Mapping the complete glycoproteome of virion-derived HIV-1 gp120 provides insights into broadly neutralizing antibody binding

Panico, Maria; Bouché, Laura; Binet, D.; O’Connor, Michael J.; Rahman, Dinah; Pang, Poh Choo; Canis, Kevin; North, Simon J.; Desrosiers, Ronald C.; Chertova, Elena; Keele, Brandon F.; Bess, Julian W.; Lifson, Jeffrey D.; Haslam, Stuart M.; Dell, Anne; Morris, Howard R.

doi:10.1038/srep32956

Cited by 72 publications

(95 citation statements)

References 53 publications

(67 reference statements)

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“…The M8166-expressed JR-FL gp150 also had a population of processed glycoforms at this site, and that population matched the majority glycoform type for all the other Env trimers reported in the tables in the supplemental material. For BAL, the Env glycopeptides were reported previously (48), but the glycopeptide coverage was low, with only four glycoforms detected at the N88 site. By comparison, we obtained between 9 and 30 For the N133-N139 glycosylation sites in the gp120 V1 variable region, only the BG505 SOSIP.664 trimer and the virion-derived BAL Env contain an outlier glycosylation profile in which complex glycans predominate.…”

Section: Resultsmentioning

confidence: 80%

“…Three of the trimer data sets are available in the literature, originally generated by the Desaire laboratory (25), the Crispin laboratory (46), and the Dell laboratory (48). Eight additional Env trimers were characterized for the present report.…”

Section: Resultsmentioning

confidence: 99%

“…Accordingly, it is unclear how these protein design and expression differences impacted the results. A subsequent analysis of glycosylation on a third genotype of Env (BAL, which is a clade B genotype), isolated from virions and purified by reverse-phase high-performance liquid chromatography (rHPLC), has been reported recently (48). Some of the glycan assignments in the latter virion study differed from ones identified in the earlier reports on the recombinant JR-FL and BG505 trimers for reasons that are not yet known.…”

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confidence: 96%

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Glycosylation Benchmark Profile for HIV-1 Envelope Glycoprotein Production Based on Eleven Env Trimers

Ding

Zhang

et al. 2017

J Virol

111

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HIV-1 envelope glycoprotein (Env) glycosylation is important because individual glycans are components of multiple broadly neutralizing antibody epitopes, while shielding other sites that might otherwise be immunogenic. The glycosylation on Env is influenced by a variety of factors, including the genotype of the protein, the cell line used for its expression, and the details of the construct design. Here, we used a mass spectrometry (MS)-based approach to map the complete glycosylation profile at every site in multiple HIV-1 Env trimers, accomplishing two goals. (i) We determined which glycosylation sites contain conserved glycan profiles across many trimeric Envs. (ii) We identified the variables that impact Env's glycosylation profile at sites with divergent glycosylation. Over half of the gp120 glycosylation sites on 11 different trimeric Envs have a conserved glycan profile, indicating that a native consensus glycosylation profile does indeed exist among trimers. We showed that some soluble gp120s and gp140s exhibit highly divergent glycosylation profiles compared to trimeric Env. We also assessed the impact of several variables on Env glycosylation: truncating the full-length Env; producing Env, instead of the more virologically relevant T lymphocytes, in CHO cells; and purifying Env with different chromatographic platforms, including nickel-nitrilotriacetic acid (Ni-NTA), 2G12, and PGT151 affinity. This report provides the first consensus glycosylation profile of Env trimers, which should serve as a useful benchmark for HIV-1 vaccine developers. This report also defines the sites where glycosylation may be impacted when Env trimers are truncated or produced in CHO cells.IMPORTANCE A protective HIV-1 vaccine will likely include a recombinant version of the viral envelope glycoprotein (Env). Env is highly glycosylated, and yet vaccine developers have lacked guidance on how to assess whether their immunogens have optimal glycosylation. The following important questions are still unanswered. (i) What is the "target" glycosylation profile, when the goal is to generate a natively glycosylated protein? (ii) What variables exert the greatest influence on Env glycosylation? We identified numerous sites on Env where the glycosylation profile does not deviate in 11 different Env trimers, and we investigated the impact on the divergent glycosylation profiles of changing the genotype of the Env sequence, the construct design, the purification method, and the producer cell type. The data presented here give vaccine developers a "glycosylation target" for their immunogens, and they show how protein production variables can impact Env glycosylation.

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Section: Resultsmentioning

confidence: 80%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 96%

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Glycosylation Benchmark Profile for HIV-1 Envelope Glycoprotein Production Based on Eleven Env Trimers

Ding

Zhang

et al. 2017

J Virol

111

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“…However, compared to the highly diverse protein component of Env, many of the potential N-glycosylation sites (PNGSs) are well conserved; their total number has also remained relatively constant despite years of viral evolution (5,6). While the glycans are derived from the host cell's glycosylation machinery, their high density leads to a large abundance of underprocessed, oligomannose-type glycans (Man [5][6][7][8][9] GlcNAc 2 ) (7-11). The glycan shield is therefore surprisingly homogenous given the extent of glycosylation, and it is itself a target for bNAbs that recognize exclusively glycan or mixed glycan/protein epitopes (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25).…”

mentioning

confidence: 99%

“…The IMP is a characteristic feature of gp120 that is conserved across all HIV-1 clades (7,8,48) and longitudinally during infection (49). Analyses of recombinant soluble, native-like trimers (40,50), membraneassociated trimers (51), and virion-derived Env (7)(8)(9) all show that their oligomannosetype glycan contents are even higher than those of gp120 monomers, implying that a "trimer-associated mannose patch" (TAMP) exists (52). However, the glycan sites forming any such TAMP have not previously been defined.…”

mentioning

confidence: 99%

Molecular Architecture of the Cleavage-Dependent Mannose Patch on a Soluble HIV-1 Envelope Glycoprotein Trimer

Behrens

Harvey

Milne

et al. 2017

J Virol

135

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The formation of a correctly folded and natively glycosylated HIV-1 viral spike is dependent on protease cleavage of the gp160 precursor protein in the Golgi apparatus. Cleavage induces a compact structure which not only renders the spike capable of fusion but also limits further maturation of its extensive glycosylation. The redirection of the glycosylation pathway to preserve underprocessed oligomannose-type glycans is an important feature in immunogen design, as glycans contribute to or influence the epitopes of numerous broadly neutralizing antibodies.Here we present a quantitative site-specific analysis of a recombinant, trimeric mimic of the native HIV-1 viral spike (BG505 SOSIP.664) compared to the corresponding uncleaved pseudotrimer and the matched gp120 monomer. We present a detailed molecular map of a trimer-associated glycan remodeling that forms a localized subdomain of the native mannose patch. The formation of native trimers is a critical design feature in shaping the glycan epitopes presented on recombinant vaccine candidates. IMPORTANCEThe envelope spike of human immunodeficiency virus type 1 (HIV-1) is a target for antibody-based neutralization. For some patients infected with HIV-1, highly potent antibodies have been isolated that can neutralize a wide range of circulating viruses. It is a goal of HIV-1 vaccine research to elicit these antibodies by immunization with recombinant mimics of the viral spike. These antibodies have evolved to recognize the dense array of glycans that coat the surface of the viral molecule. We show how the structure of these glycans is shaped by steric constraints imposed upon them by the native folding of the viral spike. This information is important in guiding the development of vaccine candidates.KEYWORDS furin, glycan, glycosylation, human immunodeficiency virus, neutralizing antibodies, oligosaccharides, structure, vaccines T he trimeric envelope (Env) glycoprotein spike of human immunodeficiency virus type 1 (HIV-1) plays a crucial role in mediating host cell infection. Its exposed position on the virus surface also makes it the target for potent, broadly neutralizing antibodies (bNAbs) that are produced in a subset of infected individuals and that now influence the design of Env immunogens intended to induce similar antibody specificities. Env is extensively glycosylated, with glycans contributing to a significant proportion of the glycoprotein's mass (1). The heavily glycosylated surface of Env has been referred to as the "silent face," with the glycans acting to shield the underlying viral protein surface from immune surveillance (2-4). The glycan shield constantly evolves to protect the virus from newly produced neutralizing antibodies. However, compared to the highly diverse protein component of Env, many of the potential

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Viral glycoproteomes: technologies for characterization and outlook for vaccine design

et al. 2018

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It has long been known that surface proteins of most enveloped viruses are covered with glycans. It has furthermore been demonstrated that glycosylation is essential for propagation and immune evasion for many viruses. The recent development of high-resolution mass spectrometry techniques has enabled identification not only of the precise structures but also the positions of such post-translational modifications on viruses, revealing substantial differences in extent of glycosylation and glycan maturation for different classes of viruses. In-depth characterization of glycosylation and other post-translational modifications of viral envelope glycoproteins is essential for rational design of vaccines and antivirals. In this Review, we provide an overview of techniques used to address viral glycosylation and summarize information on glycosylation of enveloped viruses representing ongoing public health challenges. Furthermore, we discuss how knowledge on glycosylation can be translated to means to prevent and combat viral infections.

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Mapping the complete glycoproteome of virion-derived HIV-1 gp120 provides insights into broadly neutralizing antibody binding

Cited by 72 publications

References 53 publications

Glycosylation Benchmark Profile for HIV-1 Envelope Glycoprotein Production Based on Eleven Env Trimers

Glycosylation Benchmark Profile for HIV-1 Envelope Glycoprotein Production Based on Eleven Env Trimers

Molecular Architecture of the Cleavage-Dependent Mannose Patch on a Soluble HIV-1 Envelope Glycoprotein Trimer

Viral glycoproteomes: technologies for characterization and outlook for vaccine design

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