AEGiS-07IAC: Role of N-linked glycans in the folding of HIV-1 envelope glycoproteins.

7th International AIDS Conference

Florence, Italy — June 16-21, 1991

Role of N-linked glycans in the folding of HIV-1 envelope glycoproteins.

Int Conf AIDS 1991 Jun 16-21; 7:24 (abstract no. M.A.9)
Fenouillet E, Gluckman JC; CERVI, Hopital Pitie-Salpetriere, Paris - France

OBJECTIVE: Our recent results indicate that carbohydrates (CHO) present on mature env glycoproteins (gp) do not play a major role for HIV-1 infectivity (J. Exp. Med. 1989, March; J. Virol. 1990, June). However, sugar analogs like 1 deoxynojirimycin (dNM) that interfere with the glycosylation process of nascent gp160 markedly reduce virus infectivity. To resolve this apparent discrepancy we approached the mechanisms by which glycan processing interplays with gp160 tridimensionnal structure.

METHODS: We investigated the effect of dNM (4mM) on glycosylation, production, bioactivity and immunoreactivity of gp160 produced by recombinant vaccinia virus-infected BHK21 cells. Glycosylation was studied by endoglycosidase analysis. Binding to CD4+ cells was determined by quantitative indirect immunofluorescence using anti-HIV-1 human antibodies. Because it is thought that exposed V3 loop of gp120 plays an essential role in post CD4-binding events, accessibility of V3 to mAb 110-4 (Genetic Systems) was studied by ELISA capture and by immunoaffinity purification. Non-glycosylated gp120 obtained in E. coli (ec gp120) and mannosylated gp160 from recombinant baculovirus-infected insect cells (bcv gp160) were used as controls.

RESULTS: dNM did not affect the amount of rgp160 recovered nor its secretion from the cells. As already described, dNM effect was incomplete, resulting in the production of molecules the glycosylation of which was heterogeneous with respect to their apparent MW and to sensitivity to Endoglycosidase H. Strongly reduced binding to CD4+ cells was noted with rgp160 produced in dNM treated cells, bcv gp160 bound to CD4+ cells, but not ec gp120. Similarly, dNM treatment dramatically altered (by at least 10 fold) the accessibility of V3 to 110-4, whereas ec gp120 and bcv gp160 presented unchanged immunoreactivity.

CONCLUSIONS: Proper processing of CHO of nascent gp160 is essential for normal folding. dNM profoundly affects the glycosylation, bioactivity and conformation of rgp160. This may account for impaired HIV-1 infectivity elicited by dNM, while glycans present on the mature molecule are not of paramount importance.

Keywords: AEGIS, HIV-1, Gene Products, env, Antigens, CD4, Polysaccharides, Glycosylation, Glycoproteins, Aluminum Hydroxide, Carbohydrates, Link, Human, ICA7
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