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

病毒-细胞融合后的锥形HIV-1衣壳的拆解是 建立一种终生感染。进入HIV-1病毒的这一步，被称为去涂层，仍然是关键的 人们对此知之甚少。我们最近开发了一种新的策略来可视化HIV-1揭开涂层 是基于一种衣壳结合的宿主蛋白亲环素A的荧光标记寡聚体形式 (CypA-DsRed)。CypA-DsRed，它通过高亲和力被专门包装成病毒粒子 与HIV-1衣壳结合，不会损害传染性。此探测器保持关联 病毒-细胞融合后有核，去涂层后释放。支持这一理念是我们的 发现单个融合后核心的CypA-DsRed损失率受 突变会影响核心的稳定性，并通过逆转录加速。CypA-DsRed 基于成像的分析显示HIV-1的脱壳呈两相动力学，具有大量的核心 融合后不久就会脱落衣壳蛋白。这项测试还显示出显著的差异 HeLa来源的细胞和原代人巨噬细胞的脱包被表型。我们建议 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/衣壳复合体的研究；(2)阐明CypA-DsRed/衣壳复合体的逆转关系 转录和单核脱壳；以及(3)研究宿主对HIV-1脱壳的调控 Nup358、CPSF6等因子。这些研究结合了基因、 生化和先进的成像工具，预计将提供对 活细胞中HIV-1脱壳的动态及调控。

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.