Initiation of HIV-1 Gag lattice assembly is required for recognition of the viral genome packaging signal

  1. Xiao Lei
  2. Daniel Gonçalves-Carneiro
  3. Trinity M Zang
  4. Paul D Bieniasz  Is a corresponding author
  1. Rockefeller University, United States

Abstract

The encapsidation of HIV-1 genomic RNA into virions is enabled by the binding of the nucleocapsid (NC) domain of the HIV-1 Gag polyprotein to the structured viral RNA packaging signal (Ψ) at the 5' end of the viral genome. However, the subcellular location and oligomeric status of Gag during the initial Gag-Ψ encounter remains uncertain. Domains other than NC, such as capsid (CA), may therefore indirectly affect RNA recognition. To investigate the contribution of Gag domains to Ψ recognition in a cellular environment, we performed protein-protein crosslinking and protein-RNA crosslinking immunoprecipitation coupled with sequencing (CLIP-seq) experiments. We demonstrate that NC alone does not bind specifically to Ψ in living cells, whereas full-length Gag and a CANC subdomain bind to Ψ with high specificity. Perturbation of the Ψ RNA structure or NC zinc fingers affected CANC:Ψ binding specificity. Notably, CANC variants with substitutions that disrupt CA:CA dimer, trimer or hexamer interfaces in the immature Gag lattice also affected RNA binding, and mutants that were unable to assemble a nascent Gag lattice were unable to specifically bind to Ψ. Artificially multimerized NC domains did not specifically bind Ψ. CA variants with substitutions in inositol phosphate coordinating residues that prevent CA hexamerization were also deficient in Ψ binding and second-site revertant mutants that restored CA assembly also restored specific binding to Ψ. Overall, these data indicate that the correct assembly of a nascent immature CA lattice is required for the specific interaction between Gag and Ψ in cells.

Data availability

All data generated or analysed during this study are included in the manuscript and accompanying source data files

Article and author information

Author details

  1. Xiao Lei

    Laboratory of Retrovirology, Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8641-7824
  2. Daniel Gonçalves-Carneiro

    Laboratory of Retrovirology, Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9333-1540
  3. Trinity M Zang

    Laboratory of Retrovirology, Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Paul D Bieniasz

    Laboratory of Retrovirology, Rockefeller University, New York, United States
    For correspondence
    pbieniasz@rockefeller.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2368-3719

Funding

National Institute of Allergy and Infectious Diseases (U54 AI170660)

  • Paul D Bieniasz

National Institute of Allergy and Infectious Diseases (R01AI50111)

  • Paul D Bieniasz

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

Reviewing Editor

  1. Guangxia Gao, Institute of Biophysics, Chinese Academy of Sciences, China

Publication history

  1. Received: September 18, 2022
  2. Accepted: January 20, 2023
  3. Accepted Manuscript published: January 23, 2023 (version 1)

Copyright

© 2023, Lei 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.

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  1. Xiao Lei
  2. Daniel Gonçalves-Carneiro
  3. Trinity M Zang
  4. Paul D Bieniasz
(2023)
Initiation of HIV-1 Gag lattice assembly is required for recognition of the viral genome packaging signal
eLife 12:e83548.
https://doi.org/10.7554/eLife.83548
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