HIV-1 Env protein structure and function assessed by parallel smFRET and cryoET

通过平行 smFRET 和 CryoET 评估 HIV-1 Env 蛋白结构和功能

• 批准号: 10761955
• 负责人: WALTHER H MOTHES
• 金额: $ 82.95万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-18 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10761955
• 关键词: Adopted; Affect; Antibodies; Antigens; Antiviral Therapy; Automobile Driving; Binding; Binding Proteins; Bulla; CCR5 gene; CD4 Antigens; Cell membrane; Cells; Cellular Membrane; Chimeric Proteins; Cholesterol; Chronic; Classification; Complex; Cryo-electron tomography; Data; Development; Environment; Fluorescence Resonance Energy Transfer; HIV; HIV Envelope Protein gp120; HIV-1; Image; Incubated; Laboratories; Lipids; Mediating; Membrane; Membrane Fusion; Methodology; Methods; Molecular Conformation; Monitor; Positioning Attribute; Process; Protein Conformation; Protein Dynamics; Proteins; Protomer; Receptor Activation; Resolution; Sampling; Speed; Structure; Surface; System; T-Lymphocyte; Testing; Vaccine Design; Vaccines; Viral; Viral Proteins; Virion; Virus; Virus-like particle; Visualization; Work; data exchange; env Gene Products; fluorophore; insight; instrumentation; particle; protein function; protein structure; receptor; single molecule; small molecule inhibitor; software development; therapy design; vaccine development

Summary The HIV-1 envelope protein (Env) mediates virus entry into cells while protecting functional centers from antibodies. As the only virus protein on the surface of virus particles, HIV-1 Env is a major target for vaccine development. We initiated studies on HIV-1 Env by establishing single-molecule Förster Resonance Energy Transfer (smFRET) to monitor the sampling of a single protomer in the context of a Env trimer on the surface of a virus particle. smFRET data indicated that HIV-1 Env is conformationally dynamic and samples at least three conformational states. Associating the observed FRET states with existing HIV-1 Env structural information revealed a conformational state on the surface of viruses that cannot be explained by current high-resolution structures. The controversy surrounding smFRET data prompted us to establish cryo-electron tomography (cryoET) as an orthogonal structural method. The advantage of cryoET and smFRET is that both methods allow for characterization of the structure and dynamics of Env on the surface of viruses. In addition, we identified experimental conditions that arrest a high number of Env trimers at various stages of the entry process to allow a structural characterization by cryoET and subsequent subtomogram averaging. We succeeded with a system where we incubate native HIV-1 viruses with virus-like particles (VLP) carrying receptor CD4 and coreceptor molecules. We observed Env-CD4 opposing CD4 membranes asymmetric conformational CD4 of availability on the surface of viruses that cannot be explained by current high-resolution structures. refined states. A detailed understanding of the structure and dynamics of the HIV-1 Env protein is important for the complexes to cluster at membrane-membrane interfaces thereby bringing membranes closer together. Subtomogram averaging and classification revealed that Env bound one molecule when membranes were further apart, then engaged two, and finally three CD4 molecules as moved closer together. HIV-1 Env trimers bound to one and two CD4 molecules adopted conformational states. These cryoET studies recall earlier smFRET work in our laboratory that a intermediate in the opening of the Env trimer corresponds to an asymmetric trimer with a single bound. I n this proposal, we will apply our cryoET methodology to next structurally characterize the transition Env from CD4 to coreceptor followed by activation into the pre-hairpin intermediate. Reenergized by the of cryoET and advances on smFRET, we will pursue our efforts to characterize a Finally, we are proposing smFRET methods to meet the increasingly complex structural insights into distinct Env conformational conformational state development of immunogens for vaccines and small molecule inhibitors against Env.

概括 The HIV-1 envelope protein (Env) mediates virus entry into cells while protecting functional centers from 抗体。 As the only virus protein on the surface of virus particles, HIV-1 Env is a major target for vaccine 发展。 We initiated studies on HIV-1 Env by establishing single-molecule Förster Resonance Energy Transfer (smFRET) to monitor the sampling of a single protomer in the context of a Env trimer on the surface of 病毒颗粒。 smFRET data indicated that HIV-1 Env is conformationally dynamic and samples at least three 构象状态。 Associating the observed FRET states with existing HIV-1 Env structural information revealed a conformational state on the surface of viruses that cannot be explained by current high-resolution 结构。 The controversy surrounding smFRET data prompted us to establish cryo-electron tomography （cryoET）作为一种正交结构方法。 The advantage of cryoET and smFRET is that both methods allow for characterization of the structure and dynamics of Env on the surface of viruses.此外，我们还确定了 experimental conditions that arrest a high number of Env trimers at various stages of the entry process to allow a structural characterization by cryoET and subsequent subtomogram averaging.我们通过系统取得了成功 where we incubate native HIV-1 viruses with virus-like particles (VLP) carrying receptor CD4 and coreceptor 分子。 We observed Env-CD4 反对 CD4 膜 不对称的 conformational CD4 的 可用性 on the surface of viruses that cannot be explained by current high-resolution structures. 精制 州。 A detailed understanding of the structure and dynamics of the HIV-1 Env protein is important for the complexes to cluster at membrane-membrane interfaces thereby bringing 膜靠得更近。 Subtomogram averaging and classification revealed that Env bound one molecule when membranes were further apart, then engaged two, and finally three CD4 molecules as 靠得更近了。 HIV-1 Env trimers bound to one and two CD4 molecules adopted 构象状态。 These cryoET studies recall earlier smFRET work in our laboratory that a intermediate in the opening of the Env trimer corresponds to an asymmetric trimer with a single 边界。 I n this proposal, we will apply our cryoET methodology to next structurally characterize the transition Env from CD4 to coreceptor followed by activation into the pre-hairpin intermediate. Reenergized by the of cryoET and advances on smFRET, we will pursue our efforts to characterize a 最后，我们建议 smFRET methods to meet the increasingly complex structural insights into distinct Env conformational conformational state development of immunogens for vaccines and small molecule inhibitors against Env.