The HIV-1 envelope (Env) glycans shield the surface of Env from the immune system and form integral interactions important for a functional Env. To understand how individual N-glycosylation sites (NGS) coordinate to form a dynamic shield and evade the immune system through mutations, we tracked 20 NGS in Env from HIV-transmitted/founder (T/F) and immune escape variants and their mutants involving the N262 glycan. NGS were profiled in a site-specific manner using a high-resolution mass spectrometry (MS)-based workflow. Using this site-specific quantitative heterogeneity profiling, we empirically characterized the interdependent NGS of a microdomain in the high-mannose patch (HMP). The changes (shifts) in NGS heterogeneity between the T/F and immune escape variants defined a range of NGS that we further probed for exclusive combinations of sequons in the HMP microdomain using the Los Alamos National Laboratory HIV sequence database. The resultant sequon combinations, including the highly conserved NGS N262, N448, and N301, created an immune escape map of the conserved and variable sequons in the HMP microdomain. This report provides details on how some clustered NGS form microdomains that can be identified and tracked across Env variants. These microdomains have a limited number of N-glycan-sequon combinations that may allow the anticipation of immune escape variants.IMPORTANCE The Env protein of HIV is highly glycosylated, and the sites of glycosylation can change as the virus mutates during immune evasion. Due to these changes, the glycan location and heterogeneity of surrounding N-glycosylation sites can be altered, resulting in exposure of different glycan or proteoglycan surfaces while still producing a viable HIV variant. These changes present a need for vaccine developers to identify Env variants with epitopes most likely to induce durable protective responses. Here we describe a means of anticipating HIV-1 immune evasion by dividing Env into N-glycan microdomains that have a limited number of N-glycan sequon combinations.

• MeSH
• genové produkty env - virus lidské imunodeficience chemie   genetika   metabolismus   MeSH
• glykosylace MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• HIV-1 chemie   genetika   metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• imunitní únik MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• mutace * MeSH
• polysacharidy metabolismus   MeSH
• proteinové domény MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

UNLABELLED: The HIV-1 envelope protein (Env) is heavily glycosylated, with approximately 50% of the Env molecular mass being contributed by N-glycans. HIV-1 Env N-glycans shield the protein backbone and have been shown to play key roles in determining Env structure, surface exposure, and, consequently, antigenicity, infectivity, antibody neutralization, and carbohydrate and receptor binding. Studies of HIV-1 glycosylation have focused mainly on the position of glycosylation, rather than the types of glycans. Also, the role of Env glycan moieties on HIV-1 transmission has not been systematically defined. Using viruses with modified Env glycan content and heterogeneity, we examined the effects of Env glycan moieties on the major events of HIV-1 transmission. Compared to viruses with less oligomannose and more complex Env glycans, viruses with more oligomannose and less complex glycans more efficiently (i) transcytosed across an epithelial cell monolayer, (ii) attached to monocyte-derived macrophages (MDMs), (iii) bound monocyte-derived dendritic cells (MoDCs), and (iv) trans-infected primary lymphocytes via MoDCs. However, viruses with more oligomannose and less complex glycans displayed impaired infectivity in TZMbl cells, MDMs, primary lymphocytes, and fresh human intestinal tissue. Thus, N-linked Env glycans display discordant effects on the major events of HIV-1 transmission, with mature oligosaccharide structures on Env playing a crucial role in HIV-1 infection. Env glycosylation should be taken into consideration in the development of vaccine strategies to interdict HIV-1 transmission. IMPORTANCE: HIV-1 Env N-glycans shield the protein backbone and play key roles in determining Env structure and surface exposure, thereby impacting Env antigenicity, infectivity, antibody neutralization, and carbohydrate and receptor binding. Studies of HIV-1 glycosylation have focused mainly on the position of glycosylation, rather than the types of glycans. In the study described in this report, we investigated systematically the role of Env glycan moieties on HIV-1 transmission. We show that N-linked Env glycans display discordant effects on the major events of HIV-1 transmission. These data indicate that Env glycan moieties impact HIV-1 transmission and that modulation of Env glycan moieties offers a potential strategy for the development of therapeutic or prophylactic vaccines against HIV-1.

• MeSH
• dendritické buňky virologie   MeSH
• epitelové buňky virologie   MeSH
• genové produkty env - virus lidské imunodeficience chemie   metabolismus   MeSH
• HIV-1 fyziologie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• lymfocyty virologie   MeSH
• makrofágy virologie   MeSH
• polysacharidy analýza   metabolismus   MeSH
• přichycení viru * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Darunavir is the most recently approved human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) and is active against many HIV type 1 PR variants resistant to earlier-generation PIs. Darunavir shows a high genetic barrier to resistance development, and virus strains with lower sensitivity to darunavir have a higher number of PI resistance-associated mutations than viruses resistant to other PIs. In this work, we have enzymologically and structurally characterized a number of highly mutated clinically derived PRs with high levels of phenotypic resistance to darunavir. With 18 to 21 amino acid residue changes, the PR variants studied in this work are the most highly mutated HIV PR species ever studied by means of enzyme kinetics and X-ray crystallography. The recombinant proteins showed major defects in substrate binding, while the substrate turnover was less affected. Remarkably, the overall catalytic efficiency of the recombinant PRs (5% that of the wild-type enzyme) is still sufficient to support polyprotein processing and particle maturation in the corresponding viruses. The X-ray structures of drug-resistant PRs complexed with darunavir suggest that the impaired inhibitor binding could be explained by change in the PR-inhibitor hydrogen bond pattern in the P2' binding pocket due to a substantial shift of the aminophenyl moiety of the inhibitor. Recombinant virus phenotypic characterization, enzyme kinetics, and X-ray structural analysis thus help to explain darunavir resistance development in HIV-positive patients.

• MeSH
• genové produkty env - virus lidské imunodeficience metabolismus   MeSH
• genové produkty gag - virus lidské imunodeficience metabolismus   MeSH
• HIV infekce virologie   MeSH
• HIV-1 izolace a purifikace   účinky léků   MeSH
• HIV-proteasa genetika   chemie   metabolismus   MeSH
• inhibitory HIV-proteasy farmakologie   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• missense mutace MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutační analýza DNA MeSH
• polyproteiny metabolismus   MeSH
• sekvence aminokyselin MeSH
• substituce aminokyselin MeSH
• sulfonamidy farmakologie   MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• virová léková rezistence MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH