Spatial relationships of the proteins of vesicular stomatitis virus: induction of reversible oligomers by cleavable protein cross-linkers and oxidation
Spatial relationships of the proteins of vesicular stomatitis virus: induction of reversible oligomers by cleavable protein cross-linkers and oxidation
复制标题
水泡性口炎病毒蛋白质的空间关系：通过可裂解蛋白质交联剂和氧化诱导可逆寡聚体
作者：
E. Dubovi;R. Wagner
为了描述完整的水泡性口炎（VS）病毒体的主要结构蛋白的接近性和空间排列，通过氧化或二价交联剂形成的蛋白复合物进行了分析，通过聚丙烯酰胺平板凝胶上的二维电泳。VS病毒体的H2 O2氧化在第一维凝胶上产生N-多肽二聚体（分子量，约等于110，000），其可以被还原成N个单体（分子量，约等于50，000）。从未还原和未氧化的VS病毒体中提取的蛋白质含有M-蛋白复合物的二聚体和三聚体形式以及M和N蛋白的异源二聚体。通过将VS病毒体暴露于可逆蛋白质交联剂甲基-4-巯基丁酰亚胺（MMB）、酒石酸二叠氮（TDA）和二硫代双（琥珀酰亚胺基丙酸酯）（DTBSP），生成质量相似的VS病毒蛋白复合物;通过用2-巯基乙醇（MMB或DTBSP交联）还原或通过高碘酸盐氧化（TDA交联）切割第一维凝胶上的交联复合物。除了共价连接的M和N蛋白质的同源二聚体和蛋白质M-N异源二聚体之外，蛋白质交联剂还产生G蛋白质的同源寡聚体和G-M异源二聚体。这些数据表明，VS病毒的糖蛋白刺突是由一个以上的G蛋白。N-M和G-M异源二聚体的存在与基质（M）蛋白可能在VS病毒粒子的组装中充当G和N蛋白之间的桥梁的假设一致。
To delineate the proximity and spatial arrangement of the major structural proteins of intact vesicular stomatitis (VS) virions, protein complexes formed by oxidation or by bivalent cross-linkers were analyzed by two-dimensional electrophoresis on polyacrylamide slab gels. H2O2 oxidation of VS virions produced an N-polypeptide dimer (molecular weight, approximately equal to 110,000) on a first dimension gel that could be reduced to N monomers (molecular weight, approximately equal to 50,000). Proteins extracted from unreduced and unoxidized VS virions contained dimeric and trimeric forms of M-protein complexes as well as a heterodimer of M and N protein. Qualitatively similar VS viral protein complexes were generated by exposing VS virions to the reversible protein cross-linkers methyl-4-mercaptobutyrimidate (MMB), tartryl diazide (TDA), and dithiobis(succinimidyl proprionate) (DTBSP); cross-linked complexes on first-dimension gels were cleaved by reduction with 2-mercaptoethanol (MMB or DTBSP cross-linked) or by periodate oxidation (TDA cross-linked). In addition to covalently linked homodiamers of M and N proteins and a protein M-N heterodimer, the protein cross-linkers also generated homo-oligomers of G protein and a G-M heterodimer. These data suggest that the glycoprotein spike of VS virus is composed of more than one G protein. The existence of N-M and G-M heterodimers is consistent with the hypothesis that the matrix (M) protein may serve as a bridge between the G and N proteins in assembly of the VS virion.