STRUCTURAL REARRANGEMENT OF INFECTING SINDBIS VIRIONS AT THE CELL-SURFACE - MAPPING OF NEWLY ACCESSIBLE EPITOPES
STRUCTURAL REARRANGEMENT OF INFECTING SINDBIS VIRIONS AT THE CELL-SURFACE - MAPPING OF NEWLY ACCESSIBLE EPITOPES
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DOI：
10.1128/jvi.67.9.5117-5125.1993
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发表时间：
1993-09-01
影响因子：
5.4
通讯作者：
JOHNSTON, RE
中科院分区：
文献类型：
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作者：
MEYER, WJ;JOHNSTON, RE
辛德毕斯病毒糖蛋白E1和E2在早期病毒-细胞相互作用期间在细胞表面经历构象改变（D. Flynn，W. J. Meyer，J. M.小麦肯齐和R. E.约翰斯顿，J. 64：3643-3653，1990）。天然病毒内部的某些表位在附着后但在病毒颗粒内化之前变得可接近单克隆抗体（MAb）结合。这些新暴露的表位，称为过渡表位，可能是功能重要的结构域的一部分，在改变的病毒表面可接近，以促进进入细胞。将辛德毕斯病毒粒子在51 ℃下短时间加热诱导了类似的，尽管不完全相同的E1和E2糖蛋白上的过渡表位的暴露（W. J. Meyer，S. Gidwitz，V. K.艾尔斯河Schoepp和R. E.约翰斯顿，J. 66：3504-3513，1992）。在目前的报告中，我们已经确定了几个过渡表位，成为暴露的早期病毒细胞相互作用的结果。结合重排的、加热的病毒体和病毒-细胞复合物的过渡表位MAb用于加热的辛德毕斯病毒体的抗体竞争结合测定，以绘制天然的、外部的、中和抗原位点和新暴露的过渡表位之间的空间关系。由于加热重排的颗粒保留了它们的感染性，结合过渡表位的单克隆抗体也被用于分离单克隆抗体中和逃逸突变体。对逃逸突变体的糖蛋白基因进行测序，确定了特定的E1和E2位点，其中突变阻止了MAb与过渡表位的结合。鉴定的过渡表位之一（E2残基200至202）位于E2 190-216区域，该区域含有两个主要中和位点E2 a和E2 b，以及E2 196处的N-连接糖基化位点。通过辛德毕斯病毒基因组全长cDNA克隆的定点诱变消除糖基化信号。碳水化合物部分的缺失并没有暴露映射到该位点的过渡表位，这表明在天然病毒粒子上，E2 200-202决定簇的不可及性是糖蛋白刺突结构中固有的。
Sindbis virus glycoproteins El and E2 undergo a conformational alteration during early virus-cell interaction at the cell surface (D. Flynn, W. J. Meyer, J. M. MacKenzie, Jr., and R. E. Johnston, J. Virol. 64:3643-3653, 1990). Certain epitopes normally internal on native virus become accessible to monoclonal antibody (MAb) binding after attachment but before internalization of virus particles. These newly exposed epitopes, termed transitional epitopes, may be part of functionally important domains made accessible at the surface of the altered virus to facilitate entry into cells. Heating Sindbis virions at 51-degrees-C for a short time induced a similar, although not identical, exposition of transitional epitopes on the El and E2 glycoproteins (W. J. Meyer, S. Gidwitz, V. K. Ayers, R. J. Schoepp, and R. E. Johnston, J. Virol. 66:3504-3513, 1992). In the current report, we have identified several of the transitional epitopes that become exposed as a consequence of early virus-cell interactions. Transitional epitope MAbs that bound to rearranged, heated virions and virus-cell complexes were used in antibody competition binding assays on heated Sindbis virions to map the spatial relationships between native, external, neutralizing antigenic sites and newly exposed transitional epitopes. Because the heated, rearranged particles retained their infectivity, MAbs that bound to transitional epitopes also were used to isolate MAb neutralization escape mutants. Sequencing the glycoprotein genes of the escape mutants identified specific E1 and E2 loci where mutation prevented MAb binding to transitional epitopes. One of the transitional epitopes identified (E2 residues 200 to 202) lies in the E2 190-216 region, which harbors two major neutralization sites, E2a and E2b, and an N-linked glycosylation site at E2 196. The glycosylation signal was eliminated by site-directed mutagenesis of a full-length cDNA clone of the Sindbis virus genome. The absence of a carbohydrate moiety did not expose the transitional epitopes mapped to this locus, suggesting that on native virions, the inaccessibility of the E2 200-202 determinant was inherent in the structure of the glycoprotein spike.