Initiation of HIV-1 Gag lattice assembly is required for recognition of the viral genome packaging signal
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
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
- Guangxia Gao, Institute of Biophysics, Chinese Academy of Sciences, China
Version history
- Received: September 18, 2022
- Preprint posted: October 10, 2022 (view preprint)
- Accepted: January 20, 2023
- Accepted Manuscript published: January 23, 2023 (version 1)
- Version of Record published: February 8, 2023 (version 2)
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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