Identification and characterization of inhibitors for hantavirus replication

汉坦病毒复制抑制剂的鉴定和表征

基本信息

批准号：
8309019
负责人：
Mohammad A Mir
金额：
$ 37.35万
依托单位：
UNIVERSITY OF KANSAS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-01 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8309019
关键词：
5&apos Untranslated Regions Affinity Amino Acids Binding Biological Assay Bunyaviridae C-terminal Catalytic RNA Categories Cells Cleaved cell Complex Cysteine Cytoplasm Cytoplasmic Tail Data Destinations Development Disease Family Fluorescence Fluorescence Resonance Energy Transfer Future Genetic Transcription Genome Genomics Glycoproteins Golgi Apparatus Hantavirus Hantavirus Infections Hepadnaviridae Higher Order Chromatin Structure Human Hydrogen Bonding Incubated Influenza Label Lung Mediating Membrane Messenger RNA Molecular Molecular Conformation Monitor N Domain N-terminal Nucleic Acids Nucleocapsid Nucleocapsid Proteins Nucleoproteins Nucleotides Oligonucleotides Oligoribonucleotides Play Proteins RNA RNA Caps RNA Probes RNA Recognition Motif RNA Viruses RNA annealing RNA chemical synthesis RNA-Binding Proteins RNA-Directed RNA Polymerase Regulatory Element Reporter Retroviridae Ribosomes Rodent Role Screening procedure Series Signal Transduction Structure Syndrome Tail Transcript Transcription Initiation Translating Translation Initiation Translations Trinucleotide Repeats Untranslated Regions Viral Viral Genome Viral N Protein Viral Packaging Viral Physiology Viral Proteins Virion Virus Virus Replication aerosolized base chemical synthesis hammerhead ribozyme high throughput screening infected vector rodent influenzavirus inhibitor/antagonist mRNA capping mRNA decapping member mortality novel pathogen pol Gene Products prevent promoter ribosomal protein S19 small molecule libraries stem viral RNA

项目摘要

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. !

描述（由申请人提供）：汉坦病毒，Bunyaviridae家族的成员是负链的新兴RNA病毒，类别是一种病原体，当通过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。汉坦病毒基因组分别由三种负性基因组RNA片段组成：分别编码核蛋白蛋白（N），糖蛋白前体（GPC）和病毒RNA依赖RNA的RNA聚合酶（RDRP）的S，M和L。 GPC在中间裂解，产生N端片段（GN）和C末端片段（GC）。糖蛋白GN在C末端具有142个氨基酸的细胞质尾域。汉坦病毒已经进化出了一种新型的翻译起始机制，该机制由N运行，该机制优先利用了宿主CELS中病毒mRNA的翻译。 n与核糖体蛋白S19（RPS19）结合，这是40S核糖体亚基的结构成分。另外，n还与病毒mRNA 5'帽和高度保守的三胞胎重复序列的病毒mRNA 5'UTR结合。在翻译起始期间，N端盖和5'UTR在病毒转录本上的核糖体负载同时结合了核糖体负载。越来越多的证据表明，其他阴性的RNA病毒（例如流感）使用类似的机制来翻译其mRNA。我们已经开发了一种可拖动的测定法，以研究N与mRNA帽和病毒mRNA 5'UTR的相互作用。我们想开发此测定法，以在高通量模式下筛选化学文库，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封装和包装中也起着关键作用。所有滞留的，分段的RNA病毒均具有通过基因组末端的相互作用在“ Panhandle”构象中发现的基因组段。我们发现，Panhandle结构是Hantavirus N的主要高亲和力结合底物。我们具有强大的初步数据，表明N-Panhandle复合物与糖蛋白GN的细胞质尾部特异性相互作用。我们的结果表明，N与病毒RNA（VRNA）PANHANDLE的结合产生了一种新型的核蛋白络合物，该核蛋白络合物有选择地靶向VRNA进行封装。 N-Panhandle复合物与GN细胞质尾部结构域之间的特定相互作用选择性地将核皮质传递到高尔基膜上的特定目的地，这些膜上用糖蛋白固定。因此，GN细胞质尾部结构域和N-Panhandle复合物之间的特定相互作用可能介导了将VRNA衍生的Nucleocapsids的选择性融合到病毒体中。我们已经开发了一种基于荧光的测定法，以研究N-Panhandle复合物与GN细胞质尾部结构域之间的相互作用。我们将升级该测定法，以鉴定干扰N-Panhandle复合物与GN尾域之间相互作用的分子。将来，这些测定法将用于筛选较大的化学文库，以鉴定抑制广泛的负链RNA病毒的复制的分子，包括汉坦病毒，流感病毒等，包括医学上重要的病毒等！