1. Structural Biology and Molecular Biophysics

Kinetics of HIV-1 capsid uncoating revealed by single-molecule analysis

  1. Chantal L Márquez
  2. Derrick Lau
  3. James Walsh
  4. Vaibhav Shah
  5. Conall McGuinness
  6. Andrew Wong
  7. Anupriya Aggarwal
  8. Michael W Parker
  9. David A Jacques
  10. Stuart Turville
  11. Till Böcking  Is a corresponding author
  1. University of New South Wales, Australia
  2. St Vincent's Institute, Australia
https://doi.org/10.7554/eLife.34772
Share this article
Cite this article
  1. Chantal L Márquez
  2. Derrick Lau
  3. James Walsh
  4. Vaibhav Shah
  5. Conall McGuinness
  6. Andrew Wong
  7. Anupriya Aggarwal
  8. Michael W Parker
  9. David A Jacques
  10. Stuart Turville
  11. Till Böcking
(2018)
Kinetics of HIV-1 capsid uncoating revealed by single-molecule analysis
eLife 7:e34772.
https://doi.org/10.7554/eLife.34772
  2. Download BibTeX
  3. Download .RIS

Abstract

Uncoating of the metastable HIV-1 capsid is a tightly regulated disassembly process required for release of the viral cDNA prior to nuclear import. To understand the intrinsic capsid disassembly pathway and how it can be modulated, we have developed a single-particle fluorescence microscopy method to follow the real-time uncoating kinetics of authentic HIV capsids in vitro immediately after permeabilizing the viral membrane. Opening of the first defect in the lattice is the rate-limiting step of uncoating, which is followed by rapid, catastrophic collapse. The capsid-binding inhibitor PF74 accelerates capsid opening but stabilizes the remaining lattice. In contrast, binding of a polyanion to a conserved arginine cluster in the lattice strongly delays initiation of uncoating but does not prevent subsequent lattice disassembly. Our observations suggest that different stages of uncoating can be controlled independently with the interplay between different capsid-binding regulators likely to determine the overall uncoating kinetics.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Chantal L Márquez

    EMBL Australia Node in Single Molecule Science, University of New South Wales, Sydney, Australia
    Competing interests
    The authors declare that no competing interests exist.

  2. Derrick Lau

    EMBL Australia Node in Single Molecule Science, University of New South Wales, Sydney, Australia
    Competing interests
    The authors declare that no competing interests exist.

  3. James Walsh

    EMBL Australia Node in Single Molecule Science, University of New South Wales, Sydney, Australia
    Competing interests
    The authors declare that no competing interests exist.

  4. Vaibhav Shah

    EMBL Australia Node in Single Molecule Science, University of New South Wales, Sydney, Australia
    Competing interests
    The authors declare that no competing interests exist.

  5. Conall McGuinness

    EMBL Australia Node in Single Molecule Science, University of New South Wales, Sydney, Australia
    Competing interests
    The authors declare that no competing interests exist.

  6. Andrew Wong

    The Kirby Institute, University of New South Wales, Sydney, Australia
    Competing interests
    The authors declare that no competing interests exist.

  7. Anupriya Aggarwal

    The Kirby Institute, University of New South Wales, Sydney, Australia
    Competing interests
    The authors declare that no competing interests exist.

  8. Michael W Parker

    Australian Cancer Research Foundation Rational Drug Discovery Centre, St Vincent's Institute, Fitzroy, Australia
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3101-1138

  9. David A Jacques

    EMBL Australia Node in Single Molecule Science, University of New South Wales, Sydney, Australia
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6426-4510

  10. Stuart Turville

    The Kirby Institute, University of New South Wales, Sydney, Australia
    Competing interests
    The authors declare that no competing interests exist.

  11. Till Böcking

    EMBL Australia Node in Single Molecule Science, University of New South Wales, Sydney, Australia
    For correspondence
    till.boecking@unsw.edu.au
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1165-3122

Funding

National Health and Medical Research Council (APP110071)

  • Stuart Turville
  • Till Böcking

Australian Centre for HIV and Hepatitis Virology Research

  • Till Böcking

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Wesley I Sundquist, University of Utah School of Medicine, United States

Version history

  1. Received: January 23, 2018
  2. Accepted: June 5, 2018
  3. Accepted Manuscript published: June 7, 2018 (version 1)
  4. Version of Record published: July 10, 2018 (version 2)

Copyright

© 2018, Márquez et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 5,089
    views
  • 733
    downloads
  • 76
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Chantal L Márquez
  2. Derrick Lau
  3. James Walsh
  4. Vaibhav Shah
  5. Conall McGuinness
  6. Andrew Wong
  7. Anupriya Aggarwal
  8. Michael W Parker
  9. David A Jacques
  10. Stuart Turville
  11. Till Böcking
(2018)
Kinetics of HIV-1 capsid uncoating revealed by single-molecule analysis
eLife 7:e34772.
https://doi.org/10.7554/eLife.34772

Share this article

https://doi.org/10.7554/eLife.34772

Categories and tags

Research organism

Further reading

    1. Microbiology and Infectious Disease
    2. Structural Biology and Molecular Biophysics

    IP6 is an HIV pocket factor that prevents capsid collapse and promotes DNA synthesis

    Donna L Mallery, Chantal L Márquez ... Leo C James
    Research Article Updated

    The HIV capsid is semipermeable and covered in electropositive pores that are essential for viral DNA synthesis and infection. Here, we show that these pores bind the abundant cellular polyanion IP6, transforming viral stability from minutes to hours and allowing newly synthesised DNA to accumulate inside the capsid. An arginine ring within the pore coordinates IP6, which strengthens capsid hexamers by almost 10°C. Single molecule measurements demonstrate that this renders native HIV capsids highly stable and protected from spontaneous collapse. Moreover, encapsidated reverse transcription assays reveal that, once stabilised by IP6, the accumulation of new viral DNA inside the capsid increases >100 fold. Remarkably, isotopic labelling of inositol in virus-producing cells reveals that HIV selectively packages over 300 IP6 molecules per infectious virion. We propose that HIV recruits IP6 to regulate capsid stability and uncoating, analogous to picornavirus pocket factors. HIV-1/IP6/capsid/co-factor/reverse transcription.

    1. Microbiology and Infectious Disease
    2. Structural Biology and Molecular Biophysics

    Viruses: The secrets of the stability of the HIV-1 capsid

    Martin Obr, Hans-Georg Kräusslich
    Insight

    Structural and biophysical studies help to follow the disassembly of the HIV-1 capsid in vitro, and reveal the role of a small molecule called IP6 in regulating capsid stability.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    Caenorhabditis elegans Dicer acts with the RIG-I-like helicase DRH-1 and RDE-4 to cleave dsRNA

    Claudia D Consalvo, Adedeji M Aderounmu ... Brenda L Bass
    Research Article

    Invertebrates use the endoribonuclease Dicer to cleave viral dsRNA during antiviral defense, while vertebrates use RIG-I-like Receptors (RLRs), which bind viral dsRNA to trigger an interferon response. While some invertebrate Dicers act alone during antiviral defense, Caenorhabditis elegans Dicer acts in a complex with a dsRNA binding protein called RDE-4, and an RLR ortholog called DRH-1. We used biochemical and structural techniques to provide mechanistic insight into how these proteins function together. We found RDE-4 is important for ATP-independent and ATP-dependent cleavage reactions, while helicase domains of both DCR-1 and DRH-1 contribute to ATP-dependent cleavage. DRH-1 plays the dominant role in ATP hydrolysis, and like mammalian RLRs, has an N-terminal domain that functions in autoinhibition. A cryo-EM structure indicates DRH-1 interacts with DCR-1’s helicase domain, suggesting this interaction relieves autoinhibition. Our study unravels the mechanistic basis of the collaboration between two helicases from typically distinct innate immune defense pathways.