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

The encapsidation of HIV-1 gRNA 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 Ga...

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Autores principales: Lei, Xiao, Gonçalves-Carneiro, Daniel, Zang, Trinity M, Bieniasz, Paul D
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9908077/
https://www.ncbi.nlm.nih.gov/pubmed/36688533
http://dx.doi.org/10.7554/eLife.83548
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author Lei, Xiao
Gonçalves-Carneiro, Daniel
Zang, Trinity M
Bieniasz, Paul D
author_facet Lei, Xiao
Gonçalves-Carneiro, Daniel
Zang, Trinity M
Bieniasz, Paul D
author_sort Lei, Xiao
collection PubMed
description The encapsidation of HIV-1 gRNA 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 remain 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.
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spelling pubmed-99080772023-02-09 Initiation of HIV-1 Gag lattice assembly is required for recognition of the viral genome packaging signal Lei, Xiao Gonçalves-Carneiro, Daniel Zang, Trinity M Bieniasz, Paul D eLife Microbiology and Infectious Disease The encapsidation of HIV-1 gRNA 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 remain 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. eLife Sciences Publications, Ltd 2023-01-23 /pmc/articles/PMC9908077/ /pubmed/36688533 http://dx.doi.org/10.7554/eLife.83548 Text en © 2023, Lei et al https://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Microbiology and Infectious Disease
Lei, Xiao
Gonçalves-Carneiro, Daniel
Zang, Trinity M
Bieniasz, Paul D
Initiation of HIV-1 Gag lattice assembly is required for recognition of the viral genome packaging signal
title Initiation of HIV-1 Gag lattice assembly is required for recognition of the viral genome packaging signal
title_full Initiation of HIV-1 Gag lattice assembly is required for recognition of the viral genome packaging signal
title_fullStr Initiation of HIV-1 Gag lattice assembly is required for recognition of the viral genome packaging signal
title_full_unstemmed Initiation of HIV-1 Gag lattice assembly is required for recognition of the viral genome packaging signal
title_short Initiation of HIV-1 Gag lattice assembly is required for recognition of the viral genome packaging signal
title_sort initiation of hiv-1 gag lattice assembly is required for recognition of the viral genome packaging signal
topic Microbiology and Infectious Disease
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9908077/
https://www.ncbi.nlm.nih.gov/pubmed/36688533
http://dx.doi.org/10.7554/eLife.83548
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