Elliott G, O'Hare P
Marie Curie Research Institute, Oxted, Surrey, UK.
Gene Ther. 1999 Jan;6(1):149-51. doi: 10.1038/sj.gt.3300850.
The herpes simplex virus protein VP22 exhibits the unusual property of intercellular transport whereby after being synthesised in a subpopulation of cells, in which it is largely cytoplasmic, the protein is transported to adjacent cells where it accumulates mainly in the nucleus. Here we examine the transport of a fusion protein consisting of VP22 linked to the green fluorescent protein (GFP). Intercellular transport, nuclear accumulation and chromatin binding of VP22-GFP could be detected by intrinsic GFP fluorescence in fixed cells. However, while the cytoplasmic localisation of VP22-GFP could be detected in live cells actively synthesising the protein, we were unable to detect intercellular transport by intrinsic GFP fluorescence in livecells, indicating that the levels of transported protein may be below those required for live detection, or that GFP fluorescence was quenched. The use of antibody to GFP was more sensitive than intrinsic GFP fluorescence and allowed ready detection of transport and nuclear accumulation of VP22-GFP. Intercellular transport was also confirmed in coplating experiments. Consistent with previous results showing a requirement for the C-terminus of VP22 in transport of the native protein, a fusion protein consisting of GFP linked to the N-terminal 1-192 residues of VP22 failed to transport between cells. The results support the proposal that VP22 has the ability to transport cargo proteins between cells and that it has significant potential in the field of gene therapy.
单纯疱疹病毒蛋白VP22具有细胞间运输这一非凡特性，即该蛋白在一部分细胞中合成后，主要位于细胞质中，随后被转运至相邻细胞，并主要在细胞核中积累。在此，我们研究了一种由与绿色荧光蛋白（GFP）相连的VP22组成的融合蛋白的运输情况。在固定细胞中，可通过GFP自身荧光检测到VP22-GFP的细胞间运输、核积累及与染色质的结合。然而，虽然在活跃合成该蛋白的活细胞中能检测到VP22-GFP的细胞质定位，但我们无法通过活细胞中GFP自身荧光检测到细胞间运输，这表明运输蛋白的水平可能低于活细胞检测所需水平，或者GFP荧光被淬灭了。使用抗GFP抗体比GFP自身荧光更灵敏，能够轻易检测到VP22-GFP的运输和核积累。在共培养实验中也证实了细胞间运输。与先前结果一致，先前结果表明天然蛋白运输需要VP22的C末端，由GFP与VP22的N末端1 - 192个残基相连组成的融合蛋白无法在细胞间运输。这些结果支持了VP22具有在细胞间运输货物蛋白的能力这一观点，并且它在基因治疗领域具有巨大潜力。
