HIV-1 Capsid Uncoating Is a Multistep Process That Proceeds through Defect Formation Followed by Disassembly of the Capsid Lattice

Abstract

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.

HIV-1 Vpr Functions in Primary CD4+ T Cells

“…Given that Vpr is specifically incorporated into assembled viral particles and thus exerts a biological role upon de novo infection, it is clear that the presence of Vpr plays a pivotal role early in the HIV life cycle; not only may it support viral genome integrity before, during, and after reverse transcription, but it may also simultaneously facilitate its journey into the host's nucleus. Importantly, these findings are conceptually compatible with the current paradigm change, which states that uncoating does not take place directly after viral fusion with the cellular plasma membrane, but rather during or after nuclear import [29,39]. In line with this, Vpr was shown to rapidly traverse to the nucleus after viral entry, although this has thus far only been established in cell lines [40].…”