Identification and characterization of inhibitors for hantavirus replication

描述(申请人提供)：汉坦病毒，布尼亚病毒科的成员是负性滞留的新出现的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导致严重疾病。汉坦病毒基因组由3个负义基因组片段组成：S、M和L，分别编码核衣壳蛋白(N)、糖蛋白前体(GPC)和病毒依赖RNA聚合酶(RdRp)。GPC在中间被切割，产生N-末端片段(Gn)和C-末端片段(GC)。糖蛋白Gn在C末端含有142个氨基酸的胞质尾区。汉坦病毒已经进化出一种由N控制的新的翻译启动机制，它优先有利于病毒mRNAs在宿主细胞中的翻译。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还与病毒mRNA5‘端帽子和高度保守的病毒mRNA5’端非编码区三联体重复序列结合。在翻译起始过程中，N在末端帽和5‘UTR的同时结合有利于核糖体在病毒转录物上的装载。越来越多的证据表明，其他负链RNA病毒，如流感，使用类似的机制来翻译它们的mRNAs。我们建立了一种简便的方法来研究N与mRNA帽和病毒mRNA5‘端非编码区的相互作用。我们希望建立这一方法，以高通量模式筛选化学文库，以识别抑制N-帽和N-UTR相互作用的分子。N蛋白在病毒基因组的封装和包装中也起着关键作用。所有负链、节段化的RNA病毒都有基因组片段，这些片段通过基因组末端的相互作用以“狭长柄”构象存在。我们发现PANHANDLE结构是汉坦病毒N的主要高亲和力结合底物。有强有力的初步数据表明，N-PANHANDLE复合体与糖蛋白Gn的细胞质尾部区域特异性地相互作用。我们的结果表明，N与病毒RNA(VRNA)狭长柄的结合产生了一种新型的核蛋白复合体，该复合体选择性地靶向vRNA进行包裹。N-PANHANDLE复合体与Gn胞质尾区的特异性相互作用选择性地将核衣壳运送到布满糖蛋白的高尔基体膜上的特定目的地。因此，Gn胞质尾区与N-PANHANDLE复合体之间的特异性相互作用可能介导了vRNA来源的核衣壳选择性地掺入病毒粒子。我们建立了一种基于荧光的分析方法来研究N-PANHANDLE复合体与Gn胞质尾部结构域的相互作用。我们将升级这一检测方法，以鉴定干扰N-PanHandle复合体和Gn尾部结构域相互作用的分子。将来，这些化验方法将用于筛选更大的化学库，以识别抑制广泛负链RNA病毒复制的分子，包括具有重要医学价值的病毒，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. !