Imaging of Single HIV-1 Uncoating and Transport to the nucleus

在病毒-细胞融合后，锥形HIV-1衣壳的降解是 造成终身感染HIV-1进入的这一步，被称为脱膜，是至关重要的， 不太了解。我们最近开发了一种新的策略来可视化HIV-1的脱壳， 是基于荧光标记的寡聚形式的captain结合宿主蛋白亲环素A （CypA-DsRed）。CypA-DsRed通过高亲合力特异性包装成病毒粒子， 与HIV-1衣壳结合，不损害感染性。这个探测器仍然与 在病毒-细胞融合后具有核心，并且在脱壳时释放。支持这一观点的是我们的 发现来自单个融合后核心的CypA-DsRed损失率受到以下因素的调节： 影响核心稳定性的突变，并通过逆转录加速。CypA-DsRed 基于成像分析揭示了HIV-1脱壳的双相动力学，具有大量核心 融合后不久脱落衣壳蛋白。该试验还显示， HeLa衍生细胞和原代人巨噬细胞中的未包被表型。我们提出 目的：（1）通过功能和结构描述CypA-DsRed与HIV-1核心的相互作用 CypA-DsRed/衣壳复合物的研究;（2）阐明了逆转录病毒与细胞凋亡之间的关系。 转录和单核脱壳;（3）研究宿主对HIV-1脱壳的调控 Nup 358和CPSF 6等因素。这些研究采用了基因、 生物化学和先进的成像工具，预计将提供关键的见解， HIV-1在活细胞中脱壳的动力学和调节。

Disassembly of the cone-shaped HIV-1 capsid after virus-cell fusion is a prerequisite for establishing a life-long infection. This step in HIV-1 entry, referred to as uncoating, is critical yet poorly understood. We have recently developed a novel strategy to visualize HIV-1 uncoating that is based on a fluorescently tagged oligomeric form of a capsid-binding host protein cyclophilin A (CypA-DsRed). CypA-DsRed, which is specifically packaged into virions through the high-avidity binding to the HIV-1 capsid, does not compromise the infectivity. This probe remains associated with cores after virus-cell fusion and is released upon uncoating. Supporting this notion is our finding that the rate of CypA-DsRed loss from individual post-fusion cores is modulated by mutations affecting the core stability and is accelerated by reverse transcription. The CypA-DsRed based imaging assay revealed a biphasic kinetic of HIV-1 uncoating, with a large number of cores shedding the capsid protein shortly after fusion. This assay also revealed marked differences in the uncoating phenotype in HeLa-derived cells and primary human macrophages. We propose to: (1) delineate CypA-DsRed interactions with HIV-1 core through functional and structural studies of CypA-DsRed/capsid complexes; (2) elucidate the relationship between reverse transcription and single core uncoating; and (3) investigate regulation of HIV-1 uncoating by host factors, such as Nup358 and CPSF6. These studies, employing a combination of genetic, biochemical and advanced imaging tools, are expected to provide critical insights into the dynamics and regulation of HIV-1 uncoating in living cells.