Nonrandom distribution of gp120 N-linked glycosylation sites important for infectivity of human immunodeficiency virus type 1.

Lee, William R.; Syu, Wan-Jr; Du, Bin; Matsuda, Michiko; Tan, Shencao; Wolf, Andrea; Essex, Max; Lee, Tun Hou

doi:10.1073/pnas.89.6.2213

Cited by 94 publications

(85 citation statements)

References 26 publications

(23 reference statements)

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“…In fact, when tested in U87-CD4 ϩ CXCR4 ϩ and TZM-bl cell lines, the infectivity levels of pseudovirions bearing the mutated envelopes (⌬G1, ⌬G2, ⌬G3, and ⌬G123) were enhanced, ranging from 134 to 216% as compared with those of the WT virus. These data are consistent with previous findings from other laboratories (56,57); however, whether these mutant viruses also showed comparable or slightly enhanced infectivity in primary target cells such as PBMCs remains to be determined. Hence, the minor changes in CD4 binding capacity of these mutants do not appear to seriously alter the envelope functions and virus infectivity.…”

Section: Gp120 Proteins Lacking Single or Triple N-glycans Bind To CDsupporting

confidence: 83%

Identification of an N-Linked Glycosylation in the C4 Region of HIV-1 Envelope gp120 That Is Critical for Recognition of Neighboring CD4 T Cell Epitopes

Chien

Tuen

et al. 2008

The Journal of Immunology

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The heavy glycosylation of HIV-1 envelope gp120 shields this important Ag from recognition by neutralizing Abs and cytolytic CD8 T cells. However, very little work has been done to understand the influence of glycosylation on the generation of gp120 epitopes and their recognition by MHC class II-restricted CD4 T cells. In this study, three conserved glycans (linked to N406, N448, and N463) flanking the C4 region of gp120 that contains many known CD4 T cell epitopes were disrupted individually or in combination by asparagine-to-glutamine substitutions. The mutant proteins lacking the N448 glycan did not effectively stimulate CD4 T cells specific for the nearby C4 epitopes, although the same mutants were recognized well by CD4 T cells specific for epitopes located in the distant C1 and C2 regions. The loss of recognition was not due to amino acid substitutions introduced to the mutant proteins. Data from trypsin digestion and mass spectrometry analyses demonstrated that the N448 glycan removal impeded the proteolytic cleavage of the nearby C4 region, without affecting more distant sites. Importantly, this inhibitory effect was observed only in the digestion of the native nondenatured protein and not in that of the denatured protein. These data indicate that the loss of the N448 glycan induces structural changes in the C4 region of gp120 that make this specific region more resistant to proteolytic processing, thereby restricting the generation of CD4 T cell epitopes from this region. Hence, N-linked glycans are critical determinants that can profoundly influence CD4 T cell recognition of HIV-1 gp120.

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Section: Gp120 Proteins Lacking Single or Triple N-glycans Bind To CDsupporting

confidence: 83%

Identification of an N-Linked Glycosylation in the C4 Region of HIV-1 Envelope gp120 That Is Critical for Recognition of Neighboring CD4 T Cell Epitopes

Chien

Tuen

et al. 2008

The Journal of Immunology

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“…Moreover, Fung et al (1992) have found that a broadly neutralizing MAb, reacting with a conformational epitope in the V2 region, is dependent on gpl20-associated N-linked glycans for epitope binding. Carbohydrate-induced modulation of antibody binding, as described above, has been suggested to be a viral strategy for escaping immune responses against conserved regions (Bolmstedt et al, 1991 ;Lee et al, 1992). Benjouad et al (1992) have found that hyperimmune sera raised against desialylated gpl60 show increased reactivity to a synthetic peptide representing a region of gpl20 situated close to the class I epitopes defined in the present study.…”

Section: Discussionmentioning

confidence: 89%

Carbohydrate determinant NeuAc-Galbeta(1-4) of N-linked glycans modulates the antigenic activity of human immunodeficiency virus type 1 glycoprotein gp120

Bolmstedt¹,

Olofsson

Sjögren‐Jansson³

et al. 1992

Journal of General Virology

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In the present study we investigated to what extent the peripheral carbohydrate structure of N-linked glycans influences the antigenic properties of human immunodeficiency virus type 1 glycoprotein 120 (gpl20). Recombinant gpl20 was purified from GMK cells infected with a recombinant vaccinia virus expressing gpl20. Purified gpl20 was then coated onto 96-well ELISA microplates and subjected to sequential removal of peripheral monosaccharide units. Modified or unmodified gpl20 was then incubated with monoclonal antibodies recognizing specific epitopes of gpl20 and with a reporter lectin to determine the extent of carbohydrate elimination. Antibody and lectin binding was quantified in an enzyme-linked system. We found that the carbohydrate structure NeuAc-Galfl(1-4) of N-linked glycans, defined both by lectin reactivity and by specific glycosidases, is involved in modulating the binding of antibody to a number of epitopes of peptide nature. The binding of antibody to one class of epitopes, situated in a region between amino acids 200 and 230, was strongly increased by removal of NeuAc-Galfl(1-4), whereas the binding to epitopes in the V3 region was decreased and the binding to epitopes in the far N-terminal region was not altered by the treatment. These results suggested that peripheral structures of N-glycans are involved in modulating the overall conformation of gp 120.

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“…Briefly, CEM cells (4.5 ϫ 10 5 cells per ml) were suspended in fresh culture medium and infected with HIV-1 at 100 CCID 50 (cell culture injective dose-50) per ml of cell suspension. Then 100 l of the infected cell suspension were transferred to microplate wells, mixed with 100 l of the appropriate dilutions of the test compounds, and further incubated at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…Strains-HIV-1(III B )-infected CEM cell cultures were exposed to UDA at ϳ5 g/ml (ϳ 0.6 M), which is an ϳ2-4-fold higher concentration than its EC 50 (Fig. 1).…”

Section: Selection and Genotypic Characterization Of Uda-resistant Himentioning

confidence: 99%

Carbohydrate-binding Agents Cause Deletions of Highly Conserved Glycosylation Sites in HIV GP120

Balzarini

Laethem

Hatse

et al. 2005

Journal of Biological Chemistry

110

125

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Here we demonstrated that the N-acetylglucosamine-binding protein from Urtica dioica (UDA) prevents HIV entry and eventually selects for viruses in which conserved N-glycosylation sites in GP120 were deleted. In contrast to the mannose-binding proteins, which have a 50 -100-fold decreased antiviral activity against the UDA-exposed mutant viruses, UDA has decreased anti-HIV activity to a very limited extent, even against those mutant virus strains that lack at least 9 of 22 (ϳ40%) glycosylation sites in their GP120 envelope. Therefore, UDA represents the prototype of a new conceptual class of carbohydrate-binding agents with an unusually specific and targeted drug resistance profile. It forces HIV to escape drug pressure by deleting the indispensable glycans on its GP120, thereby obligatorily exposing previously hidden immunogenic epitopes on its envelope.The glycoproteins GP120 and GP41 that are present on the envelope of HIV 2 mediate the entry of the virus into its host cells. The envelope glycoproteins bind sequentially to the cellular receptor protein CD4 and a co-receptor, mainly CXCR4 or CCR5. The receptor-binding events trigger conformational changes in the GP120 and GP41 that lead to membrane fusion and virus entry. After infection, the host cell synthesizes the viral envelope glycoproteins encoded by the HIV env gene. The env precursor becomes N-glycosylated to form the GP160 glycoprotein by addition of preassembled Glc 3 Man 9 GlcNAc 2 entities in the rough endoplasmic reticulum. After oligomerization to a trimer, GP160 is cleaved in the Golgi apparatus by a cellular protease (leading to noncovalently linked GP120 and GP41), and then the glycans are further processed (3). A large fraction of the predicted accessible surface of GP120 in the trimer is composed of heavily glycosylated structures that surround the receptor-binding regions (4). In GP120 of HIV-1(III B ), all 24 potential N-linked glycosylation sites are utilized as follows: 13 sites containing complex-type oligosaccharides and 11 sites containing hybrid and/or high mannose-type structures (5, 6). In GP41, there are seven potential N-glycosylation sites but only four of them seem to be glycosylated. All N-linked glycoprotein carbohydrates in GP120 share a common pentasaccharide Man 3 GlcNAc 2 linked to the amide nitrogen of asparagine through the reducing hydroxyl group of GlcNAc. In HIV-1 GP120, 33% of the oligosaccharides are of the high mannose type,

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Nonrandom distribution of gp120 N-linked glycosylation sites important for infectivity of human immunodeficiency virus type 1.

Cited by 94 publications

References 26 publications

Identification of an N-Linked Glycosylation in the C4 Region of HIV-1 Envelope gp120 That Is Critical for Recognition of Neighboring CD4 T Cell Epitopes

Identification of an N-Linked Glycosylation in the C4 Region of HIV-1 Envelope gp120 That Is Critical for Recognition of Neighboring CD4 T Cell Epitopes

Carbohydrate determinant NeuAc-Galbeta(1-4) of N-linked glycans modulates the antigenic activity of human immunodeficiency virus type 1 glycoprotein gp120

Carbohydrate-binding Agents Cause Deletions of Highly Conserved Glycosylation Sites in HIV GP120

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