The RER-localized rotavirus intracellular receptor: a truncated purified soluble form is multivalent and binds virus particles.

A budding event transfers the immature, single-shelled rotavirus particle (SSP) across the RER membrane prior to assembly of mature virions in the ER lumen. Budding is triggered by the interaction of the SSP with a viral receptor glycoprotein (NS28) which is located in the RER membrane. We have expressed the cytoplasmic domain of the NS28 receptor as a glutathione S-transferase fusion protein to generate a soluble polypeptide that in turn can be cleaved to yield a carboxy-terminal receptor domain. The soluble terminal domain (delta 1-85 NS28) has been purified to homogeneity and retains SSP-binding activity when immobilized on a solid matrix. Integral membrane status therefore is not an essential prerequisite for ligand binding. The Kd for the interaction between immobilized delta 1-85 NS28 and purified particles is 4.6 x 10(-11) M, a value indistinguishable from the value obtained for the full-length and membrane-anchored receptor. Cross-linking with the bifunctional reagent dimethylsuberimidate indicates that delta 1-85 NS28 is a tetramer. When delta 1-85 NS28 is added to a monodisperse suspension of purified virus, the particles aggregate, indicating that the receptor is multivalent. The rotavirus intracellular receptor therefore provides a model for the detailed analysis of the early events that trigger the budding of cytoplasmically located particles across cell membranes.

Transient expression and mutational analysis of the rotavirus intracellular receptor: the C-terminal methionine residue is essential for ligand binding.

Maturation of rotavirus involves an intracellular membrane budding event in which the single-shelled icosahedral particle interacts with a virus-encoded receptor glycoprotein, NS28, that is located in the rough endoplasmic reticulum membrane. The receptor is a tetramer and is oriented with the C-terminal 131 amino acids on the cytoplasmic side of the membrane (A.R. Bellamy and G.W. Both, Adv. Virus Res. 38:1-48, 1990). We have used the T7-vaccinia virus transient expression system to deliver mutant variants of the NS28 gene to CV1 cells in order to assess the effects of site-specific modifications on receptor function. Three types of mutant proteins have been constructed by altering the extreme C-terminal methionine, cysteine residues within the third hydrophobic domain, and internal residues located within the cytoplasmic portion of the receptor, respectively. Deletion or conservative substitution of the C-terminal methionine completely abolishes receptor activity. Substitution of cysteine residues has no effect on receptor activity or on the ability of the receptor to adopt its native oligomeric state. Internal deletions result only in a reduction in the level of binding. An N-terminally truncated form of the receptor, containing only the cytoplasmic domain, retains full receptor activity and can form membrane-associated tetramers.