Meyer W J, Johnston R E
Department of Microbiology and Immunology, University of North Carolina, Chapel Hill 27599-7290.
J Virol. 1993 Sep;67(9):5117-25. doi: 10.1128/JVI.67.9.5117-5125.1993.
Sindbis virus glycoproteins E1 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 E1 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.
辛德毕斯病毒糖蛋白E1和E2在病毒与细胞早期相互作用期间，于细胞表面发生构象改变（D. Flynn、W. J. Meyer、J. M. MacKenzie, Jr.和R. E. Johnston，《病毒学杂志》64:3643 - 3653, 1990）。某些在天然病毒上通常位于内部的表位，在病毒颗粒附着后但内化前，可被单克隆抗体（MAb）识别。这些新暴露的表位，称为过渡性表位，可能是功能重要结构域的一部分，在改变后的病毒表面暴露出来，以促进病毒进入细胞。将辛德毕斯病毒粒子在51℃加热短时间，可诱导E1和E2糖蛋白上出现类似但不完全相同的过渡性表位暴露（W. J. Meyer、S. Gidwitz、V. K. Ayers、R. J. Schoepp和R. E. Johnston，《病毒学杂志》66:3504 - 3513, 1992）。在本报告中，我们鉴定了一些因病毒与细胞早期相互作用而暴露的过渡性表位。与重排的、加热的病毒粒子及病毒 - 细胞复合物结合的过渡性表位单克隆抗体，用于对加热的辛德毕斯病毒粒子进行抗体竞争结合试验，以绘制天然、外部、中和性抗原位点与新暴露的过渡性表位之间的空间关系。由于加热、重排的粒子保留了其感染性，与过渡性表位结合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E1和E2的特定基因座，在这些基因座发生的突变可阻止单克隆抗体与过渡性表位结合。鉴定出的一个过渡性表位（E2的200至202位氨基酸残基）位于E2的190 - 216区域，该区域包含两个主要中和位点E2a和E2b，以及E2 196位的一个N - 连接糖基化位点。通过对辛德毕斯病毒基因组全长cDNA克隆进行定点诱变消除了糖基化信号。碳水化合物部分的缺失并未暴露定位于该基因座的过渡性表位，这表明在天然病毒粒子上，E2 200 - 202决定簇的不可接近性是糖蛋白刺突结构所固有的。
