Intracellular trafficking of adeno-associated virus vectors: Routing to the late endosomal compartment and proteasome degradation
Intracellular trafficking of adeno-associated virus vectors: Routing to the late endosomal compartment and proteasome degradation
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DOI：
10.1128/jvi.75.4.1824-1833.2001
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发表时间：
2001-02-01
影响因子：
5.4
通讯作者：
Danos, O
中科院分区：
文献类型：
--
作者：
Douar, AM;Poulard, K;Danos, O
腺相关病毒（AAV）感染的早期步骤涉及附着于多种细胞表面受体（硫酸乙酰肝素、整联蛋白和成纤维细胞生长因子受体1），随后进行网格蛋白依赖性或独立性内化。在此，我们研究了AAV颗粒从内体区室到细胞核的后续细胞内运输。在影响运输的试剂存在下，用携带报告基因（荧光素酶或绿色荧光蛋白）的重组AAV（rAAV）转导人细胞系。测定了巴弗洛霉素A（1）、布雷菲德菌素A和MG-132的作用。这些药物分别作用于内体酸化、早期至晚期内体转变和蛋白酶体活性水平。我们观察到转导病毒粒子需要被路由到晚期内体隔室。这种行为与腺病毒颗粒观察到的行为明显不同。MG-132的抗蛋白酶体治疗导致转导效率提高50倍。这种作用伴随着单链DNA AAV基因组的10倍细胞内积累，表明转导增强的机制不同于辅助腺病毒介导的机制，辅助腺病毒促进rAAV单链DNA基因组转化为其复制形式，MG-132是目前临床使用的药物，可以实际用于增强rAAV介导的治疗基因的递送。
The early steps of adeno-associated virus (AAV) infection involve attachment to a variety of cell surface receptors (heparan sulfate, integrins, and fibroblast growth factor receptor 1) followed by clathrin-dependent or independent internalization. Here we have studied the subsequent intracellular trafficking of AAV particles from the endosomal compartment to the nucleus, Human cell lines were transduced with a recombinant AAV (rAAV) carrying a reporter gene (luciferase or green fluorescent protein) in the presence of agents that affect trafficking. The effects of bafilomycin A(1), brefeldin A, and MG-132 were measured. These drugs act at the level of endosome acidification, early-to-late endosome transition, and proteasome activity, respectively. We observed that the transducing virions needed to be routed as far as the late endosomal compartment. This behavior was markedly different from that observed with adenovirus particles. Antiproteasome treatments with MG-132 led to a 50-fold enhancement in transduction efficiency. This effect was accompanied by a 10-fold intracellular accumulation of single-stranded DNA AAV genomes, suggesting that the mechanism of transduction enhancement was different from the one mediated by a helper adenovirus, which facilitates the conversion of the rAAV single-stranded DNA genome into its replicative form, MG-132, a drug currently in clinical use, could be of practical use for potentializing rAAV-mediated delivery of therapeutic genes.