Identification and characterization of inhibitors for hantavirus replication

描述（由申请方提供）：汉坦病毒属布尼亚病毒科成员，是一种负链新兴RNA病毒和A类病原体，当通过受感染啮齿动物的排泄物雾化传播给人类时，会引起严重疾病。汉坦病毒基因组由三个负义RNA片段组成：S、M和L，分别编码核衣壳蛋白（N）、糖蛋白前体（GPC）和病毒RNA依赖性RNA聚合酶（RdRp）。GPC在中间裂解，产生N-末端片段（Gn）和C-末端片段（Gc）。糖蛋白Gn的C末端含有一个由142个氨基酸组成的细胞质尾区。汉坦病毒已经进化出一种新的翻译起始机制，由N操作，其优先支持病毒mRNA在宿主细胞中的翻译。N与核糖体蛋白S19（RPS 19）结合，RPS 19是40 S核糖体亚基的结构组分。此外，N还结合病毒mRNA 5'帽和病毒mRNA 5' UTR的高度保守的三联体重复序列。N在末端帽和5' UTR处的同时结合有利于在翻译起始期间核糖体加载到病毒转录物上。越来越多的证据表明，其他负链RNA病毒，如流感病毒，使用类似的机制来翻译它们的mRNA。我们已经开发了一种易于操作的测定来研究N与mRNA帽和病毒mRNA 5' UTR的相互作用。我们希望开发这种用于以高通量模式筛选化学文库的测定，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组装和包装中也起着关键作用。所有负链、分段RNA病毒都具有通过基因组末端的相互作用以“锅柄”构象存在的基因组片段。我们发现锅柄结构是汉坦病毒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-柄状复合物特异性地与糖蛋白Gn的胞质尾区相互作用。我们的研究结果表明，N的病毒RNA（vRNA）柄结合产生一种新的核蛋白复合物，选择性地针对vRNA的双核苷化。N-柄状复合物与Gn胞质尾区之间的特异性相互作用选择性地将核衣壳转运到布满糖蛋白的高尔基体膜上的特定目的地。因此，Gn胞质尾域和N-柄状复合物之间的特异性相互作用可能介导vRNA衍生的核衣壳选择性掺入病毒体中。我们建立了一种基于荧光的方法来研究N-柄状复合物与Gn胞质尾区之间的相互作用。我们将升级该测定以鉴定干扰N-柄状复合物和Gn尾结构域之间相互作用的分子。将来，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. !