The trimeric envelope glycoprotein complex (Env) is the focus of vaccine development programs aimed at generating protective humoral responses to human immunodeficiency virus type 1 (HIV-1). N-Linked glycans, which constitute almost half of the molecular mass of the external Env domains, produce considerable structural heterogeneity and are a major impediment to crystallization studies. Moreover, by shielding the peptide backbone, glycans can block attempts to generate neutralizing antibodies against a substantial subset of potential epitopes when Env proteins are used as immunogens. Here, we describe the partial deglycosylation of soluble, cleaved recombinant Env trimers by inhibition of the synthesis of complex N-glycans during Env production, followed by treatment with glycosidases under conditions that preserve Env trimer integrity. The partially deglycosylated trimers are stable, and neither abnormally sensitive to proteolytic digestion nor prone to aggregation. Moreover, the deglycosylated trimers retain or increase their ability to bind CD4 and antibodies that are directed to conformational epitopes, including the CD4-binding site and the V3 region. However, as expected, they do not react with glycan-dependent antibodies 2G12 and PGT123, or the C-type lectin receptor DC-SIGN. Electron microscopic analysis shows that partially deglycosylated trimers have a structure similar to fully glycosylated trimers, indicating that removal of glycans does not substantially perturb the structural integrity of the trimer. The glycan-depleted Env trimers should be useful for structural and immunogenicity studies. Background: The heterogeneity and flexibility of HIV-1 envelope glycoprotein N-glycans interfere with structural and vaccine studies. Results: HIV-1 envelope trimers can be partially deglycosylated without affecting trimer integrity. Conclusion: HIV-1 envelope glycoprotein N-glycans do not contribute to trimer integrity once the protein is folded. Significance: Deglycosylated HIV-1 envelope trimers should be useful for structural and vaccine studies.

The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man9 at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully glycosylated gp120 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short β-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificify. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface.

Variable regions 1 and 2 (V1/V2) of human immunodeficiency virus-1 (HIV-1) gp120 envelope glycoprotein are critical for viral evasion of antibody neutralization, and are themselves protected by extraordinary sequence diversity and N-linked glycosylation. Human antibodies such as PG9 nonetheless engage V1/V2 and neutralize 80% of HIV-1 isolates. Here we report the structure of V1/V2 in complex with PG9. V1/V2 forms a four-stranded β-sheet domain, in which sequence diversity and glycosylation are largely segregated to strand-connecting loops. PG9 recognition involves electrostatic, sequence-independent and glycan interactions: the latter account for over half the interactive surface but are of sufficiently weak affinity to avoid autoreactivity. The structures of V1/V2-directed antibodies CH04 and PGT145 indicate that they share a common mode of glycan penetration by extended anionic loops. In addition to structurally defining V1/V2, the results thus identify a paradigm of antibody recognition for highly glycosylated antigens, which—with PG9—involves a site of vulnerability comprising just two glycans and a strand. The crystal structure of V1/V2, the only unresolved portion of the HIV-1 gp120 envelope glycoprotein, is reported in complex with human antibody PG9 and reveals a paradigm of antibody recognition with implications for vaccine development. The V1/V2 variable region of the gp120 envelope glycoprotein of HIV-1, with its extraordinary sequence diversity and N-linked glycosylation, exemplifies the ability of the virus to evade antibody recognition. It has also resisted structural determination. Now Peter Kwong and colleagues have determined the atomic-level structure of gp120 V1/V2 by using an antibody called PG9, which can neutralize most strains of HIV. Instead of being confounded by the N-linked glycan that shields most of gp120 from immune recognition, PG9 uses N-linked glycan for binding through a mechanism shared by a number of antibodies capable of effective HIV neutralization. The structure shows that the antibody recognizes glycopeptide conjugates and avoids diversity in V1/V2 by making sequence-independent interactions, such as hydrogen bonds.