HIV-1核心由由衣壳蛋白(CA)六聚体和包封病毒基因组的五聚体制成的锥形衣壳壳壳组成。HIV-1衣壳拆解,称为退涂,对生产性感染很重要;然而,位置,定时,和未涂覆的规定仍然存在争议。这里,我们采用琥珀密码子抑制直接标记CA。此外,将液相荧光探针掺入病毒核心中以检测衣壳晶格中的小缺陷。这种双重标记策略可以在体外和活细胞中可视化单个核心的未涂层,我们发现总是通过至少两个不同的步骤进行-在衣壳晶格中形成缺陷,从而引发CA逐渐损失至可检测水平以下。重要的是,含有液相和CA荧光标记的完整岩心进入和脱出岩心,这两个标记的连续丢失证明,在建立生产性感染之前。在不同的细胞中观察到这种两步脱膜过程,包括巨噬细胞系。值得注意的是,在液相标记物的释放和CA的末端丧失之间的滞后似乎与细胞类型或逆转录无关,并且与无细胞核心或细胞溶质中的核心相比,核衣壳的时间更长(>5倍)。这表明衣壳晶格是由衣壳结合核因子稳定的。我们的结果表明,完整的HIV-1核心进入细胞核,并且在CA整体丧失之前,通过衣壳晶格中的局部缺陷开始脱膜。
The HIV-1 core consists of a cone-shaped capsid shell made of capsid protein (CA) hexamers and pentamers encapsulating the viral genome. HIV-1 capsid disassembly, referred to as uncoating, is important for productive infection; however, the location, timing, and regulation of uncoating remain controversial. Here, we employ amber codon suppression to directly label CA. In addition, a fluid phase fluorescent probe is incorporated into the viral core to detect small defects in the capsid lattice. This double-labeling strategy enables the visualization of uncoating of single cores in vitro and in living cells, which we found to always proceed through at least two distinct steps─the formation of a defect in the capsid lattice that initiates gradual loss of CA below a detectable level. Importantly, intact cores containing the fluid phase and CA fluorescent markers enter and uncoat in the nucleus, as evidenced by a sequential loss of both markers, prior to establishing productive infection. This two-step uncoating process is observed in different cells, including a macrophage line. Notably, the lag between the release of fluid phase marker and terminal loss of CA appears to be independent of the cell type or reverse transcription and is much longer (>5-fold) for nuclear capsids compared to cell-free cores or cores in the cytosol, suggesting that the capsid lattice is stabilized by capsid-binding nuclear factors. Our results imply that intact HIV-1 cores enter the cell nucleus and that uncoating is initiated through a localized defect in the capsid lattice prior to a global loss of CA.