HIV-1 envelope protein gp120. Glycovirology of the V3 loop

Abstract

The human immunodeficiency virus type 1 (HIV-1) infection usually is lethal as a consequence of the acquired immunodeficiency syndrome (AIDS). HIV-1 is an enveloped virus and the trimeric envelope glycoprotein complex, gp120/gp41, is responsible for binding of the virus both to the primary cellular receptor CD4 and the coreceptors, CXCR4 and CCR5. Antibody interference with virion attachment to target cell appears to be a major mechanism of neutralisation by gp120-specific antibodies, and is therefore a major target for vaccine development. However, the extremely high carbohydrate content of gp120 interferes with not only induction of an antibody immune response but also with the ability of antibodies to neutralise HIV-1. The aim of the present study was to characterise the mechanisms by which carbohydrates participate in immune escape mechanisms, involving gp120-V3 region.N-linked oligosaccharides were eliminated by site-directed mutagenesis of N-glycosylation sites. Mutagenised gp120 was cloned into a proviral vector for production of modified virus after transfection of permissive cells. The ability of modified gp120 to interact with monoclonal antibodies to the V3 domain was investigated with molecular immunology methods, including virus neutralisation assays, affinity assays, and radioimmunoprecipitation techniques on gp120 monomers and gp120/gp41 trimers. It was found that epitopes of the V3 region were shielded from neutralising antibodies by N-linked glycans located in the V3 and V1 regions. However, this carbohydrate-induced shielding of neutralisation epitopes was strictly dependent on the oligomeric organisation of the envelope glycoprotein. Mutant virus lacking the N-glycan in the V3 region was subjected to a strong immunoselection pressure by propagating virus in the presence of antibodies to V3 region. Several escape mutants were isolated and sequenced and most of the resistant clones had regained the N-glycosylation site. The conclusion that carbohydrate-induced protection of gp120 V3 is an important escape mechanism also in vivo is supported by sequence data from early and late patient isolates of HIV-1. The present work defines N-glycans with the ability to interfere with the neutralising antibody response to HIV-1 and elucidates the mechanisms for shielding of gp120 epitopes. This may have implications for development of improved vaccines.