Identification and characterization of inhibitors for hantavirus replication

描述（由申请人提供）：汉坦病毒，布尼亚病毒科的成员，是负链新兴RNA病毒和A类病原体，当通过受感染啮齿动物的雾化排泄物传播给人类时，会引起严重疾病。汉坦病毒基因组由3个负义基因组RNA片段S、M和L组成，分别编码核衣壳蛋白(N)、糖蛋白前体（GPC）和病毒RNA依赖RNA聚合酶（RdRp）。GPC在中间断裂，产生一个n端片段（Gn）和一个c端片段（Gc）。糖蛋白Gn在c端有一个由142个氨基酸组成的细胞质尾部结构域。汉坦病毒已经进化出一种新的翻译起始机制，该机制由N操作，优先支持宿主细胞中病毒mrna的翻译。N与核糖体蛋白S19 （RPS19）结合，RPS19是40S核糖体亚基的结构成分。此外，N还结合病毒mRNA 5‘帽和病毒mRNA 5’ UTR高度保守的三联体重复序列。在翻译起始过程中，N在末端帽和5' UTR上的同时结合有利于核糖体装载在病毒转录本上。越来越多的证据表明，其他负链RNA病毒，如流感病毒，也使用类似的机制来翻译它们的mrna。我们开发了一种易于操作的实验来研究N与mRNA cap和病毒mRNA 5' UTR的相互作用。我们希望开发这种高通量模式筛选化学文库的方法，以鉴定抑制N-cap和N-UTR相互作用的分子。N蛋白在病毒基因组的衣壳和包装中也起着关键作用。所有负链、分段RNA病毒都有基因组片段，通过基因组末端的相互作用形成“长柄”构象。我们发现长柄结构是汉坦病毒n的主要高亲和力结合底物。我们有强有力的初步数据表明n -长柄复合物特异性地与糖蛋白Gn的细胞质尾部结构域相互作用。我们的研究结果表明，N与病毒RNA （vRNA）长柄的结合产生了一种新的核蛋白复合物，该复合物选择性地靶向vRNA进行衣壳化。n -长柄复合物和Gn细胞质尾结构域之间的特异性相互作用选择性地将核衣壳转运到点缀有糖蛋白的高尔基膜上的特定目的地。因此，Gn细胞质尾结构域和n -长柄复合物之间的特异性相互作用可能介导了vRNA衍生的核衣壳选择性结合到病毒粒子中。我们建立了一种基于荧光的方法来研究n -长柄复合物与Gn细胞质尾结构域之间的相互作用。我们将对该方法进行升级，以鉴定干扰n -长柄复合物与Gn尾结构域相互作用的分子。未来，这些检测将用于筛选更大的化学文库，以鉴定抑制广谱负链RNA病毒复制的分子，包括医学上重要的病毒，如汉坦病毒、流感病毒等！

DESCRIPTION (provided by applicant): Hantaviruses, members of the Bunyaviridae family are negative stranded emerging RNA viruses and category A pathogens that cause serious illness when transmitted to humans through aerosolized excreta of infected rodents. The hantaviral genome is composed of three negative sense genomic RNA segments: S, M and L that encode nucleocapsid protein (N), glycoprotein precursor (GPC) and viral RNA dependent RNA polymerase (RdRp), respectively. The GPC is cleaved in the middle, generating an N-terminal fragment (Gn) and a C-terminal fragment (Gc). The glycoprotein Gn harbors a cytoplasmic tail domain of 142 amino acids at the C-terminus. Hantaviruses have evolved a novel translation initiation mechanism, operated by N, which preferentially favors the translation of viral mRNAs in the host cels. N binds to the ribosomal protein S19 (RPS19), a structural component of 40S ribosomal subunit. In addition, N also binds to both the viral mRNA 5' cap and a highly conserved triplet repeat sequence of viral mRNA 5' UTR. The simultaneous binding of N at both the terminal cap and 5' UTR favors ribosome loading on viral transcripts during translation initiation. There is growing evidence that other negative stranded RNA viruses such as influenza use similar mechanisms for the translation of their mRNAs. We have developed a tractable assay to study the interaction of N with mRNA cap and viral mRNA 5' UTR. We would like to develop this assay for screening chemical libraries in high throughput mode for the identification of molecules that inhibit N-cap and N-UTR interaction. N protein also plays a key role in encapsidation and packaging of viral genome. All minus stranded, segmented RNA viruses have genome segments that are found in "panhandle" conformation via the interaction of the genome termini. We have found that panhandle structure is the primary high affinity binding substrate for hantavirus N. We have strong preliminary data showing that N-panhandle complex specifically interacts with the cytoplasmic tail domain of glycoprotein Gn. Our results demonstrate that binding of N to the viral RNA (vRNA) panhandle generates a novel nucleoprotein complex that selectively targets vRNA for encapsidation. The specific interaction between N-panhandle complex and Gn cytoplasmic tail domain selectively transports the nucleocapsids to specific destinations on Golgi membranes that are studded with the glycoproteins. Thus, the specific interaction between Gn cytoplasmic tail domain and N-panhandle complex likely mediates the selective incorporation of vRNA derived nucleocapsids into virions. We have developed a fluorescence based assay to study the interaction between N-panhandle complex and Gn cytoplasmic tail domain. We will upgrade this assay for the identification of molecules that interfere in the interaction between N-panhandle complex and Gn tail domain. In future, these assays will be used for screen larger chemical libraries for the identification of molecules that inhibit the replication of a broad spectrum of negative stranded RNA viruses, including medically important viruses such as hantaviruses, influenza virus etc. !