Glycosylation Patterns of HIV-1 gp120 Depend on the Type of Expressing Cells and Affect Antibody Recognition

Department of Immunology, Palacky University in Olomouc, Olomouc 77100, Czech Republic.
Journal of Biological Chemistry (Impact Factor: 4.57). 05/2010; 285(27):20860-9. DOI: 10.1074/jbc.M109.085472
Source: PubMed


Human immunodeficiency virus type 1 (HIV-1) entry is mediated by the interaction between a variably glycosylated envelope
glycoprotein (gp120) and host-cell receptors. Approximately half of the molecular mass of gp120 is contributed by N-glycans, which serve as potential epitopes and may shield gp120 from immune recognition. The role of gp120 glycans in the
host immune response to HIV-1 has not been comprehensively studied at the molecular level. We developed a new approach to
characterize cell-specific gp120 glycosylation, the regulation of glycosylation, and the effect of variable glycosylation
on antibody reactivity. A model oligomeric gp120 was expressed in different cell types, including cell lines that represent
host-infected cells or cells used to produce gp120 for vaccination purposes. N-Glycosylation of gp120 varied, depending on the cell type used for its expression and the metabolic manipulation during expression.
The resultant glycosylation included changes in the ratio of high-mannose to complex N-glycans, terminal decoration, and branching. Differential glycosylation of gp120 affected envelope recognition by polyclonal
antibodies from the sera of HIV-1-infected subjects. These results indicate that gp120 glycans contribute to antibody reactivity
and should be considered in HIV-1 vaccine design.

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    • "The enormous relevance of glycans in HIV-1 vaccine design is underscored by the isolation of numerous distinct families of potent bNAbs whose binding is dependent on Env glycans (Blattner et al., 2014; Falkowska et al., 2014; Garces et al., 2014; Huang et al., 2014; Kong et al., 2013; McLellan et al., 2011; Mouquet et al., 2012; Pancera et al., 2013; Pejchal et al., 2011; Scharf et al., 2014; Walker et al., 2009, 2011). Studies on monomeric gp120 proteins have consistently identified two major subgroups of glycan structures: underprocessed oligomannose and processed complex glycans (Bonomelli et al., 2011; Doores et al., 2010; Go et al., 2013; Leonard et al., 1990; Raska et al., 2010). The underprocessed glycans contain multiple terminal mannose sugars (typically five to nine, referred to as Man 5 GlcNAc 2 to Man 9 GlcNAc 2 ). "
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    ABSTRACT: A highly glycosylated, trimeric envelope glycoprotein (Env) mediates HIV-1 cell entry. The high density and heterogeneity of the glycans shield Env from recognition by the immune system, but paradoxically, many potent broadly neutralizing antibodies (bNAbs) recognize epitopes involving this glycan shield. To better understand Env glycosylation and its role in bNAb recognition, we characterized a soluble, cleaved recombinant trimer (BG505 SOSIP.664) that is a close structural and antigenic mimic of native Env. Large, unprocessed oligomannose-type structures (Man8-9GlcNAc2) are notably prevalent on the gp120 components of the trimer, irrespective of the mammalian cell expression system or the bNAb used for affinity purification. In contrast, gp41 subunits carry more highly processed glycans. The glycans on uncleaved, non-native oligomeric gp140 proteins are also highly processed. A homogeneous, oligomannose-dominated glycan profile is therefore a hallmark of a native Env conformation and a potential Achilles' heel that can be exploited for bNAb recognition and vaccine design. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 06/2015; 11(10). DOI:10.1016/j.celrep.2015.05.017 · 8.36 Impact Factor
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    • "To elucidate the role of Env glycans in binding of gp120 by HIV-1-specific antibodies, we evaluated the binding of recombinant gp120 produced in four cell lines (HEK 293T, Jurkat, HepG2, and CHO) representing four differentially glycosylated gp120 variants [21]. "
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    ABSTRACT: Background HIV-1 entry into host cells is mediated by interactions between the virus envelope glycoprotein (gp120/gp41) and host-cell receptors. N-glycans represent approximately 50% of the molecular mass of gp120 and serve as potential antigenic determinants and/or as a shield against immune recognition. We previously reported that N-glycosylation of recombinant gp120 varied, depending on the producer cells, and the glycosylation variability affected gp120 recognition by serum antibodies from persons infected with HIV-1 subtype B. However, the impact of gp120 differential glycosylation on recognition by broadly neutralizing monoclonal antibodies or by polyclonal antibodies of individuals infected with other HIV-1 subtypes is unknown. Methods Recombinant multimerizing gp120 antigens were expressed in different cells, HEK 293T, T-cell, rhabdomyosarcoma, hepatocellular carcinoma, and Chinese hamster ovary cell lines. Binding of broadly neutralizing monoclonal antibodies and polyclonal antibodies from sera of subtype A/C HIV-1-infected subjects with individual gp120 glycoforms was assessed by ELISA. In addition, immunodetection was performed using Western and dot blot assays. Recombinant gp120 glycoforms were tested for inhibition of infection of reporter cells by SF162 and YU.2 Env-pseudotyped R5 viruses. Results We demonstrated, using ELISA, that gp120 glycans sterically adjacent to the V3 loop only moderately contribute to differential recognition of a short apex motif GPGRA and GPGR by monoclonal antibodies F425 B4e8 and 447-52D, respectively. The binding of antibodies recognizing longer peptide motifs overlapping with GPGR epitope (268 D4, 257 D4, 19b) was significantly altered. Recognition of gp120 glycoforms by monoclonal antibodies specific for other than V3-loop epitopes was significantly affected by cell types used for gp120 expression. These epitopes included CD4-binding site (VRC03, VRC01, b12), discontinuous epitope involving V1/V2 loop with the associated glycans (PG9, PG16), and an epitope including V3-base-, N332 oligomannose-, and surrounding glycans-containing epitope (PGT 121). Moreover, the different gp120 glycoforms variably inhibited HIV-1 infection of reporter cells. Conclusion Our data support the hypothesis that the glycosylation machinery of different cells shapes gp120 glycosylation and, consequently, impacts envelope recognition by specific antibodies as well as the interaction of HIV-1 gp120 with cellular receptors. These findings underscore the importance of selection of appropriately glycosylated HIV-1 envelope as a vaccine antigen.
    AIDS Research and Therapy 08/2014; 11(1):23. DOI:10.1186/1742-6405-11-23 · 1.46 Impact Factor
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    • "It has previously been reported that gp120 is able to repress TLR9-triggered responses in pDCs, while gp120-induced no effect on TLR7-induced pDC responses (Martinelli et al., 2007). In the former report recombinant gp120 was employed, which later on has been shown not to be glycosylated in a manner similar to viral particles existing in vivo (Doores et al., 2010; Raska et al., 2010). Therefore we decided to use complete viral particles to assay our hypothesis of MR-mediated immune dampening effects. "
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    ABSTRACT: Plasmacytoid dendritic cells (pDCs) play a vital role in activation of anti-HIV-1 immunity, and suppression of pDCs might mitigate immune responses against HIV-1. HIV-1 gp120 high-mannose has been attributed immunosuppressive roles in human myeloid DCs, but no receptors for high-mannose have so far been reported on human pDCs. Here we show that upon activation with HIV-1 or by a synthetic compound triggering the same receptor in human pDCs as single-stranded RNA, human pDCs upregulate the mannose receptor (MR, CD206). To examine the functional outcome of this upregulation, inactivated intact or viable HIV-1 particles with various degrees of mannosylation were cultured with pDCs. Activation of pDCs was determined by assaying secretion of IFN-alpha, viability, and upregulation of several pDC-activation markers: CD40, CD86, HLA-DR, CCR7, and PD-L1. The level of activation negatively correlated with degree of mannosylation, however, subsequent reduction in the original mannosylation level had no effect on the pDC phenotype. Furthermore, two of the infectious HIV-1 strains induced profound necrosis in pDCs, also in a mannose-independent manner. We therefore conclude that natural mannosylation of HIV-1 is not involved in HIV-1-mediated immune suppression of pDCs.
    Molecular Immunology 06/2014; 59(2):180–187. DOI:10.1016/j.molimm.2014.02.009 · 2.97 Impact Factor
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