VP5* cleavage product selectively permeabilizes membranes and liposomes and contains an internal hydrophobic domain that is required for membrane permeability. Here we investigate VP5* domains (residues 248 to 474) that direct membrane binding. We determined that expressed VP5 fragments containing residues 248 to 474 or 265 to 474, including the internal hydrophobic domain, bind to cellular membranes but are not present in Triton X-100-resistant membrane rafts. Expressed VP5 partitions into aqueous but not detergent phases of Triton X-114, suggesting that VP5 is not integrally inserted into membranes. Since high-salt or alkaline conditions eluted VP5 from membranes, our findings demonstrate that VP5 is peripherally associated with membranes. Interestingly, mutagenesis of residue 394 (W-->R) within the VP5 hydrophobic domain, which abolishes VP5-directed permeability, had no effect on VP5's peripheral membrane association. In contrast, deletion of N-terminal VP5 residues (residues 265 to 279) abolished VP5 binding to membranes. Alanine mutagenesis of two positively charged residues within this domain (residues 274R and 276K) dramatically reduced (>95%) binding of VP5 to membranes and suggested their potential interaction with polar head groups of the lipid bilayer. Mutations in either the VP5 hydrophobic or basic domain blocked VP5-directed permeability of cells. These findings indicate that there are at least two discrete domains within VP5* required for pore formation: an N-terminal basic domain that permits VP5* to peripherally associate with membranes and an internal hydrophobic domain that is essential for altering membrane permeability. These results provide a fundamental understanding of interactions between VP5* and the membrane, which are required for rotavirus entry.

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Figures

FIG. 1.
FIG. 1.
Membrane association of the rotavirus VP5 proteins. HEK293 cells were transfected with pcDNA4 constructs expressing VP5N248, VP5N248(394R), or VP5N265. Cellular membranes were fractionated by sucrose gradient centrifugation. His-tagged VP5N248, VP5N248(394R), or VP5N265 proteins were precipitated from total cell lysates (Total), membrane fractions (Membrane), or soluble fractions (Soluble) using Ni-NTA agarose. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotted using an anti-HisG antibody.
FIG. 2.
FIG. 2.
(A) Triton X-100 treatment of VP5-containing membrane fractions. COS7 cells were transfected with plasmids expressing VP5N248 or VP5N248(394R), and membranes containing VP5N248 or VP5N248(394R) proteins were fractionated by sucrose gradient centrifugation and treated with 1% Triton X-100 at 4°C (+) or not treated (−). Samples were subjected to a second sucrose gradient centrifugation, and caveolin was detected by Western blotting using anti-caveolin 1 antibody. VP5N248 and VP5N248(394R) proteins from the membrane (M) or soluble (S) fraction were precipitated and analyzed as described in the legend to Fig. 1. (B) Triton X-114 phase partitioning of VP5. COS7 cells were transfected with plasmid expressing VP5N248 or VP5N248(394R) and fractionated by sucrose gradient centrifugation. Membrane fractions containing VP5N248 (lanes 1 and 3) or VP5N248(394R) (lanes 2 and 4) were subjected to Triton X-114 phase partitioning, and aliquots were analyzed for caveolin by Western blotting. VP5 proteins from aqueous (lanes 1 and 2) or detergent (lanes 3 and 4) phases were analyzed and detected as described in the legend to Fig. 1.
FIG. 3.
FIG. 3.
(A) High-salt treatment of membrane fractions containing VP5. COS7 cells were transfected with plasmids expressing VP5N248 or VP5N248(394R). Membrane fractions containing VP5N248 or VP5N248(394R) were extracted with 1 M NaCl for 45 min at 4°C (+) or not treated (−). Following treatment, membranes were refloated on a second sucrose gradient, and caveolin was detected by Western blotting as described in the legend to Fig. 2. VP5N248 or VP5N248(394W→R) proteins from the membrane (M) or soluble (S) fractions were analyzed as described in the legend to Fig. 1. (B) Alkaline treatment of VP5-containing membrane fractions. HEK293 cells were transfected with plasmid expressing VP5N248 or VP5N248(394R). Membrane (M) fractions containing VP5N248 (+) or VP5N248(394R) (+) were treated with 0.1 M Na2CO3 (pH 11.5) for 45 min at 4°C (+) or not treated (−). Treated or untreated membrane fractions were refloated on a second sucrose gradient, and membrane fractions were Western blotted for VP5N248 or VP5N248(394R) protein.
FIG. 4.
FIG. 4.
Influence of N-terminal mutations on membrane association of VP5 proteins. HEK293 cells were transfected with plasmids expressing VP5N248, VP5N280, or VP5N248(274A,276A). Cellular membranes were fractionated by sucrose gradient centrifugation, and six-His-tagged VP5 proteins were precipitated from total cell lysates (Total), membrane fractions (Membrane), or soluble fractions (Soluble) using Ni-NTA agarose. Precipitated proteins were analyzed by Western blotting.
FIG. 5.
FIG. 5.
VP5-directed changes in intracellular Ca2+ concentration. HEK293 cells were transfected with plasmids expressing VP5N248, VP5N265, VP5N248(394R), or VP5N248(274A,276A) or not transfected. Cells were loaded with the calcium-sensitive fluorophore fluo-3, and fluorescence was monitored at 526 nm after extracellular [Ca2+] was increased to 5 to 8 mM for 30 to 60 s. Intracellular (in) [Ca2+] was calculated as described in Materials and Methods. The averages ± standard errors (error bars) of three separate measurements are shown.
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