Vellinga Jort, Uil Taco G, de Vrij Jeroen, Rabelink Martijn J W E, Lindholm Leif, Hoeben Rob C
Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
J Gene Med. 2006 Feb;8(2):147-54. doi: 10.1002/jgm.844.
The adenovirus 14.3 kDa hexon-associated protein IX (pIX) functions in the viral capsid as 'cement' and assembles the hexons in stable groups-of-nine (GONs). Although viruses lacking pIX do not form GONs, and are less heat-stable than wild-type (wt) viruses, they can be propagated with the same kinetics and yields as the wt viruses. To facilitate 'pseudotyping' of adenoviral vectors we have set up an efficient system for the generation of pIX-producing helper cell lines.
With a lentiviral pIX-expression cassette, monoclonal and polyclonal helper cell lines were generated, which express wt or modified pIX genes at levels equivalent to wt HAdV-5 infected cells. The incorporation efficiency into pIX gene deleted viruses was examined by Western analysis, immuno-affinity electron microscopy, and heat-stability assays.
Immuno-affinity electron microscopy on viruses lacking the pIX gene demonstrated that more than 96% of the particles contain pIX protein in their capsids after propagation on the pIX-expressing helper cell lines. In addition, the pIX level in the helper cells was sufficient to generate heat-stable particles. Finally, the ratio between pIX and fiber was equivalent to that found in wt particles. The pIX-producing cell lines are very stable, demonstrating that pIX is not toxic to cells.
These data demonstrate that lentivirus vectors can be used for the establishment of pIX-complementing helper cell lines.
腺病毒14.3 kDa六邻体相关蛋白IX（pIX）在病毒衣壳中起“黏合剂”的作用，将六邻体组装成稳定的九聚体（GONs）。虽然缺乏pIX的病毒不形成GONs，且热稳定性低于野生型（wt）病毒，但它们能够以与wt病毒相同的动力学和产量进行增殖。为便于腺病毒载体的“假型化”，我们建立了一个高效的系统来产生表达pIX的辅助细胞系。
利用慢病毒pIX表达盒，产生了单克隆和多克隆辅助细胞系，这些细胞系表达的野生型或修饰型pIX基因水平与wt HAdV-5感染的细胞相当。通过蛋白质免疫印迹分析、免疫亲和电子显微镜和热稳定性测定，检测了其整合到缺失pIX基因的病毒中的效率。
对缺乏pIX基因的病毒进行免疫亲和电子显微镜分析表明，在表达pIX的辅助细胞系上增殖后，超过96%的病毒颗粒在其衣壳中含有pIX蛋白。此外，辅助细胞中的pIX水平足以产生热稳定颗粒。最后，pIX与纤维的比例与wt颗粒中的比例相当。产生pIX的细胞系非常稳定，表明pIX对细胞无毒。
这些数据表明慢病毒载体可用于建立pIX互补辅助细胞系。
