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.
Centre for Gene Technology, School of Biological Sciences, University of Auckland, New Zealand.
J A
JA
Taylor
J A
JA
O'Brien
V J
VJ
Lord
J C
JC
Meyer
A R
AR
Bellamy
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