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.
Taylor
J A
JA
Centre for Gene Technology, School of Biological Sciences, University of Auckland, New Zealand.
Meyer
J C
JC
Legge
M A
MA
O'Brien
J A
JA
Street
J E
JE
Lord
V J
VJ
Bergmann
C C
CC
Bellamy
A R
AR
D000818:Animals; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004721:Endoplasmic Reticulum / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008024:Ligands; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011991:Receptors, Virus / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D012401:Rotavirus / Q000378:metabolism*; D014162:Transfection; D014763:Viral Matrix Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D006023:Glycoproteins; D008024:Ligands; D011991:Receptors, Virus; D014763:Viral Matrix Proteins; D008715:Methionine