Meulenbroek Robert A, Sargent Kathy L, Lunde John, Jasmin Bernard J, Parks Robin J
Molecular Medicine Program, Ottawa Health Research Institute, Room, 501 Smyth Road, Ottawa, ON K1H 8L6, Canada.
Mol Ther. 2004 Apr;9(4):617-24. doi: 10.1016/j.ymthe.2004.01.012.
The adenovirus (Ad) protein IX (pIX) is a minor component of the Ad capsid and associates with the hexons that make up the facets of the icosahedron. In this study, we investigated whether a large protein tag could be fused to pIX without compromising the Ad vector itself. As proof-of-principle, we generated a pIX-green fluorescent protein (GFP) fusion protein. We show that a virus encoding the pIX-GFP can be generated and that pIX-GFP fusion protein was incorporated into the Ad capsid as efficiently as native pIX. In tissue culture, translocation of Ad/pIX-GFP from the outside of the cell to the nucleus could be followed using fluorescence microscopy, and the timing of migration to the nucleus was similar to that previously reported for Ad. We also could track the virus after injection into the tibialis anterior muscle of mice. Shortly after injection, the majority of the Ad/pIX-GFP accumulated in pockets adjacent to the muscle fibers, with some migration of the virus between fibers. Our ability to attach GFP to the Ad virion, through fusion to pIX, provides a valuable tool for virus tracking in vitro and in vivo. Moreover, our data indicate that pIX can be used as a platform to anchor proteins to the Ad capsid, such as large ligands for cell-type-specific targeting of the vector.
腺病毒（Ad）蛋白IX（pIX）是Ad衣壳的次要成分，与构成二十面体小面的六邻体相关联。在本研究中，我们调查了是否可以在不影响Ad载体本身的情况下将大的蛋白标签融合到pIX上。作为原理验证，我们生成了一种pIX-绿色荧光蛋白（GFP）融合蛋白。我们表明，可以产生编码pIX-GFP的病毒，并且pIX-GFP融合蛋白与天然pIX一样有效地整合到Ad衣壳中。在组织培养中，使用荧光显微镜可以追踪Ad/pIX-GFP从细胞外部向细胞核的转运，并且向细胞核迁移的时间与先前报道的Ad相似。在将病毒注射到小鼠胫前肌后，我们也能够追踪病毒。注射后不久，大多数Ad/pIX-GFP积聚在与肌纤维相邻的囊袋中，病毒在纤维之间有一些迁移。我们通过与pIX融合将GFP连接到Ad病毒体上的能力，为体外和体内病毒追踪提供了一种有价值的工具。此外，我们的数据表明，pIX可以用作将蛋白质锚定到Ad衣壳上的平台，例如用于载体细胞类型特异性靶向的大配体。
