Reverse Transcription Mechanically Initiates HIV-1 Capsid Disassembly

Abstract

The HIV-1 core consists of the viral genomic RNA and several viral proteins encased within a conical capsid. After cell entry, the core disassembles in a process termed uncoating. Although HIV-1 uncoating has been linked to reverse transcription of the viral genome in target cells, the mechanism by which uncoating is initiated is unknown. Using time-lapse atomic force microscopy, we analyzed the morphology and physical properties of isolated HIV-1 cores during the course of reverse transcription in vitro. We found that, during an early stage of reverse transcription the pressure inside the capsid increases, reaching a maximum after 7 h. Highresolution mechanical mapping reveals the formation of a stiff coiled filamentous structure underneath the capsid surface. Subsequently, this coiled structure disappears, the stiffness of the capsid drops precipitously to a value below that of a prereverse transcription core, and the capsid undergoes partial or complete rupture near the narrow end of the conical structure. We propose that the transcription of the relatively flexible single-stranded RNA into a more rigid filamentous structure elevates the pressure within the core, which triggers the initiation of capsid disassembly.IMPORTANCE For successful infection, the HIV-1 genome, which is in the form of a single-stranded RNA 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 that triggers uncoating is a pivotal question of long standing. By using atomic force microscopy, we found that during reverse transcription the pressure inside the capsid increases until the internal stress exceeds the strength of the capsid structure and the capsid breaks open. The application of AFM technologies to study purified HIV-1 cores represents a new experimental platform for elucidating additional aspects of capsid disassembly and HIV-1 uncoating.KEYWORDS HIV-1, atomic force microscopy, capsid, reverse transcription, uncoating F ollowing fusion between the HIV-1 membrane and the target cell membrane, the viral core is released into the host cell cytoplasm. The core consists of the singlestranded viral RNA (ssRNA) genome and associated proteins encapsulated within a cone-shaped capsid composed of ϳ250 CA (p24) protein hexamers and 12 CA pentamers (1-3). Successful infection requires reverse transcription of the ssRNA into double-stranded DNA (dsDNA) that is then transported into the nucleus, where it integrates into the target cell genome. Since the intact HIV-1 core is too large to cross the nuclear pore (reviewed in reference 4), the capsid is thought to disassemble prior to nuclear import in a process known as uncoating. This is supported by a study which shows by superresolution microscopy differences in size of reverse transcription complexes between the cytoplasm and the nucleus in infected cells (5). Several studies

Permeability of the HIV-1 capsid to metabolites modulates viral DNA synthesis

“…Changes in capsid stiffness and morphologies during reverse transcription were monitored in realtime by AFM ( Fig. 4) 11,24,25 . The effect of IP6 binding on the stiffness of intact HIV-1 capsids was analyzed using AFM operated in the nanoindentation mode.…”

“…While reverse transcription likely initiates within the capsid and is affected by uncoating, the mechanisms connecting the two viral processes remain a long-standing question in HIV-1 biology [7][8][9][10] . We and others have proposed that reverse transcription mechanically induces changes in capsid morphology and triggers uncoating 11,12 . To support vDNA synthesis, RT requires adequate concentrations of deoxynucleotide triphosphates (dNTPs) within the lumen of the capsid.…”

Virological characterization of HIV‐1 CA‐NTD mutants constructed in a virus‐lineage reflected manner

“…It is also necessary to define in detail how virion-Gag disassembles at the early post-entry stage. This subject is now under vigorous investigation by many researchers (26)(27)(28)(29)(30)(31). For better understanding the HIV-1 virology and for therapeutic purpose, the above issues need to be precisely elucidated.…”

Localized Phosphorylation of a Kinesin-1 Adaptor by a Capsid-Associated Kinase Regulates HIV-1 Motility and Uncoating

“…However, recent live cell imaging supports the notion that uncoating begins in the cytoplasm (Francis et al, 2016). Notably, uncoating and reverse transcription are intricately coupled processes (Cosnefroy et al, 2016; Hulme et al, 2011; Rankovic et al, 2017; Stultz et al, 2017; Yang et al, 2013) that are further intertwined with the bi-directional movement of HIV-1 particles. Interfering with MT or dynein function suppresses HIV-1 trafficking and delays uncoating during early infection (Arhel et al, 2006; Delaney et al, 2017; Lukic et al, 2014; Malikov et al, 2015; McDonald et al, 2002; Pawlica and Berthoux, 2014; Sabo et al, 2013).…”