Abstract:The analysis of HIV-1 envelope carbohydrates is critical to understanding their roles in HIV-1 transmission as well as in binding of envelope to HIV-1 antibodies. However, direct analysis of protein glycosylation by glycopeptide-based mass mapping approaches involves structural simplification of proteins with the use of a protease followed by an isolation and/or enrichment step before mass analysis. The successful completion of glycosylation analysis is still a major analytical challenge due to the complexity … Show more
“…Consistent with the notion that uninterpretable glycan density results from sample heterogeneity rather than from refinement problems, we found unambiguous density at positions that should be homogenous, e.g. at N332 gp120 , a high-mannose-only site (Behrens et al, 2016), whereas sites predicted to be more heterogeneous, such as N156 gp120 (Behrens et al, 2016;Go et al, 2011), exhibited some uninterpretable heterogeneous electron density. Although the relatively low resolution of our crystal structures and heterogeneous glycosylation compounded inherent difficulties in making unambiguous glycan assignments, we built coordinates into very extensive densities (e.g.…”
Section: Glycan Interpretation and Refinementsupporting
confidence: 70%
“…In other cases, the interpretation of our electrondensity maps was partially based on supporting experimental data: e.g. for the N301 gp120 glycan a core fucose was not ordered in our maps, but the N301 gp120 glycan was modeled as complextype in our structures based on mass-spectrometric data (Go et al, 2011). As expected given the large degree of glycan heterogeneity in HIV-1 Env (Doores, 2015), the glycan density was sometimes ambiguous.…”
Section: Glycan Interpretation and Refinementmentioning
confidence: 96%
“…Glycans were assigned as complextype if there was density for a core fucose and/or based on mass-spectrometry assignments (Behrens et al, 2016;Go et al, 2011). A core fucose was sometimes visible in one structure but not in the other.…”
Section: Glycan Interpretation and Refinementmentioning
The structural and biochemical characterization of broadly neutralizing anti-HIV-1 antibodies (bNAbs) has been essential in guiding the design of potential vaccines to prevent infection by HIV-1. While these studies have revealed critical mechanisms by which bNAbs recognize and/or accommodate N-glycans on the trimeric envelope glycoprotein (Env), they have been limited to the visualization of high-mannose glycan forms only, since heterogeneity introduced from the presence of complex glycans makes it difficult to obtain high-resolution structures. 3.5 and 3.9 Å resolution crystal structures of the HIV-1 Env trimer with fully processed and native glycosylation were solved, revealing a glycan shield of high-mannose and complex-type N-glycans that were used to define the complete epitopes of two bNAbs. Here, the refinement of the N-glycans in the crystal structures is discussed and comparisons are made with glycan densities in glycosylated Env structures derived by single-particle cryo-electron microscopy.
“…Consistent with the notion that uninterpretable glycan density results from sample heterogeneity rather than from refinement problems, we found unambiguous density at positions that should be homogenous, e.g. at N332 gp120 , a high-mannose-only site (Behrens et al, 2016), whereas sites predicted to be more heterogeneous, such as N156 gp120 (Behrens et al, 2016;Go et al, 2011), exhibited some uninterpretable heterogeneous electron density. Although the relatively low resolution of our crystal structures and heterogeneous glycosylation compounded inherent difficulties in making unambiguous glycan assignments, we built coordinates into very extensive densities (e.g.…”
Section: Glycan Interpretation and Refinementsupporting
confidence: 70%
“…In other cases, the interpretation of our electrondensity maps was partially based on supporting experimental data: e.g. for the N301 gp120 glycan a core fucose was not ordered in our maps, but the N301 gp120 glycan was modeled as complextype in our structures based on mass-spectrometric data (Go et al, 2011). As expected given the large degree of glycan heterogeneity in HIV-1 Env (Doores, 2015), the glycan density was sometimes ambiguous.…”
Section: Glycan Interpretation and Refinementmentioning
confidence: 96%
“…Glycans were assigned as complextype if there was density for a core fucose and/or based on mass-spectrometry assignments (Behrens et al, 2016;Go et al, 2011). A core fucose was sometimes visible in one structure but not in the other.…”
Section: Glycan Interpretation and Refinementmentioning
The structural and biochemical characterization of broadly neutralizing anti-HIV-1 antibodies (bNAbs) has been essential in guiding the design of potential vaccines to prevent infection by HIV-1. While these studies have revealed critical mechanisms by which bNAbs recognize and/or accommodate N-glycans on the trimeric envelope glycoprotein (Env), they have been limited to the visualization of high-mannose glycan forms only, since heterogeneity introduced from the presence of complex glycans makes it difficult to obtain high-resolution structures. 3.5 and 3.9 Å resolution crystal structures of the HIV-1 Env trimer with fully processed and native glycosylation were solved, revealing a glycan shield of high-mannose and complex-type N-glycans that were used to define the complete epitopes of two bNAbs. Here, the refinement of the N-glycans in the crystal structures is discussed and comparisons are made with glycan densities in glycosylated Env structures derived by single-particle cryo-electron microscopy.
“…We and others have reported that founder viruses generally have fewer glycosylation sites and more compact Envs (9,(31)(32)(33), and our earlier studies also demonstrated that newly transmitted viruses were not escape variants from PL-neutralizing antibodies in the donor (9). Recent studies have suggested that subtype A and C founder viruses bind efficiently to the gut homing receptor α4β7 expressed on a subset of highly susceptible CD4 T lymphocytes and that this property can be reduced with evolution of the virus after transmission (34).…”
The predominant mode of HIV-1 infection is heterosexual transmission, where a genetic bottleneck is imposed on the virus quasispecies. To probe whether limited genetic diversity in the genital tract (GT) of the transmitting partner drives this bottleneck, viral envelope sequences from the blood and genital fluids of eight transmission pairs from Rwanda and Zambia were analyzed. The chronically infected transmitting partner's virus population was heterogeneous with distinct genital subpopulations, and the virus populations within the GT of two of four women sampled longitudinally exhibited evidence of stability over time intervals on the order of weeks to months. Surprisingly, the transmitted founder variant was not derived from the predominant GT subpopulations. Rather, in each case, the transmitting variant was phylogenetically distinct from the sampled locally replicating population. Although the exact distribution of the virus population present in the GT at the time of transmission cannot be unambiguously defined in these human studies, it is unlikely, based on these data, that the transmission bottleneck is driven in every case by limited viral diversity in the donor GT or that HIV transmission is solely a stochastic event.
“…Pal et al showed that gp41 from viruses prepared in MOLT-3 cells was Endo H resistant, but when prepared in the presence of an ER mannosidase inhibitor, 1-deoxymannojirimycin, gp41 became sensitive to Endo H digestion (59). More recent work by us and by Go and colleagues has shown that the gp41 glycans on recombinant gp140 trimers (13,60,61), including the SOSIP trimer (14), are predominantly complex type and that these structures are generally more dependent on producer cells. In this study, we were able to record the chromatograms of labeled glycans from PGT151-isolated pseudovirion-derived material and cell surface-associated Env to provide a more detailed description of glycan structures present on native virion-derived gp41.…”
Section: Oligomannose Isomers Are the Same On Pbmc-derived Env And Rementioning
The gp120/gp41 HIV-1 envelope glycoprotein (Env) is highly glycosylated, with up to 50% of its mass consisting of N-linked glycans. This dense carbohydrate coat has emerged as a promising vaccine target, with its glycans acting as epitopes for a number of potent and broadly neutralizing antibodies (bnAbs). Characterizing the glycan structures present on native HIV-1 Env is thus a critical goal for the design of Env immunogens. In this study, we used a complementary, multistep approach involving ion mobility mass spectrometry and high-performance liquid chromatography to comprehensively characterize the glycan structures present on HIV-1 gp120 produced in peripheral blood mononuclear cells (PBMCs). The capacity of different expression systems, including pseudoviral particles and recombinant cell surface trimers, to reproduce native-like glycosylation was then assessed. A population of oligomannose glycans on gp120 was reproduced across all expression systems, supporting this as an intrinsic property of Env that can be targeted for vaccine design. In contrast, Env produced in HEK 293T cells failed to accurately reproduce the highly processed complex-type glycan structures observed on PBMC-derived gp120, and in particular the precise linkage of sialic acid residues that cap these glycans. Finally, we show that unlike for gp120, the glycans decorating gp41 are mostly complex-type sugars, consistent with the glycan specificity of bnAbs that target this region. These findings provide insights into the glycosylation of native and recombinant HIV-1 Env and can be used to inform strategies for immunogen design and preparation.
IMPORTANCEDevelopment of an HIV vaccine is desperately needed to control new infections, and elicitation of HIV bnAbs will likely be an important component of an effective vaccine. Increasingly, HIV bnAbs are being identified that bind to the N-linked glycans coating the HIV envelope glycoproteins gp120 and gp41, highlighting them as important targets for vaccine design. It is therefore important to characterize the glycan structures present on native, virion-associated gp120 and gp41 for development of vaccines that accurately mimic native-Env glycosylation. In this study, we used a number of analytical techniques to precisely study the structures of both the oligomannose and complex-type glycans present on native Env to provide a reference for determining the ability of potential HIV immunogens to accurately replicate the glycosylation pattern on these native structures.T he HIV-1 envelope glycoprotein (Env) consists of a noncovalently linked trimer of gp120/gp41 heterodimers. Interaction of this trimer with CD4 and its subsequent rearrangement is essential for infection of target cells and therefore is the sole target for broadly neutralizing antibodies (bnAbs). The surface protein, gp120, is extensively modified with host-derived glycans, having a median of 25 potential N-linked glycosylation sites (PNGSs) (1) and thus making it one of the most densely glycosylated proteins known. The t...
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