Evidence for biphasic uncoating during HIV-1 infection from a novel imaging assay

Xu, Hongzhan; Franks, Tamera; Gibson, Gregory A.; Huber, Kimberly; Rahm, Nadia; Strambio‐De‐Castillia, Caterina; Luban, Jeremy; Aiken, Christopher; Watkins, Simon C.; Sluis‐Cremer, Nicolas; Ambrose, Zandrea

doi:10.1186/1742-4690-10-70

Cited by 74 publications

(129 citation statements)

References 46 publications

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“…Interestingly, in the E45A capsid mutant the peak stiffness appeared sooner, after 4 h of transcription. This kinetic difference may be related to the observation that E45A cores appear to be more permeable to small molecules than WT cores (24), suggesting that reverse transcription may occur more rapidly owing to enhanced dNTP influx. Recent data suggest that nucleotides are transported into the core through electrostatic channels that were found in CA hexamers (25).…”

Section: Discussionmentioning

confidence: 99%

Reverse Transcription Mechanically Initiates HIV-1 Capsid Disassembly

Rankovic

Varadarajan

Ramalho

et al. 2017

J Virol

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112

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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

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Section: Discussionmentioning

confidence: 99%

Reverse Transcription Mechanically Initiates HIV-1 Capsid Disassembly

Rankovic

Varadarajan

Ramalho

et al. 2017

J Virol

Self Cite

112

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“…The use of GFP-Vpr for analysis of viral postentry events, pioneered by Hope and colleagues (33), has provided numerous valuable insights into postentry events during HIV-1 replication (22,24,(34)(35)(36). However, GFP-Vpr dissociates from the RTCs/PICs shortly after infection and has not been observed in the nuclei of infected cells (33,36,37). To date, only a small number of studies have detected PICs in the nuclei of infected cells by using virions labeled with fluorescently tagged IN (38)(39)(40)(41), and one recent study showed that there are nuclear IN complexes that are not associated with viral cDNA (42), suggesting that fluorescently tagged IN may not be a suitable marker for labeling PICs in the nuclei.…”

Section: Discussionmentioning

confidence: 99%

Nuclear import of APOBEC3F-labeled HIV-1 preintegration complexes

Burdick

Pathak

2013

Proc. Natl. Acad. Sci. U.S.A.

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Human cytidine deaminases APOBEC3F (A3F) and APOBEC3G (A3G) are host factors that incorporate into virions and restrict virus replication. We labeled HIV-1 particles with yellow fluorescent protein (YFP)-tagged APOBEC3 proteins and examined their association with preintegration complexes (PICs) in infected cells. Labeling of PICs with A3F-YFP, and to a lesser extent A3G-YFP, could be used to visualize PICs in the nuclei, which was dependent on nuclear pore protein Nup153 but not TNPO3. We show that reverse transcription is not required for nuclear import of PICs, indicating that a viral core uncoating event associated with reverse transcription, and the central DNA flap that forms during reverse transcription, are not required for nuclear import. We also quantify association of cytoplasmic PICs with nuclear envelope (NE) and report that capsid mutations that increase or decrease core stability dramatically reduce NE association and nuclear import of PICs. In addition, we find that nuclear PICs remain close to the NE and are not distributed throughout the nuclei. These results provide tools for tracking retroviral PICs in infected cells and reveal insights into HIV-1 replication.retrovirus | microscopy | early events

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“…Interestingly, several imaging-based experiments have shown that a minor amount of viral capsid protein (CA) is associated with the RTC, indicating disassembly of some viral cores in the cytoplasm (Xu et al 2013;Campbell and Hope 2015). Uncoating may occur when the viral cores pass through the NPC (Lahaye et al 2013;Campbell and Hope 2015).…”

Section: Canonical Nuclear Import Mechanismsmentioning

confidence: 99%

Cellular and viral determinants of retroviral nuclear entry

2016

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Retroviruses must integrate their cDNA into the host genome to generate proviruses. Viral DNA-protein complexes interact with cellular proteins and produce pre-integration complexes, which carry the viral genome and cross the nuclear pore channel to enter the nucleus and integrate viral DNA into host chromosomal DNA. If the reverse transcripts fail to integrate, linear or circular DNA species such as 1-and 2-long terminal repeats are generated. Such complexes encounter numerous cellular proteins in the cytoplasm, which restrict viral infection and protect the nucleus. To overcome host cell defenses, the pathogens have evolved several evasion strategies. Viral proteins often contain nuclear localization signals, allowing entry into the nucleus. Among more than 1000 proteins identified as required for HIV infection by RNA interference screening, karyopherins, cleavage and polyadenylation specific factor 6, and nucleoporins have been predominantly studied. This review discusses current opinions about the synergistic relationship between the viral and cellular factors involved in nuclear import, with focus on the unveiled mysteries of the host-pathogen interaction, and highlights novel approaches to pinpoint therapeutic targets.

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Evidence for biphasic uncoating during HIV-1 infection from a novel imaging assay

Cited by 74 publications

References 46 publications

Reverse Transcription Mechanically Initiates HIV-1 Capsid Disassembly

Reverse Transcription Mechanically Initiates HIV-1 Capsid Disassembly

Nuclear import of APOBEC3F-labeled HIV-1 preintegration complexes

Cellular and viral determinants of retroviral nuclear entry

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