Chen D Y, Estes M K, Ramig R F
Division of Molecular Virology, Baylor College of Medicine, Houston, Texas 77030.
J Virol. 1992 Jan;66(1):432-9. doi: 10.1128/JVI.66.1.432-439.1992.
We previously reported that the expression of rotavirus phenotypes by reassortants was affected by recipient genetic background and proposed specific interactions between the outer capsid proteins VP4 and VP7 as the basis for the phenotypic effects (D. Chen, J. W. Burns, M. K. Estes, and R. F. Ramig, Proc. Natl. Acad. Sci. USA 86:3743-3747, 1989). A neutralizing, cross-reactive VP4-specific monoclonal antibody (MAb), 2G4, was used to probe the protein-protein interactions. The VP4 specificity of 2G4 was confirmed by immunoblot analysis. MAb 2G4 reacted with both standard (SA11-C13) and variant rotavirus SA11 (SA11-4F) but did not react with bovine rotavirus B223 as determined by plaque reduction neutralization (PRN) and enzyme-linked immunosorbent assay (ELISA). When a panel of SA11-4F/B223 and SA11-Cl3/B223 reassortants in purified or crude lysate form that had been grown in the presence or absence of trypsin was analyzed with MAb 2G4 by PRN and ELISA, the results with some reassortants were unexpected. That is, MAb 2G4 reacted with VP4 of SA11 parental origin (4F or C13) when it was assembled into capsids with the homologous SA11 VP7 but failed to react with VP4 of SA11 assembled into capsids with heterologous B223 VP7. Conversely, MAb 2G4 failed to react with VP4 of B223 parental origin when it was assembled into capsids with homologous B223 VP7 but did react with B223 VP4 assembled into capsids with the heterologous SA11 VP7. Similar reactivity was observed when 2G4 was used to immunoprecipitate purified double-shelled virions. When soluble unassembled viral proteins were analyzed by ELISA, the 2G4 reactive pattern was as predicted from the parental origin of VP4. That is, 2G4 reacted with the soluble VP4 of reassortants having VP4 from SA11-Cl3 or SA11-4F and failed to react with VP4 of B223 origin, regardless of the origin of VP7. PRN and ELISA results obtained with nonglycosylated viruses revealed that the unexpected reactivity of 2G4 with virus particles was not the result of differential glycosylation of VP7 and epitope masking. These results indicate that the 2G4 epitope existed in the soluble form of VP4 encoded by SA11-Cl3 or SA11-4F but not in soluble B223 VP4. On the other hand, in assembled virions, the presentation of the 2G4 epitope on VP4 was unexpected in some reassortants and was affected by the specific interactions between VP4 and VP7 of heterologous parental origin.
我们之前报道过，重配病毒轮状病毒表型的表达受受体遗传背景的影响，并提出外衣壳蛋白VP4和VP7之间的特定相互作用是表型效应的基础（D. Chen、J. W. Burns、M. K. Estes和R. F. Ramig，《美国国家科学院院刊》86:3743 - 3747，1989年）。一种具有中和作用、交叉反应性的VP4特异性单克隆抗体（MAb）2G4被用于探究蛋白质 - 蛋白质相互作用。通过免疫印迹分析证实了2G4的VP4特异性。通过蚀斑减少中和试验（PRN）和酶联免疫吸附测定（ELISA）确定，单克隆抗体2G4与标准轮状病毒（SA11 - C13）和变异轮状病毒SA11（SA11 - 4F）均发生反应，但不与牛轮状病毒B223发生反应。当用PRN和ELISA用单克隆抗体2G4分析一组以纯化或粗裂解物形式存在的、在有或无胰蛋白酶存在的情况下培养的SA11 - 4F/B223和SA11 - Cl3/B223重配病毒时，一些重配病毒的结果出乎意料。也就是说，当SA11亲本来源（4F或C13）的VP4与同源SA11 VP7组装成衣壳时，单克隆抗体2G4与之发生反应，但当SA11的VP4与异源B223 VP7组装成衣壳时，2G4不与之反应。相反，当B223亲本来源的VP4与同源B223 VP7组装成衣壳时，单克隆抗体2G4不与之反应，但当B223 VP4与异源SA11 VP7组装成衣壳时，2G4与之反应。当用2G4免疫沉淀纯化的双层病毒粒子时，观察到类似的反应性。当通过ELISA分析可溶性未组装病毒蛋白时，2G4反应模式与VP4的亲本来源预测一致。也就是说，2G4与具有来自SA11 - Cl3或SA11 - 4F的VP4的重配病毒的可溶性VP4发生反应，而不与B223来源的VP4发生反应，无论VP7的来源如何。用非糖基化病毒获得的PRN和ELISA结果表明，2G4与病毒颗粒的意外反应性不是VP7差异糖基化和表位掩盖的结果。这些结果表明，2G4表位存在于由SA11 - Cl3或SA11 - 4F编码的VP4的可溶性形式中，而不存在于可溶性B223 VP4中。另一方面，在组装的病毒粒子中，VP4上2G4表位的呈现在一些重配病毒中出乎意料，并且受异源亲本来源的VP4和VP7之间的特定相互作用影响。
