Rotavirus proteins VP7, NS28, and VP4 form oligomeric structures.
Sucrose gradient sedimentation analysis of rotavirus SA11-infected Ma104 cells revealed the presence of oligomers of VP7, the structural glycoprotein, and NS28, the nonstructural glycoprotein. Cross-linking the proteins, either before or after sucrose gradient centrifugation, stabilizes oligomers, which can be analyzed by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after immunoprecipitation. The major NS28 oligomer was tetrameric, though dimers and higher-order structures were observed as well. VP7 formed predominantly dimers, and again tetramers and higher oligomeric forms were present. Each oligomer of VP7 and NS28 sedimented at the same characteristic rate through the sucrose gradient either in the presence or absence of cross-linking. Monomers could not be cross-linked to form oligomers, demonstrating that cross-linked oligomers were not artifactually derived from monomers. Reversing the cross-linking of immunoprecipitated VP7 on reducing SDS-PAGE resulted in the appearance of only the monomeric form of VP7. Dissociation of the NS28 oligomers resulted in stable dimers as well an monomers. In the faster-sedimenting fractions, a 16S to 20S complex, which contained the rotavirus outer shell proteins VP7 and VP4 cross-linked to NS28, was observed. These complexes were shown not to be associated with any known subviral particle. The association of VP4, NS28, and VP7 may represent sites on the endoplasmic reticulum membrane that participate in the budding of the single-shelled particles into the lumen of the endoplasmic reticulum, where maturation to double-shelled particles occurs.
Maass
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DR
Department of Developmental Biology and Cancer, Albert Einstein College of Medicine, Bronx, New York 10461.
Atkinson
P H
PH
D000118:Acetylglucosaminidase; D000818:Animals; D000956:Antigens, Viral; D002213:Capsid / Q000302:isolation & purification / Q000378:metabolism*; D036022:Capsid Proteins; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D046911:Macromolecular Substances; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008970:Molecular Weight; D012401:Rotavirus / Q000235:genetics / Q000378:metabolism*; D013388:Succinimides / Q000378:metabolism; D014162:Transfection; D014758:Viral Core Proteins / Q000302:isolation & purification / Q000378:metabolism*; D017361:Viral Nonstructural Proteins
D000956:Antigens, Viral; D036022:Capsid Proteins; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D013388:Succinimides; C060339:VP4 protein, Rotavirus; C050779:VP7 protein, Rotavirus; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; C011240:dithiobis(succinimidylpropionate)