Binley JM, Sanders RW, Clas B, Schuelke N, Grasp A, Guo Y, Kajumo F, Anselma DJ, Maddon PJ, Olson WC, Moore JP
Binley JM, Sanders RW, Clas B, Schuelke N, Grasp A, Guo Y, Kajumo F, Anselma DJ, Maddon PJ, Olson WC, Moore JP. ABSTRACT HIV-1 envelope (Env) glycoprotein gp160 exists as a trimer of heterodimers around 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 Smoc2 region, residues 513 and 515 were also sensitive to Asp substitution, suggesting that this fusion peptide may not be fully uncovered in native Env. gp41 is usually metastable in the context of native trimer. Introduction of Asp at residues that are uncovered in the prefusion state but buried in the postfusion state is usually expected to destabilize the postfusion state and any intermediate says where the residue is usually 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 comparative 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 around the virion surface is not known. This knowledge may be useful for stabilizing Env in its prefusion Y-33075 conformation and will also help to improve understanding of the viral entry process. Burial of the charged residue Asp in a protein structure is usually 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 Y-33075 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. KEYWORDS: charge burial, electrostatics, fusion mechanism, immunogen design, protein stability, vaccine INTRODUCTION HIV-1 is the causative agent of AIDS. The computer virus Y-33075 infects target cells through its envelope (Env) glycoprotein. This envelope glycoprotein is usually synthesized as a gp160 precursor protein inside the cell. During the course of transport to the virion surface, it is cleaved by the host cell protease furin into surface-exposed gp120 and membrane-anchored gp41. The three gp120 and gp41 monomers are associated noncovalently, forming a trimer of heterodimers (1, 2). The integrity of this trimer is usually important for HIV-1 viral entry into the host cell. Since a significant fraction of gp120 is usually surface exposed, it is the primary target of the host humoral immune response. gp120 binds to the primary host cell receptor CD4; this induces conformational changes in the envelope that in turn expose the coreceptor binding site for binding to CXCR4/CCR5, ultimately leading to virus-cell fusion (1, 3,C6). Around the virion surface, there are Y-33075 also other nonfunctional Env derivatives, such as gp41 Y-33075 stumps and misfolded gp120 termed junk envelope (7, 8), that elicit nonneutralizing antibodies. Monomeric gp120, when used as a vaccine, also primarily elicits nonneutralizing immune responses (9,C11). In order to drive elicitation of broadly.
