Metzner Christoph, Mostegl Meike M, Günzburg Walter H, Salmons Brian, Dangerfield John A
Institute of Virology, University of Veterinary Medicine, Veterinärplatz 1, A-1210 Vienna, Austria.
FASEB J. 2008 Aug;22(8):2734-9. doi: 10.1096/fj.08-108217. Epub 2008 May 13.
We describe for the first time the association of glycosylphosphatidylinositol (GPI) -anchored proteins with retroviral and lentiviral particles, similar to a process well established for cells, termed "painting." The aim of the study was to assess the feasibility of modification of retroviral vectors by exogenous addition of recombinant protein, removing the need for genetic engineering of virus producer cell lines. The recombinant GPI protein CD59his was purified via fast protein liquid chromatography and associated with concentrated virus stock in a controlled incubation procedure. Reaction mixtures were purified in order to remove nonassociated GPI protein and endogenous protein. Analysis of samples by immunoblotting revealed that CD59his was only detectable in the presence of viral particles. From this, we conclude that CD59his could be stably associated with retroviral particles. In addition, we demonstrated by flow cytometry that virus particles remain infectious after these procedures. As well as suggesting a novel possibility for interaction between enveloped virus and host, we believe that the stable association of recombinant GPI proteins to retroviral particles can be developed into an important tool for both research and clinical applications, especially in the fields of gene therapy and vaccine development.
我们首次描述了糖基磷脂酰肌醇（GPI）锚定蛋白与逆转录病毒和慢病毒颗粒的关联，这类似于在细胞中已确立的一种过程，称为“描绘”。本研究的目的是评估通过外源添加重组蛋白来修饰逆转录病毒载体的可行性，从而无需对病毒生产细胞系进行基因工程改造。重组GPI蛋白CD59his通过快速蛋白质液相色谱法纯化，并在可控的孵育过程中与浓缩病毒原液结合。反应混合物经过纯化以去除未结合的GPI蛋白和内源性蛋白。通过免疫印迹分析样品发现，只有在存在病毒颗粒的情况下才能检测到CD59his。由此，我们得出结论，CD59his可以与逆转录病毒颗粒稳定结合。此外，我们通过流式细胞术证明，这些操作后病毒颗粒仍具有感染性。除了提示包膜病毒与宿主之间相互作用的新可能性外，我们认为重组GPI蛋白与逆转录病毒颗粒的稳定结合可发展成为研究和临床应用的重要工具，特别是在基因治疗和疫苗开发领域。
