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Probing the Structure of the HIV-1 Envelope Trimer Using Aspartate Scanning Mutagenesis

Das, R and Datta, R and Varadarajan, R (2020) Probing the Structure of the HIV-1 Envelope Trimer Using Aspartate Scanning Mutagenesis. In: Journal of Virology, 94 (21).

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Official URL: https://doi.org/10.1128/JVI.01426-20


HIV-1 envelope (Env) glycoprotein gp160 exists as a trimer of heterodimers on the viral surface. In most structures of the soluble ectodomain of trimeric HIV-1 envelope glycoprotein, the regions from 512 to 517 of the fusion peptide and from 547 to 568 of the N-heptad repeat are disordered. We used aspartate scanning mutagenesis of subtype B strain JRFL Env as an alternate method to probe residue burial in the context of cleaved, cell surface-expressed Env, as buried residues should be intolerant to substitution with Asp. The data are inconsistent with a fully disordered 547 to 568 stretch, as residues 548, 549, 550, 555, 556, 559, 562, and 566 to 569 are all sensitive to Asp substitution. In the fusion peptide region, residues 513 and 515 were also sensitive to Asp substitution, suggesting that the fusion peptide may not be fully exposed in native Env. gp41 is metastable in the context of native trimer. Introduction of Asp at residues that are exposed in the prefusion state but buried in the postfusion state is expected to destabilize the postfusion state and any intermediate states where the residue is buried. We therefore performed soluble CD4 (sCD4)-induced gp120 shedding experiments to identify Asp mutants at residues 551, 554 to 559, 561 to 567, and 569 that could prevent gp120 shedding. We also observed similar mutational effects on shedding for equivalent mutants in the context of clade C Env from isolate 4-2J.41. These substitutions can potentially be used to stabilize native-like trimer derivatives that are used as HIV-1 vaccine immunogens. IMPORTANCE In most crystal structures of the soluble ectodomain of the HIV-1 Env trimer, some residues in the fusion and N-heptad repeat regions are disordered. Whether this is true in the context of native, functional Env on the virion surface is not known. This knowledge may be useful for stabilizing Env in its prefusion conformation and will also help to improve understanding of the viral entry process. Burial of the charged residue Asp in a protein structure is highly destabilizing. We therefore used Asp scanning mutagenesis to probe the burial of apparently disordered residues in native Env and to examine the effect of mutations in these regions on Env stability and conformation as probed by antibody binding to cell surface-expressed Env, CD4-induced shedding of HIV-1 gp120, and viral infectivity studies. Mutations that prevent shedding can potentially be used to stabilize native-like Env constructs for use as vaccine immunogens. Copyright © 2020 American Society for Microbiology. All Rights Reserved.

Item Type: Journal Article
Publication: Journal of Virology
Publisher: American Society for Microbiology
Additional Information: The copyright for this article belongs to The Authors.
Keywords: aspartic acid; CD4 antigen; glycoprotein gp 120; virus envelope protein; aspartic acid; CD4 antigen; glycoprotein gp 120; glycoprotein gp 160; glycoprotein gp 41; gp120 protein, Human immunodeficiency virus 1; gp160 protein, Human immunodeficiency virus 1; gp41 protein, Human immunodeficiency virus 1; intrinsically disordered protein; recombinant protein; soluble CD4 antigen, Article; controlled study; human; human cell; Human immunodeficiency virus 1; mutation; nonhuman; priority journal; protein cleavage; protein expression; protein structure; scanning mutagenesis; virus isolation; virus shedding; virus strain; amino acid substitution; chemistry; gene expression; gene vector; genetics; HEK293 cell line; metabolism; molecular cloning; mutagenesis; protein degradation; protein multimerization; protein stability, Amino Acid Substitution; Aspartic Acid; CD4 Antigens; Cloning, Molecular; Gene Expression; Genetic Vectors; HEK293 Cells; HIV Envelope Protein gp120; HIV Envelope Protein gp160; HIV Envelope Protein gp41; HIV-1; Humans; Intrinsically Disordered Proteins; Mutagenesis; Protein Multimerization; Protein Stability; Proteolysis; Recombinant Proteins
Department/Centre: Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited: 11 Jan 2023 05:12
Last Modified: 11 Jan 2023 05:12
URI: https://eprints.iisc.ac.in/id/eprint/79038

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