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（由适用提供）：竞技病毒负责全球发病率高和死亡率的严重出血性发烧。在缺乏疫苗或特定疗法的情况下，这些病毒被认为是对公共卫生和生物形式构成重大威胁的优先病原体。针对体育症病毒蛋白病毒糖蛋白复合物（GPC）和病毒进入宿主细胞的干预策略，可以打击这些致命感染。与其他I类病毒融合蛋白不同，GPC包含三个亚基：GP1，GP2和独特的稳定信号肽（SSP）。我们已经证明，SSP与GP2融合亚基一起作用，以在成熟的内体中感知酸性pH，从而触发导致病毒细胞膜融合的结构过渡。我们已经表征了六种化学上不同的小分子体育病毒融合抑制剂，它们的选择性与新世界（NW）和旧世界（OW）体育疾病病毒的选择性不同，但在GPC上共享一个共同的结合位点。我们的遗传研究表明，这些化合物在SSP和GP2的pH值界面上结合。在初步研究中，我们表明一种这种抑制剂lassamycin-1的光性衍生物可以在重组LASSA病毒（LASV）GPC中特异性标记SSP和GP2亚基，该GPC从SSP-GP2界面的抑制剂结合中纯化。因此，我们是独特的定位，可以剖析轨道病毒融合激活的分子基础及其抑制作用。我们将通过使用生化，药物，遗传和最先进的质谱方法来实现这些目标，以追求以下目的：（1）确定LASV GPC中的光贴氨基酸性保留的保留，以阐明抑制剂结合点。我们将使用合并的化学生物学和高分辨率串联质谱测序来识别通过lassamycin衍生物修饰的GPC中的氨基酸残留物。标记的SSP和GP2亚基和Pepperides将使用稳定的异位素标记的可裂解的生物素接头富含质谱法，并单击化学以使共价绑定的抑制剂功能功能化。在其他位点具有光片基组的新抑制剂将用于识别结合口袋中其他地方的残留物。 （2）在JUNV GPC中识别并表征同源抑制剂结合位点。拉菌素衍生物还显示出对NWJunín（JUNV）病毒的抑制活性，我们将使用质谱法来鉴定JUNV GPC中的同源抑制剂结合位点。 （3）开发了SSP和GP2的界面表面的结构模型，以识别融合激活及其抑制作用的常见和规格的确定剂。作为结构信息 完整的GPC不可用，我们将利用结果来构建SSP-GP2接口的空间模型。将采用扫描诱变和功能分析来研究保守和不同的氨基酸侧卫生在控制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敏感性和融合活化的分子基础的理解，以及在此过程中如何干扰药物样分子。