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

 描述(申请人提供)：ARENAV是严重出血热的罪魁祸首，在世界范围内发病率和死亡率都很高。在缺乏疫苗或特定疗法的情况下，这些病毒被公认为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ArenaVirus包膜糖蛋白复合体(GPC)和病毒进入宿主细胞的干预策略有望对抗这些致命的感染。与其他I类病毒融合蛋白不同，GPC含有三个亚基：GP1、GP2和一个独特的稳定信号肽(SSP)。我们已经证明了SSP与GP2融合亚基一起作用于成熟的内小体中的酸性pH，从而触发导致病毒-细胞膜融合的结构转变。我们鉴定了六类化学上不同的小分子阿拉伯病毒融合抑制剂，它们对新世界(NW)和旧世界(OW)阿拉伯病毒的选择性不同，但在GPC上有一个共同的结合位点。我们的遗传学研究表明，这些化合物结合在SSP和GP2的pH敏感界面上。在初步研究中，我们发现拉沙霉素-1的光亲和衍生物可以特异性地标记从昆虫细胞膜中纯化的重组拉萨病毒(LASV)GPC中的SSP和GP2亚基，与SSP-GP2界面上的抑制剂结合一致。因此，我们在剖析ArenaVirus融合激活及其抑制的分子基础方面具有独特的地位。我们将通过生物化学、药理学、遗传学和最新的质谱学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，以阐明抑制剂结合部位。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GP2亚基和多肽将使用稳定同位素标记的可切割生物素连接物和点击化学来使共价结合的抑制物官能化，以用于质谱学。在其他位置具有光敏基团的新的抑制剂将被用来识别结合口袋中的其他残基。(2)鉴定和鉴定JUNV GPC中的同源抑制物结合位点。拉沙霉素衍生物也显示出对NW Junín(JUNV)病毒的抑制活性，我们将使用质谱学来鉴定JUNV GPC中的同源抑制物结合部位。(3)建立SSP和GP2界面的结构模型，以确定共同的和物种特有的融合激活及其抑制的决定因素。作为关于的结构信息 完整的GPC不可用，我们将利用我们的结果来构建SSP-GP2界面的空间模型。扫描突变和功能分析将被用来研究保守的和发散的氨基酸侧链在控制GPC融合活性和抑制物选择性中的作用。这些研究将加深我们对GPC中pH传感和融合激活的分子基础的理解，以及类药物分子如何干预这一过程。

 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.