Wild-type and central DNA flap defective HIV-I lentiviral vector genomes: intracellular visualization at ultrastructural resolution levels
Wild-type and central DNA flap defective HIV-I lentiviral vector genomes: intracellular visualization at ultrastructural resolution levels
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DOI：
10.1186/1742-4690-3-38
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发表时间：
2006-06-26
期刊：
影响因子：
3.3
通讯作者：
Charneau, Pierre
中科院分区：
文献类型：
--
作者：
Arhel, Nathalie J.;Souquere-Besse, Sylvie;Charneau, Pierre
HIV-1和其他慢病毒在逆转录病毒中具有独特的在非分裂细胞中有效复制的能力，这是由于它们的DNA基因组通过期间核膜活跃的核输入的结果。先前的研究表明，在HIV-1逆转录过程中合成的三链DNA结构，称为中央DNA瓣，作为HIV-1基因组核输入的顺式决定因素。与此同时，在慢病毒衍生的基因治疗载体中重新插入DNA皮瓣可以刺激基因转移效率，并补充了到野生型水平的核输入水平，在迄今为止所检测的所有细胞类型和组织中，与野生型病毒在数量上无法区分。为了明确DNA flap突变病毒复制缺陷的确切性质，我们在电镜下进行了原位DNA杂交实验，以确定DNA flap突变型和野生型HIV-1基因组的亚细胞定位。我们发现皮瓣缺陷DNA基因组在核膜的细胞质表面积累，在核膜上没有重叠，而野生型基因组在整个核室中定位。这些数据明确证实了DNA瓣在HIV-1核输入中的作用，并进一步证实了DNA瓣控制着通过核孔易位之前的一个步骤。此外，野生型基因组在开放染色质内的广泛分布证实了最近HIV-1 cDNA整合位点的全基因组定位，并指出了HIV-1预整合复合物的核内运输的一个尚不清楚的步骤。
HIV-1 and other lentiviruses have the unique ability among retroviruses to efficiently replicate in non-dividing cells as a result of the active nuclear import of their DNA genome across an interphasic nuclear membrane. Previous work has shown that a three-stranded DNA structure synthesized during HIV-1 reverse transcription, called the central DNA flap, acts as a cis-determinant of HIV-1 genome nuclear import. Concordantly, DNA Flap re-insertion in lentiviralderived gene therapy vectors stimulates gene transfer efficiencies and complements the level of nuclear import to wild-type levels quantitatively indistinguishable from wild-type virus in all cell types and tissues examined so far. In order to define the precise nature of the replicative defect of DNA flap mutant viruses, we carried out in situ DNA hybridization experiments with electron microscopy to determine the subcellular localization of DNA flap mutant and wild-type HIV-1 genomes. We found that Flap defective DNA genomes accumulate at the cytoplasmic face of the nuclear membrane with no overlap across the nuclear membrane, whereas wild-type genomes localize throughout the nuclear compartment. These data provide an unequivocal confirmation of the role of the DNA flap in HIV-1 nuclear import and further establish that the DNA flap controls a step that immediately precedes translocation through the nuclear pore. Further, the widespread distribution of wild-type genomes within the open chromatin confirms the recent genome-wide mapping of HIV-1 cDNA integration sites and points to an as-yet poorly understood step of intranuclear transport of HIV-1 pre-integration complexes.