Detection of orientation-specific anti-gp120 antibodies by a new N-glycanase protection assay.

Several functions have been assigned to the extensive glycosylation of HIV-1 envelope glycoprotein gp120, especially immune escape mechanisms, but the intramolecular interactions between gp120 and its carbohydrate complement are not well understood. To analyse this phenomenon we established a new microwell deglycosylation assay for determining N-linked glycan accessibility after binding of gp120-specific agents. Orientation-specific exposition of gp120 in ELISA microplates was achieved by catching with either anti-C5 antibody D7324 or anti-V3 antibody NEA-9205. We found that soluble CD4 inhibited the deglycosylation of gp120 only when gp120 was caught by D7324 and not by NEA9205. In contrast, antibodies from HIV-infected individuals inhibited the deglycosylation best when gp120 was caught by NEA9205. These results demonstrated that both the CD4-binding site and the epitopes recognised by antibodies from HIV-infected individuals have N-glycans in the close vicinity. However, the difference in gp120 orientation indicates that antibodies in HIV-infected individuals, at least partly, bind to epitopes different from the CD4-binding site. Finally, we determined the structural class of the glycan of one V1 glycosylation site of prototype HIV-1 LAI gp120, which remained unsolved from previous studies, and found that it belonged to the complex type of glycans.

Enhanced immunogenicity of a human immunodeficiency virus type 1 env DNA vaccine by manipulating N-glycosylation signals. Effects of elimination of the V3 N306 glycan.

DNA encoding HIV-1 env is a poorly efficient B-cell immunogen and one probable explanation is that the numerous gp120 N-linked glycans gp120 may interfere with B-cell epitope presentation. The N306 glycan in gp120 shields HIV-1 from neutralizing antibodies. A DNA immunogen lacking the N306 glycosylation signal (T308A) was constructed to determine whether this glycan affected the immune response. Mice were immunized intranasally twice with DNA containing either the wild type or the mutant env. Two additional groups were primed with wild type or mutant env and boosted with rgp160 protein, containing the complete set of N-linked glycans. Immunization with DNA alone resulted in priming of B-cell clones but was not sufficient to induce a complete antibody response. Animals primed with the N306 mutant and subsequently boosted with rgp160 protein displayed higher serum IgG-binding titers to gp120 than animals primed with wild type env DNA. The manipulation of the glycosylation sites of the env DNA strongly primes antibody responses (but non-neutralizing) as well as T-cell responses to the wild type strain gp160. However, priming with mutant plasmid did not result in higher neutralization titers to wild type or T308A-mutated virus than did the wild type plasmid. With the N306 mutant DNA we thus immunized a non-neutralization epitope, but obtained strong env-binding IgG after rgp160 boosting.

Early steps in O-linked glycosylation and clustered O-linked glycans of herpes simplex virus type 1 glycoprotein C: effects on glycoprotein properties.

The pathogenesis of herpes simplex virus type 1 (HSV-1) implies the sequential infection of many cell types from mucosal cells to neurons, each having a unique pattern of protein glycosylation. The HSV-1 glycoprotein gC-1 is highly glycosylated and contains not only N-linked glycans but also a large number of O-linked glycans, some of which are clustered into two pronase-resistant arrays in the vicinity of the HSV-1 receptor-binding domain of gC-1. The aim of the present study was to characterize gC-1 signals for addition of clustered glycans, to determine the efficacy of synthetic peptides, representing putative O-glycosylation signals, as substrates for a panel of GalNAc transferases, and to identify possible effects of early O-linked glycosylation on the biological functions of gC-1. Gel filtration analysis of the pronase-resistant gC-1 O-glycan clusters from a glycoprotein mutant, lacking a site for N-linked glycosylation at Asn 73 in the vicinity of the O-glycosylation signal, suggested that one function of this N-linked glycan was to modulate the access for GalNAc transferases to one particular O-glycosylation peptide signal (aa 80-104). The ability of four GalNAc-transferase isoenzymes with different cell type expression patterns to initialize O-glycosylation of synthetic gC-1 derived peptides was analyzed. Two synthetic gC-1 peptides (aa 55-69 and aa 80-104) were excellent substrates for all four GalNAc-transferases, suggesting that cell types expressing less frequent GalNAc transferase species with unusual acceptor peptide sequence specificities may also produce a highly O-glycosylated gC-1 after HSV-1 infection. The O-linked glycans were not essential for cell surface expression of gC-1, but monoclonal antibody-assisted epitope analysis of N-acetylgalactosaminidase-treated gC-1 showed that the O-linked monosaccharide GalNAc contributed to expression of a three-dimensional epitope overlapping the heparan sulfate-binding domain of gC-1.

The N-linked glycan of the V3 region of HIV-1 gp120 and CXCR4-dependent multiplication of a human immunodeficiency virus type 1 lymphocyte-tropic variant.

We have previously shown that an N-glycosylation site of N306 of HIV-1 gp120 is not necessary for the HIV-1 infectivity but protects HIV-1 from neutralising antibodies. In contrast Nakayama et al. [FEBS Lett. (1998) 426, 367-372], using a virus with an identical V3 region, suggested that elimination of this particular glycan reduced the ability of T-tropic HIV to bind to CXCR4 and hence its ability to infect T cell lines. We therefore re-examined the ability of a mutant virus, lacking the N306 glycan, to replicate in various types of cells and found no change in co-receptor usage for mutant virus. The ability of mutant virus to replicate or to induce syncytia in infected cells was similar to that of wild type virus. These results corroborate our original observation, confirming that the induced mutation in the N306 glycosylation site neither impairs nor improves the ability of mutant virus to replicate in permissive cells.

The role of a single N-linked glycosylation site for a functional epitope of herpes simplex virus type 1 envelope glycoprotein gC.

A monoclonal antibody, B1C1, binding to an epitope of antigenic site II of the herpes simplex virus type 1 (HSV-1) glycoprotein gC-1, is a potent inhibitor of two important biological functions of gC-1: its binding to cell surface heparan sulfate and its binding to the receptor for complement factor C3b. Here, we have analyzed a B1C1-resistant HSV-1 variant (HSV-12762/B1C1B4.2), obtained after passage of wild type HSV-1 (HSV-12762) in the presence of high concentrations of B1C1. The transport of newly synthesized mutant gC-1 to the cell surface was comparable to that of wild type glycoprotein, but no binding of surface-associated mutant gC-1 to B1C1 was detected. However, mutant and wild type gC-1 bound equally well to other site II Mabs. Attachment of wild type but not mutant virus was inhibited by B1C1. Sequencing of the mutant gC-1 gene revealed only one nucleotide change, resulting in replacement of Thr150 by an Ile, in turn destroying an N-glycosylation site at Asn148. Loss of one complex type N-linked glycan was confirmed by endoglycosidase digestion and subsequent SDS-polyacrylamide gel electrophoresis. Circular dichroism analysis of purified gC-1 from cells infected with mutant or wild type virus did not reveal any difference in secondary structure between mutant and wild type gC-1. It was not possible to obtain a B1C1-resistant phenotype by nucleotide-directed mutagenesis of gC-1 where Asn148 was changed to a glutamine. These data demonstrated that the threonine of the glycosylation site and not the N-linked glycan in itself was essential for B1C1 binding

Simplified procedure for fractionation and structural characterisation of complex mixtures of N-linked glycans, released from HIV-1 gp120 and other highly glycosylated viral proteins.

HIV-1 gp120 is heavily glycosylated containing 24 N-glycosylation sites, and this makes elucidation of the significance of glycans at individual glycosylation sites a difficult task. A procedure is described where a complex mixture of biologically radiolabelled glycans of gp120, derived from a relatively small number of virus-infected cells may be characterized by a combination of N-glycanase release, single lectin separation, and normal phase HPLC (NP-HPLC). The method was applied in analysis of three N-linked glycosylation sites essential for the in vivo priming of T-cells, specific for an epitope in their vicinity (Sjölander, S., Bolmstedt, A., Akerblom, 1996. Virology 215, 124-133.). The carbohydrate compositions of wild type gp120 and of mutant variants gp120 lacking one, two, or all of these three active N-linked glycans were analysed. Cells were infected with r-vaccinia virus expressing wild-type gp120 or mutated gp120, or were infected with HIV-1BRU (wild type) or mutant virus variants. HIV-1 glycoproteins were purified by immunosorbent affinity chromatography and released glycans were separated on lectins, then analysed with NP-HPLC. Our data showed that the structural composition of glycans occupying two of the three glycosylation sites was heterogeneous but the site located adjacent to the T-cell epitope was equipped with one large, high mannose-type structure (> 11 units) with the capacity to cover a substantial part of the gp120 surface.

Demonstration of peripheral fucose units in N-linked glycans of human immunodeficiency virus type 1 gp 120: effects on glycoprotein conformation.

Fucosylated N-linked glycans are important constituents of membrane glycoproteins, owing to their significance as biologically active ligands for several selectins and their role in modulating protein conformation of viral glycoproteins. The human immunodeficiency virus type 1 (HIV-1) glycoprotein contains more than 30 different glycan structures but so far fucose was found associated solely with the innermost GlcNAc of N-linked glycans. In the present report we determined whether fucose units also were linked to the distal GlcNAc via alpha(1-3) or alpha(1-4) linkages in N-linked glycans of gp 120. [3H]-fucose labelled gp 120 was subjected to endoglycosidase F digestion, releasing diantennary complex type N-linked glycans, but leaving the inner polypeptide-bound carbohydrates, GlcNAc and possibly associated fucose units, intact. Gel filtration of the digested material revealed that [3H]-fucose label was released from gp 120 by this treatment, indicating presence of peripheral fucose units. Furthermore, [3H]-focuse label was also released by treatment of the labelled gp 120 with an alpha-L-fucosidase specifically removing fucose in alpha(1-3) and alpha(1-4) linkages. Altogether the results indicated presence of fucose units linked to peripheral GlcNAc of gp 120 N-linked glycans. We have earlier shown that other peripheral carbohydrate determinants, i.e. beta(1-4)-galactose on N-linked glycans, maintain a correct antigenic conformation of gp 120. Using a coupled ELISA system, where changes in antigenic behaviour of a viral glycoprotein were correlated to stepwise elimination of peripheral monosaccharides from N-linked glycans, we found that treatment of gp 120 with a pan-specific alpha-fucosidase as well as an enzyme specific for alpha(1-3)- or alpha(1-4)-linked fucose disclosed a hidden linear epitope situated in the gp 120 C2 region. The effects of the general fucosidase on epitope exposure was more prominent than those obtained with the enzyme with narrow specificity, suggesting that peripheral and inner fucose units co-operate in the maintenance of gp 120 conformation.