Carleton M, Brown D T
The Cell Research Institute and Department of Microbiology, University of Texas at Austin, 78713-7640, USA.
J Virol. 1996 Aug;70(8):5541-7. doi: 10.1128/JVI.70.8.5541-5547.1996.
The Sindbis virus envelope is composed of 80 E1-E2 (envelope glycoprotein) heterotrimers organized into an icosahedral protein lattice with T=4 symmetry. The structural integrity of the envelope protein lattice is maintained by E1-E1 interactions which are stabilized by intramolecular disulfide bonds. Structural domains of the envelope proteins sustain the envelope's icosahedral lattice, while functional domains are responsible for virus attachment and membrane fusion. We have previously shown that within the mature Sindbis virus particle, the structural domains of the envelope proteins are significantly more resistant to the membrane-permeative, sulfhydryl-reducing agent dithiothreitol (DTT) than are the functional domains (R. P. Anthony, A. M. Paredes, and D. T. Brown, Virology 190:330-336, 1992). We have used DTT to probe the accessibility of intramolecular disulfides within PE2 (the precursor to E2) and E1, as these proteins fold and are assembled into the spike heterotrimer. We have determined through pulse-chase analysis that intramolecular disulfide bonds within PE2 are always sensitive to DTT when the glycoproteins are in the endoplasmic reticulum. The reduction of these disulfides results in the disruption of PE2-E1 associations. E1 acquires increased resistance to DTT as it folds through a series of disulfide intermediates (E1alpha, -beta, and -gamma) prior to assuming its native and most compact conformation (E1epsilon). The transition from a DTT-sensitive form into a form which exhibits increased resistance to DTT occurs after E1 has folded into its E1beta conformation and correlates temporally with the dissociation of BiP-E1 complexes and the formation of PE2-E1 heterotrimers. We propose that the disulfide bonds within E1 which stabilize the protein domains required for maintaining the structural integrity of the envelope protein lattice form early within the folding pathway of E1 and become inaccessible to DTT once the heterotrimer has formed.
辛德毕斯病毒包膜由80个E1-E2（包膜糖蛋白）异源三聚体组成，这些异源三聚体组装成具有T = 4对称性的二十面体蛋白质晶格。包膜蛋白质晶格的结构完整性通过E1-E1相互作用得以维持，这种相互作用由分子内二硫键稳定。包膜蛋白的结构域维持着包膜的二十面体晶格，而功能域则负责病毒的附着和膜融合。我们之前已经表明，在成熟的辛德毕斯病毒颗粒中，包膜蛋白的结构域比功能域对膜渗透性的巯基还原剂二硫苏糖醇（DTT）具有显著更高的抗性（R.P.安东尼、A.M.帕雷德斯和D.T.布朗，《病毒学》190:330 - 336，1992）。我们使用DTT来探测PE2（E2的前体）和E1内分子内二硫键的可及性，因为这些蛋白质折叠并组装成刺突异源三聚体。我们通过脉冲追踪分析确定，当糖蛋白在内质网中时，PE2内的分子内二硫键始终对DTT敏感。这些二硫键的还原导致PE2-E1关联的破坏。E1在折叠通过一系列二硫键中间体（E1α、-β和-γ）后，在呈现其天然且最紧密构象（E1ε）之前，对DTT的抗性增加。从对DTT敏感的形式转变为对DTT抗性增加的形式发生在E1折叠成其E1β构象之后，并且在时间上与BiP-E1复合物的解离以及PE2-E1异源三聚体的形成相关。我们提出，E1内稳定维持包膜蛋白晶格结构完整性所需蛋白质结构域的二硫键在E1折叠途径的早期形成，一旦异源三聚体形成，DTT就无法触及这些二硫键。
