Green fluorescent protein-tagged adeno-associated virus particles allow the study of cytosolic and nuclear trafficking
Green fluorescent protein-tagged adeno-associated virus particles allow the study of cytosolic and nuclear trafficking
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DOI：
10.1128/jvi.79.18.11776-11787.2005
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发表时间：
2005-09-01
影响因子：
5.4
通讯作者：
Büning, H
中科院分区：
文献类型：
--
作者：
Lux, K;Goerlitz, N;Büning, H
为了实现病毒运输的直接可视化，我们通过用 GFP-VP2 融合蛋白替换野生型 VP2，将增强型绿色荧光蛋白 (GFP) 基因整合到腺相关病毒 (AAV) 衣壳中。获得高滴度病毒后代并用于阐明进入核的过程。在没有腺病毒 5 (Ad5) 的情况下，AAV 衣壳的核转位是一个缓慢且低效的过程：在感染后 2 小时和 4 小时 (p.i.)，在核周区域和核内陷中发现了 GFP-VP2-AAV 颗粒，但在核内没有发现。在 Ad5 共感染的细胞中，注射后 2 小时，细胞核中已可检测到分离的 GFP-VP2-AAV 颗粒，表明 Ad5 增强了 AAV 衣壳的核转位。细胞核内展示病毒衣壳的细胞数量随着时间的推移略有增加，与辅助病毒水平无关，但在所有分析条件下，大多数 AAV 衣壳仍保留在核周区域。相比之下，独立于辅助病毒，并且在注射后 2 小时观察到每个细胞的病毒粒子减少了 10 倍，病毒基因组在细胞核内可见。在这些条件下，即使延长孵育时间（注射后长达 11 小时），细胞核内也检测不到完整的病毒衣壳。总之，结果表明 GFP 标记的 AAV 颗粒可用于研究 AAV 的细胞运输和入核。此外，我们的研究结果反对完整 AAV 衣壳的有效入核机制，并支持在入核之前或期间发生病毒脱壳。
To allow the direct visualization of viral trafficking, we genetically incorporated enhanced green fluorescent protein (GFP) into the adeno-associated virus (AAV) capsid by replacement of wild-type VP2 by GFP-VP2 fusion proteins. High-titer virus progeny was obtained and used to elucidate the process of nuclear entry. In the absence of adenovirus 5 (Ad5), nuclear translocation of AAV capsids was a slow and inefficient process: at 2 h and 4 h postinfection (p.i.), GFP-VP2-AAV particles were found in the perinuclear area and in nuclear invaginations but not within the nucleus. In Ad5-coinfected cells, isolated GFP-VP2-AAV particles were already detectable in the nucleus at 2 h p.i., suggesting that Ad5 enhanced the nuclear translocation of AAV capsids. The number of cells displaying viral capsids within the nucleus increased slightly over time, independently of helper virus levels, but the majority of the AAV capsids remained in the perinuclear area under all conditions analyzed. In contrast, independently of helper virus and with 10 times less virions per cell already observed at 2 h p.i., viral genomes were visible within the nucleus. Under these conditions and even with prolonged incubation times (up to 11 h p.i.), no intact viral capsids were detectable within the nucleus. In summary, the results show that GFP-tagged AAV particles can be used to study the cellular trafficking and nuclear entry of AAV. Moreover, our findings argue against an efficient nuclear entry mechanism of intact AAV capsids and favor the occurrence of viral uncoating before or during nuclear entry.