HIV-1 Uncoating Occurs via a Series of Rapid Biomechanical Changes in the Core Related to Individual Stages of Reverse Transcription

Abstract

The HIV core consists of the viral genome and associated proteins encased by a cone-shaped protein shell termed the capsid. Successful infection requires reverse transcription of the viral genome and disassembly of the capsid shell within a cell in a process known as uncoating. The integrity of the viral capsid is critical for reverse transcription, yet the viral capsid must be breached to release the nascent viral DNA prior to integration. We employed atomic force microscopy to study the stiffness changes in HIV-1 cores during reverse transcription in vitro in reactions containing the capsid-stabilizing host metabolite IP6. Cores exhibited a series of stiffness spikes, with up to three spikes typically occurring between 10-30, 40-80, and 120-160 minutes after initiation of reverse transcription. Addition of the reverse transcriptase (RT) inhibitor efavirenz eliminated the appearance of these spikes and the subsequent disassembly of the capsid, thus establishing that both result from reverse transcription. Using timed addition of efavirenz, and analysis of an RNAseH-defective RT mutant, we established that the first stiffness spike requires minus-strand strong stop DNA synthesis, with subsequent spikes requiring later stages of reverse transcription. Additional rapid AFM imaging experiments revealed repeated morphological changes in cores that were temporally correlated with the observed stiffness spikes. Our study reveals discrete mechanical changes in the viral core that are likely related to specific stages of reverse transcription. These reverse-transcription-induced changes in the capsid progressively remodel the viral core to prime it for temporally accurate uncoating in target cells. Importance For successful infection, the HIV-1 genome, which is enclosed inside a capsid shell, must be reverse transcribed into double-stranded DNA and released from the capsid (in a process known as uncoating) before it can be integrated into the target cell genome. The mechanism of HIV-1 uncoating is a pivotal question of long standing. Using atomic force microscopy to analyze individual HIV-1 cores during reverse transcription, we observe a reproducible pattern of stiffness spikes. These spikes were shown to be associated with distinct stages of the reverse transcription reaction. Our findings suggest that these reverse-transcription-induced alterations gradually prepared the core for uncoating at the right time and location in target cells.

The HIV-1 capsid and reverse transcription

“…Recently we analyzed the effects of IP6 on the dynamics of HIV-1 core stiffness changes during ERT [ 66 ]. Addition of IP6 to purified cores increased their stiffness even in the absence of reverse transcription, consistent with its capsid stabilizing effect.…”

“…Moreover, addition of IP6 and the CA-targeting inhibitor PF74 stiffens the capsid. Using AFM operating in the ultrafast scanning mode, we have observed that capsid stiffening is associated with rounding of the core, which returns to an elongated shape upon relaxation [ 66 ]. This suggests that the flexibility of the capsid endows it with elastic properties which allow it to accommodate the stresses during reverse transcription without breaking.…”

The Role of Capsid in the Early Steps of HIV-1 Infection: New Insights into the Core of the Matter

“…It is therefore likely that the observed changes in sedimentation velocity of the RTCs at different times post-infection [ 34 ] reflect the different capsid core morphologies brought forward by the conversion of the viral genome from RNA into a double-stranded DNA. This notion is supported by recent atomic force microscopy analysis of the capsid core in the presence of IP 6 , showing step-wise, discrete core deformations that coincide with the three steps of reverse transcription, with progressive loss of core integrity [ 25 ].…”

“…Near-atomic reconstruction of the HIV-1 capsid core by cryo-EM also revealed that the NTD–CTD linker domains are flexible, allowing some degree of asymmetry of the capsid hexamer, which can curve at an angle of about 20°, thus contributing to the overall curvature of the capsid core itself [ 16 , 23 ]. In agreement with this notion, molecular dynamics simulations predicted that the capsid core oscillates, suggesting that structural flexibility might allow the core potentially to adopt different conformations in response to allosteric cues, such as co-factor binding [ 24 ] or reverse transcription [ 25 ].…”

The Role of Capsid in HIV-1 Nuclear Entry

“…In vitro studies using atomic force microscopy (AFM) suggested that reverse transcription increases the pressure inside the core triggering uncoating [ 17 ]; this agrees with our previous assessment that uncoating is initiated after first strand transfer during reverse transcription [ 16 ]. Rankovic et al have recently suggested that different stages of reverse transcription induce mechanical changes in the capsid that progressively remodel the viral core to prime it for uncoating [ 62 ]. By using an impressive in vitro replication system, Christensen et al imaged viral cDNA loops extruding from partially uncoated cores [ 63 ].…”