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- Losman, Britt, 1963
(författare)
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HIV-1 envelope protein gp120. Glycovirology of the V3 loop
- 2000
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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.
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| 2. |
- Losman, Britt, et al.
(författare)
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Protection of neutralization epitopes in the V3 loop of oligomeric human immunodeficiency virus type I glycoprotein 120 by N-linked oligosaccharides in the V1 region
- 2001
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Ingår i: AIDS Research and Human Retroviruses. - : Mary Ann Liebert Inc. - 1931-8405 .- 0889-2229. ; 17:11, s. 1067-1076
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Tidskriftsartikel (refereegranskat)abstract
- The V3 region of the human immunodeficiency virus type 1 envelope protein gp120 constitutes a potential neutralization target, but the oligosaccharide of one conserved N-glycosylation site in this region protects it from neutralizing antibodies. Here, we determined whether N-linked glycans of other gp120 domains were also involved in protection of V3 neutralization epitopes. Two molecular clones of HIV-1, one lacking three N-Iinked glycans of the V1 region (HIV-1(3N/V1)) and another lacking three N-linked glycans of the C2 region (HIV-1(3N/C2)), were created and characterized. gp120 from both mutated viral clones had higher electrophoretic mobilities than gp120 from wild-type virus, confirming loss of N-linked glycans. Wild-type virus and both mutant clones replicated equally well in established T cell lines and all three viruses were able to utilize CXCR4 but not CCR5 as a coreceptor. The induced mutations increased gp120 affinity for CXCR4 but caused no corresponding increase in viral ability to replicate in T cell lines. HIV-1(3N/V1) was neutralized at about 25 times lower concentrations of an antibody to the V3 region than were wild-type virus and HIV-1(3N/C2). Soluble, monomeric gp120 from HIV-1(3N/V1) and wild type virus had identical avidity for the V3 antibody, indicating that the V1 glycans were able to shield V3 only in oligomeric but not monomeric gp120. In conclusion, one or more N-linked glycans of gp120 V1 is engaged in protection of the V3 region from potential neutralizing antibodies, and this effect is dependent on the oligomeric organization of gp120/gp4l.
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