Kremser Leopold, Petsch Martina, Blaas Dieter, Kenndler Ernst
Institute of Analytical Chemistry, University of Vienna, Austria.
Electrophoresis. 2006 Jul;27(13):2630-7. doi: 10.1002/elps.200600066.
Human rhinoviruses (HRVs), the main etiologic agents of the common cold, transform into subviral B- or 80S particles (they sediment at 80S upon sucrose density gradient centrifugation) during infection and, in vitro, upon exposure to a temperature between 50 and 56 degrees C. With respect to the native virion they lack the genomic RNA and the viral capsid protein VP4. 80S particles are unstable and easily disintegrate into their components, VP1, VP2, and VP3 in buffers containing SDS. However, this detergent was found to be a necessary constituent of the BGE for the analysis of these viruses and their complexes with receptors and antibodies by CE. We here demonstrate that dodecylpoly(ethyleneglycol ether) (D-PEG) a nonionic detergent, is suitable for analysis of subviral particles as it preserves their integrity, in contrast to SDS. Electrophoresis of the 80S particles in borate buffer (pH 8.3, 100 mM) containing 10 mM D-PEG resulted in a well-defined electrophoretic peak. The identity of the peak was confirmed, among other means, by complexation with mAb 2G2, which recognizes a structural epitope exclusively present on subviral particles but not on native virus. Upon incubation of the 80S particles with mAb 2G2 the peak disappeared, but a new peak, attributed to the antibody complex emerged. The separation system allowed following the time course of the transformation of intact HRV serotype 2 into 80S particles upon incubation at temperatures between 40 and 65 degrees C. We also demonstrate that subviral particles derived from HRV2 labeled with the fluorescence dyes FITC or Cy3.5 were stable in the separation system containing D-PEG. Dye-modified particles were still recognized by mAb 2G2, suggesting that the exposed lysines that are derivatized by the reagent do not form part of the epitope of the antibody.
人鼻病毒（HRVs）是普通感冒的主要病原体，在感染期间以及在体外暴露于50至56摄氏度的温度下会转化为亚病毒B颗粒或80S颗粒（它们在蔗糖密度梯度离心时以80S沉降）。相对于天然病毒体，它们缺乏基因组RNA和病毒衣壳蛋白VP4。80S颗粒不稳定，在含有十二烷基硫酸钠（SDS）的缓冲液中很容易分解成其组成成分VP1、VP2和VP3。然而，发现这种去污剂是用于通过毛细管电泳（CE）分析这些病毒及其与受体和抗体复合物的硼酸盐凝胶电解质（BGE）的必要成分。我们在此证明，与SDS不同，非离子去污剂十二烷基聚（乙二醇醚）（D-PEG）适用于分析亚病毒颗粒，因为它能保持其完整性。在含有10 mM D-PEG的硼酸盐缓冲液（pH 8.3，100 mM）中对80S颗粒进行电泳，得到了一个清晰明确的电泳峰。通过与单克隆抗体2G2（mAb 2G2）结合等方法确认了该峰的身份，mAb 2G2识别仅存在于亚病毒颗粒而非天然病毒上的结构表位。80S颗粒与mAb 2G2孵育后，该峰消失，但出现了一个归因于抗体复合物的新峰。该分离系统能够追踪完整的HRV血清型2在40至65摄氏度孵育时转化为80S颗粒的时间进程。我们还证明，用荧光染料异硫氰酸荧光素（FITC）或Cy3.5标记的源自HRV2 的亚病毒颗粒在含有D-PEG的分离系统中是稳定的。染料修饰的颗粒仍能被mAb 2G2识别，这表明被试剂衍生化的暴露赖氨酸不是抗体表位的一部分。
