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.
Taylor
J A
JA
Centre for Gene Technology, School of Biological Sciences, University of Auckland, New Zealand.
O'Brien
J A
JA
Lord
V J
VJ
Meyer
J C
JC
Bellamy
A R
AR
D001692:Biological Transport; D003432:Cross-Linking Reagents; D004252:DNA Mutational Analysis; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008961:Models, Structural; D011991:Receptors, Virus / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012401:Rotavirus / Q000378:metabolism* / Q000648:ultrastructure; D014779:Virus Replication
D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase