Characterization of the pH-sensing interface of the arenavirus envelope glycoprotein GPC

沙粒病毒包膜糖蛋白 GPC pH 传感界面的表征

• 批准号: 9089864
• 负责人: ALEX S. EVERS
• 金额: $ 18.48万
• 依托单位: UNIVERSITY OF MONTANA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-15 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9089864
• 关键词: Address; Alkynes; Amino Acids; Animal Model; Arenavirus; Arenavirus Infections; Azides; Binding; Binding Sites; Biochemical; Biological Assay; Biology; Biotin; Categories; Cell membrane; Cells; Chemicals; Chemistry; Collaborations; Complex; Copper; Development; Endosomes; GP2 gene; GTPBP1 gene; Genetic; Genetic study; Glycoproteins; Health; Human; Infection; Insecta; Intervention; Junin virus; Knowledge; Label; Lassa virus; Life; Mass Spectrum Analysis; Membrane Fusion; Modeling; Molecular; Morbidity - disease rate; Mutagenesis; Old World Arenaviruses; Pattern; Peptide Signal Sequences; Peptides; Pharmaceutical Preparations; Photoaffinity Labels; Population; Positioning Attribute; Process; Public Health; Recombinants; Research Institute; Research Personnel; Resolution; Rodent; Role; Scanning; Site; Streptavidin; Structural Models; Structure-Activity Relationship; Surface; Tacaribe Complex Viruses; Therapeutic; Vaccines; Viral; Viral Fusion Proteins; Viral Hemorrhagic Fevers; Virus; base; biodefense; combat; cycloaddition; design; inhibitor/antagonist; insight; interfacial; mortality; novel; pathogen; prevent; small molecule; small molecule inhibitor; stable isotope; tandem mass spectrometry

 DESCRIPTION (provided by applicant): Arenaviruses are responsible for severe hemorrhagic fevers with high morbidity and mortality worldwide. In the absence of vaccines or specific therapies, these viruses are recognized as Category A priority pathogens that pose significant threats to public health and biodefense. Intervention strategies that target the arenavirus envelope glycoprotein complex (GPC) and virus entry into the host cell hold promise for combating these lethal infections. Unlike other Class I viral fusion proteins, GPC contains three subunits: GP1, GP2 and a unique stable signal peptide (SSP). We have demonstrated that SSP acts in conjunction with the GP2 fusion subunit to sense acidic pH in the maturing endosome and thereby trigger the structural transitions leading to virus-cell membrane fusion. We have characterized six chemically distinct classes of small-molecule arenavirus fusion inhibitors that differ in their selectivities against New World (NW) and Old World (OW) arenaviruses, but share a common binding site on GPC. Our genetic studies suggest that these compounds bind at the pH-sensing interface of SSP and GP2. In preliminary studies, we show that photoaffinity derivatives of one such inhibitor, lassamycin-1, can specifically label SSP and GP2 subunits in recombinant Lassa virus (LASV) GPC purified from insect-cell membranes, consistent with inhibitor binding at the SSP-GP2 interface. Thus, we are uniquely positioned to dissect the molecular basis of arenavirus fusion activation and its inhibition. We will accomplish these objectives by using biochemical, pharmacological, genetic and state-of-the-art mass spectrometry approaches to pursue the following aims: (1) Identify photolabeled amino-acid residues in LASV GPC to elucidate the inhibitor-binding site. We will utilize combined chemical biology and high-resolution tandem mass spectrometric sequencing to identify amino-acid residues in GPC modified by lassamycin derivatives. Labeled SSP and GP2 subunits and peptides will be enriched for mass spectrometry using a stable-isotope- tagged, cleavable biotin linker and click chemistry to functionalize the covalently bound inhibitor. New inhibitors with photolabile groups at other sites will be used to identify residues elsewhere in the binding pocket. (2) Identify and characterize the homologous inhibitor-binding site in JUNV GPC. Lassamycin derivatives also show inhibitory activity against the NW Junín (JUNV) virus, and we will use mass spectrometry to identify the homologous inhibitor-binding site in JUNV GPC. (3) Develop a structural model of the interfacial surfaces of SSP and GP2 to identify common and species-specific determinants of fusion activation and its inhibition. As structural information on intact GPC is not available, we will utilize our results to construct a spatial model of the SSP-GP2 interface. Scanning mutagenesis and functional assays will be employed to investigate the role of conserved and divergent amino-acid sidechains in controlling GPC fusion activity and inhibitor selectivity. These studies will enhance our understanding of the molecular basis for pH sensing and fusion activation in GPC, and how drug-like molecules can interfere in this process.

 描述（由申请方提供）：沙粒病毒是全球范围内发病率和死亡率高的严重出血热的原因。在缺乏疫苗或特定疗法的情况下，这些病毒被认为是A类优先病原体，对公共卫生和生物防御构成重大威胁。针对沙粒病毒包膜糖蛋白复合物（GPC）和病毒进入宿主细胞的干预策略有望对抗这些致命感染。与其他I类病毒融合蛋白不同，GPC包含三个亚基：GP 1，GP 2和一个独特的稳定信号肽（SSP）。我们已经证明，SSP与GP 2融合亚基一起作用，以感知成熟内体中的酸性pH，从而触发导致病毒-细胞膜融合的结构转变。我们已经表征了六种化学上不同的小分子沙粒病毒融合抑制剂，它们对新世界（NW）和旧世界（OW）沙粒病毒的选择性不同，但在GPC上有一个共同的结合位点。我们的遗传研究表明，这些化合物结合在SSP和GP 2的pH传感界面。在初步研究中，我们表明，光亲和衍生物的一个这样的抑制剂，拉沙霉素-1，可以特异性地标记SSP和GP 2亚基的重组拉沙病毒（LASV）GPC从昆虫细胞膜纯化，与抑制剂结合在SSP-GP 2接口。因此，我们处于独特的地位，剖析沙粒病毒融合激活及其抑制的分子基础。我们将利用生物化学、药理学、遗传学和最先进的质谱方法来实现这些目标，以实现以下目标：（1）鉴定LASV GPC中的光标记氨基酸残基，以阐明底物结合位点。我们将结合化学生物学和高分辨率串联质谱测序来鉴定拉沙霉素衍生物修饰的GPC中的氨基酸残基。标记的SSP和GP 2亚基和肽将使用稳定同位素标记的可裂解生物素接头和点击化学富集用于质谱分析，以使共价结合的抑制剂官能化。新的抑制剂与光不稳定基团在其他网站将被用来确定其他地方的结合口袋中的残基。(2)识别和表征JUNV GPC中的同源抑制剂结合位点。拉沙霉素衍生物也显示出对NW Junín（JUNV）病毒的抑制活性，我们将使用质谱法鉴定JUNV GPC中的同源底物结合位点。(3)开发SSP和GP 2界面的结构模型，以确定融合激活及其抑制的共同和物种特异性决定因素。作为结构信息， 完整的GPC是不可用的，我们将利用我们的结果来构建一个空间模型的SSP-GP 2接口。将采用扫描诱变和功能测定来研究保守和趋异氨基酸侧链在控制GPC融合活性和抑制剂选择性中的作用。这些研究将增强我们对GPC中pH传感和融合激活的分子基础以及药物样分子如何干扰这一过程的理解。