Saccharomyces cerevisiae strain:AWY101
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ABSTRACT:
PROVIDER: PRJNA678709 | ENA |
REPOSITORIES: ENA
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Project description:HIV-1 infection begins with binding of the viral envelope glycoprotein Env to the host receptor CD4, triggering a series of conformational changes that lead to fusion of the virus and cell membranes. Env, a trimer of gp120 and gp41 subunits, occupies a ‘closed’ conformation with contacts between gp120 subunits at the apex, and transitions through an ‘open’ conformation with the gp120 subunits spread apart following CD4 binding. Using deep mutational scanning, sequence-fitness landscapes were mapped for full-length Env from the clade B BaL strain interacting with CD4, and broadly neutralizing antibodies VRC01 and PG16, which preferentially bind closed Env. Contacting residues are conserved for CD4 binding, and glycosylation at N262 is critical for accessing the high-affinity CD4-bound state. By comparison, VRC01 binding is resistant to most single amino acid substitutions, an ideal quality in a broadly neutralizing antibody. Also in contrast to CD4 interaction, Env interfacial residues are under tight selection for PG16 binding to maintain a closed conformation. Screening for mutations that enhanced PG16 binding, we identified several important sites, in particular neutralization of the electropositive apical cavity that we hypothesize promotes trimer opening by electrostatic repulsion. Mutations were combined to generate Quaternary Epitope Stabilized (QES) mutants with enhanced presentation of the PG16 epitope, and the mutations were partially transferable to other HIV-1 strains. These mutational analyses offer insight into Env conformational stabilization that may assist immunogen design.
2019-02-03 | GSE102276 | GEO
Project description:Buffering of deleterious mutations by molecular chaperones and degradation of aberrant proteins by quality control systems are both major factors that can impact the mutational landscape available to a client protein. The impacts of the proteostasis network on protein evolution are not limited to just endogenous clients, but can also shape the mutational landscapes accessible to rapidly evolving viral proteins. Here, we test the hypothesis that the composition of the host cell’s endoplasmic reticulum (ER) proteostasis network shapes the evolution of RNA viruses by focusing on human immunodeficiency virus-1 envelope (Env), a membrane glycoprotein that folds and matures in the host cell’s secretory pathway. We apply chemical genetic methods to activate the IRE1-XBP1s and/or the ATF6 transcriptional arms of the unfolded protein response in a stress-independent manner. We then quantitatively assess the impact of the resulting altered host cell ER proteostasis environments on the relative enrichment of all Env single amino acid substitutions using deep mutational scanning. We find that upregulation of host ER proteostasis factors globally reduces the mutational tolerance of HIV-1 Env, particularly upon induction of the IRE1-XBP1s transcriptional arm of the UPR. The effects of ATF6 activation are less global, but still significant at particular Env sites. The impact of the XBP1s-induced ER proteostasis environment is disparate for diverse structural elements of Env. Conserved, functionally important regions generally exhibit the largest decreases in mutational tolerance upon XBP1s activation. In contrast, specific regions of Env, including regions targeted by broadly neutralizing antibodies, display greatly enhanced mutational tolerance when XBP1s is activated. Altogether, these data reveal a new set of host factors that specifically shape the mutational space accessible to HIV Env and, more generally, provide compelling evidence that UPR-regulated proteostasis mechanisms play critical roles in membrane protein evolution.
2022-01-30 | GSE171356 | GEO
Project description:Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus
| PRJNA202933 | ENA
Project description:Phase-I trial of broadly neutralizing anti-HIV-1 antibody 10-1074
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Project description:Tissue-like memory, activated memory and resting memory B cells were sorted by FACS from the individual living with HIV (EC17) who was aviremic and transcriptomes generated using the SmartSeq2 protocol. This was to provide a reference set for each memory B cell subset in the context of HIV. Next, HIV-specific memory B cells from the individual with broadly neutralizing plasma were then also sorted by FACS and single cell transcriptomes generated using the SmartSeq2 protocol. The phenotypes of memory B cells from the individual with broadly neutralizing plasma (T125) were then inferred from the reference set using Glmnet and Celltypist packages.
2023-12-24 | E-MTAB-13592 | biostudies-arrayexpress
Project description:Many human monoclonal antibodies that neutralize multiple clades of HIV-1 are polyreactive and bind avidly to mammalian autoantigens. Indeed, the generation of neutralizing antibodies to the 2F5 and 4E10 epitopes of HIV-1 gp41 in man may be proscribed by immune tolerance since mice expressing the VH and VL regions of 2F5 have a block in B-cell development characteristic of central tolerance. This developmental blockade implies the presence of tolerizing autoantigens that are mimicked by the membrane-proximal external region of HIV-1 gp41. Here we identify human kynureninase (KYNU) and splicing factor 3b subunit 3 (SF3B3) as the primary conserved, vertebrate self-antigens recognized by the 2F5 and 4E10 antibodies, respectively. 2F5 binds the H4 domain of KYNU which contains the complete 2F5 linear epitope (ELDKWA). 4E10 recognizes a conformational epitope of SF3B3 that is strongly dependent on hydrophobic interactions. Opossums carry a rare KYNU H4 domain that abolishes 2F5 binding, but retain all SF3B3 4E10 epitopes. Immunization of opossums with HIV-1 gp140 induced extraordinary titers of serum antibody to the 2F5 ELDKWA epitope but little or nothing to the 4E10 determinant. Identification of structural motif shared by vertebrates and HIV-1 provides direct evidence that immunological tolerance can impair humoral responses to HIV-1. The invitrogen protoarray that contains >9,400 recombinant human proteins was used to identify self-ligands that are recognized by broadly neutralizing HIV-1 antibodies 2F5 and 4E10. An isotype-matched human myeloma protein (151K, Southern Biotech) was used as control.
2012-12-03 | E-GEOD-42673 | biostudies-arrayexpress
Project description:A protective HIV-1 vaccine has been hampered by a limited understanding of how B cells acquire neutralizing activity. Our previous vaccines expressing two different HIV-1 envelopes elicited robust antigen specific serum IgG titers in 20 rhesus macaques; yet serum from only two animals neutralized the autologous virus. Here, we used high throughput immunoglobulin receptor and single cell RNA sequencing to characterize the overall expansion, recall, and maturation of antigen specific B cells longitudinally over 90 weeks. Diversification and expansion of many B cell clonotypes occurred broadly in the absence of serum neutralization. However, in one animal that developed neutralization, two neutralizing B cell clonotypes arose from the same immunoglobulin germline and were tracked longitudinally. Early antibody variants with high identity to germline neutralized the autologous virus while later variants acquired somatic hypermutation and increased neutralization potency. The early engagement of precursors capable of neutralization with little to no SHM followed by prolonged affinity maturation allowed the two neutralizing lineages to successfully persist despite many other antigen specific B cells. The findings provide new insight into B cells responding to HIV-1 envelope during heterologous prime and boost immunization in rhesus macaques and the development of selected autologous neutralizing antibody lineages
2023-10-11 | GSE244293 | GEO
Project description:Fc-mediated effector function contributes to the in vivo antiviral effect of an HIV broadly neutralizing antibody.
| PRJNA564087 | ENA