ROTAVIRUS PROTEIN-VP7, PROTEIN-NS28, AND PROTEIN-VP4 FORM OLIGOMERIC STRUCTURES
ROTAVIRUS PROTEIN-VP7, PROTEIN-NS28, AND PROTEIN-VP4 FORM OLIGOMERIC STRUCTURES
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DOI：
10.1128/jvi.64.6.2632-2641.1990
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发表时间：
1990-06-01
影响因子：
5.4
通讯作者：
ATKINSON, PH
中科院分区：
文献类型：
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作者：
MAASS, DR;ATKINSON, PH
蔗糖梯度沉降分析表明，轮状病毒sa11感染的Ma104细胞存在结构糖蛋白VP7和非结构糖蛋白NS28的低聚物。在蔗糖梯度离心之前或之后，将蛋白质交联可以稳定低聚物，免疫沉淀后可以通过非还原性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行分析。主要的NS28寡聚物为四聚体，但也观察到二聚体和高阶结构。VP7主要形成二聚体，四聚体和更高的低聚体形式也存在。无论是否存在交联，VP7和NS28的每个低聚物都以相同的特征速率通过蔗糖梯度沉积。单体不能交联形成低聚物，这表明交联低聚物不是人工地从单体中衍生出来的。逆转免疫沉淀的VP7在还原SDS-PAGE上的交联，导致VP7只出现单体形式。NS28低聚物的解离产生稳定的二聚体和单体。在快速沉淀的馏分中，观察到含有轮状病毒外壳蛋白VP7和VP4与NS28交联的16S - 20S复合物。这些复合物被证明与任何已知的亚病毒颗粒无关。VP4、NS28和VP7的结合可能代表内质网膜上的位点，但参与单壳颗粒出芽进入内质网管腔，并在管腔成熟为双壳颗粒。
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 as 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 but participate in the budding of the single-shelled particles into the lumen of the endoplasmic reticulum, where maturation to double-shelled particles occurs.