Molecular Basis of Host-Filovirus Interactions in Pathogenesis

发病机制中宿主-丝状病毒相互作用的分子基础

• 批准号: 8745559
• 负责人: Hideki Ebihara
• 金额: $ 45.43万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8745559
• 关键词: Alanine; Amino Acids; Animal Model; Animals; Bioinformatics; Biological; Biological Process; Biomedical Research; Case Fatality Rates; Cavia; Cell Culture Techniques; Cells; Clinical; Clinical Data; Coagulation Process; Communicable Diseases; Communities; Confocal Microscopy; Data; Development; Disease; Disease Outbreaks; Ebola Hemorrhagic Fever; Ebola Vaccines; Ebola virus; Electron Microscopy; Emerging Communicable Diseases; Expressed Sequence Tags; Family; Filoviridae; Filovirus; Foundations; Future; Gene Expression Profile; Genetic Transcription; Hantavirus Pulmonary Syndrome; Henipavirus Infections; Human; Immune; Immune response; Immunoprecipitation; In Vitro; Infection; Libraries; Licensing; Mesocricetus auratus; Modeling; Molecular; Monitor; Mus; Mutate; Nucleocapsid; Nucleoproteins; Organ; Pathogenesis; Play; Process; Proteins; RNA; Reading; Research; Resources; Rodent; Rodent Model; Role; Series; Signal Pathway; Syndrome; System; Techniques; Therapeutic; Transcript; Tropism; Vaccines; Viral; Viral Hemorrhagic Fevers; Viral Proteins; Virulence; Virus; Virus Diseases; Virus-like particle; Work; base; cDNA Library; effective therapy; functional genomics; functional outcomes; genome annotation; improved; in vivo; interest; member; mutant; nonhuman primate; novel; positional cloning; protein function; protein protein interaction; response; tool; transcriptome sequencing

Viral hemorrhagic fevers caused by viruses belonging to the genus Ebolavirus and Marburgvirus, both members of the Filoviridae family, are among the most severe infectious diseases in human and nonhuman primates (NHPs), and no licensed vaccines or effective therapeutics are currently available. Ebola virus (EBOV), in particular, has been responsible for multiple Ebola hemorrhagic fever (EHF) outbreaks with case-fatality rates ranging from 65 to 90%. Studies with animal models and limited clinical data from EHF outbreaks suggest that interdependent pathogenic processes, including both the host immune and pathophysiological responses, induced by EBOV infection trigger the severe hemorrhagic syndrome. In order to develop effective treatments for EHF, it is necessary to better understand the mechanisms of viral and host interactions at the molecular and cellular levels and how these interactions contribute to the in vivo pathogenic process. Our research for this year is therefore focused on elucidating the functions of viral proteins in the viral replication cycle and pathogenesis. To accomplish this, we have four ongoing projects: (1) characterization of the pathogenic processes in the Syrian hamster model that recapitulates EHF and (2) characterization of EBOV protein interactions. (1) Characterization of pathogenic processes in the Syrian hamster model, which recapitulates EHF. While the NHP model is used to evaluate the efficacy of EBOV vaccines and therapeutics because it accurately recapitulates disease, rodent models (mice and guinea pigs) are convenient and suitable for elucidating the roles of specific viral proteins in the pathogenic process and have been widely used in numerous aspects of EBOV research. However, rodent models produce only limited and inconsistent coagulation abnormalities, which are a hallmark clinical feature of EHF. Recently, we have developed and characterized a novel lethal Syrian hamster model of EHF based on infection with mouse-adapted EBOV that manifests many of the clinical and pathological findings observed in EBOV-infected NHPs and humans, including coagulation abnormalities. To determine the mechanisms of pathogenesis in this model, we have started to develop the tools for monitoring the host immune responses in infected animals. As an initial step, we performed Syrian hamster transcriptome sequencing for a cDNA library generated from pooled RNA isolated from the major organs of Syrian hamsters. The sequence reads were assembled into larger non-overlapping contigs (sequences), and expressed sequence tags (ESTs) were annotated based on other rodent species transcriptomes. We identified the most highly covered and highly expressed transcripts in our library and performed a functional enrichment analysis to identify which biological functions and canonical signaling pathways were significantly represented. This EST library improves our understanding of the Syrian hamster transcriptome, especially in the context of emerging infectious diseases, including Ebola hemorrhagic fever, hantavirus pulmonary syndrome, and henipavirus infections. Moreover, this library is a significant resource for the wider biomedical research community to help improve genome annotation of the Syrian hamster and closely related species. Finally, these data provide the basis for development of microarrays that can be utilized in functional genomics studies to understand the pathogenesis of Ebola hemorrhagic fever. (2) Characterization of EBOV protein interactions. Relatively little information exists regarding the molecular details that govern interactions between EBOV proteins. As such, we are actively interested in understanding the determinants of EBOV protein interaction and the functional outcomes of those interactions. The EBOV nucleoprotein (NP) and viral protein (VP) 24, both constituents of the viral nucleocapsid, are the sole factors responsible for EBOV virulence in mice, suggesting that these two proteins play a critical role in the induction of disease. Given their contribution to EBOV virulence, we sought to characterize the physical relationship between NP and VP24. We used confocal microscopy and immunoprecipitations to demonstrate that wild-type NP both co-localizes and interacts with VP24. A series of NP deletion mutants were then constructed to refine the determinants of this interaction. Ultimately, the N-terminus of NP was identified as the region necessary and sufficient for interaction with VP24. To determine the region on VP24 responsible for the interaction with NP, we performed bioinformatics analysis to identify the amino acids most likely to be involved in protein-protein interactions. Based on this prediction, we generated a series of VP24 mutants each with up to eight consecutive amino acids mutated to alanines throughout the protein. Assessing these mutants for their ability to interact with NP revealed a region near the C-terminus of VP24 that likely plays a critical role in the ability of VP24 to interact with NP. The work to elucidate the physical relationship between NP and VP24 has laid the foundation for understanding the functional relationship between these two proteins. Indeed, based on this and other work, we hypothesize that VP24 plays a critical role in condensing the nucleocapsid, thereby restricting viral replication and transcription and promoting nucleocapsid packaging and egress. Future work will focus on validating this hypothesis using a variety of techniques, including electron microscopy, reverse genetics, and minigenome or transcription/replication competent virus like particle systems.

由属于埃博拉病毒属和马尔堡病毒属的病毒（两者都是丝状病毒科的成员）引起的病毒性出血热是人类和非人类灵长类动物（NHP）中最严重的传染病之一，并且目前没有获得许可的疫苗或有效的治疗剂。特别是埃博拉病毒（EBOV）是造成多起埃博拉出血热（EHF）暴发的原因，病死率为65%至90%。EHF暴发的动物模型研究和有限的临床数据表明，EBOV感染诱导的相互依赖的致病过程，包括宿主免疫和病理生理反应，触发了严重出血综合征。为了开发EHF的有效治疗方法，有必要更好地了解病毒和宿主在分子和细胞水平上相互作用的机制，以及这些相互作用如何促进体内致病过程。因此，我们今年的研究重点是阐明病毒蛋白在病毒复制周期和发病机制中的功能。为了实现这一目标，我们有四个正在进行的项目：（1）表征叙利亚仓鼠模型中的致病过程，重现EHF和（2）表征EBOV蛋白质相互作用。 (1)叙利亚仓鼠模型中致病过程的表征，该模型概括了EHF。虽然NHP模型用于评估EBOV疫苗和治疗剂的功效，因为它准确地再现了疾病，但啮齿动物模型（小鼠和豚鼠）方便且适合于阐明特定病毒蛋白在致病过程中的作用，并且已广泛用于EBOV研究的许多方面。然而，啮齿动物模型仅产生有限且不一致的凝血异常，这是EHF的标志性临床特征。最近，我们已经开发并表征了一种新的致死性EHF叙利亚仓鼠模型，该模型基于感染小鼠适应性EBOV，其表现出在EBOV感染的NHP和人类中观察到的许多临床和病理学发现，包括凝血异常。为了确定该模型中的发病机制，我们已经开始开发用于监测感染动物中宿主免疫应答的工具。作为第一步，我们对从叙利亚仓鼠的主要器官分离的合并RNA产生的cDNA文库进行叙利亚仓鼠转录组测序。将序列读数组装成较大的非重叠群（序列），并基于其他啮齿动物物种转录组注释表达的序列标签（EST）。我们在我们的文库中鉴定了最高度覆盖和高度表达的转录本，并进行了功能富集分析，以鉴定哪些生物学功能和典型信号通路被显著代表。这个EST库提高了我们对叙利亚仓鼠转录组的理解，特别是在新兴传染病的背景下，包括埃博拉出血热，汉坦病毒肺综合征和亨尼帕病毒感染。此外，该文库是更广泛的生物医学研究社区的重要资源，有助于改善叙利亚仓鼠和密切相关物种的基因组注释。最后，这些数据为开发可用于功能基因组学研究的微阵列提供了基础，以了解埃博拉出血热的发病机制。 (2)EBOV蛋白相互作用的表征。关于控制EBOV蛋白之间相互作用的分子细节的信息相对较少。因此，我们对理解EBOV蛋白相互作用的决定因素以及这些相互作用的功能结果非常感兴趣。EBOV核蛋白（NP）和病毒蛋白（VP）24（两者都是病毒核衣壳的组成部分）是负责EBOV在小鼠中毒力的唯一因子，表明这两种蛋白质在诱导疾病中起关键作用。鉴于它们对EBOV毒力的贡献，我们试图表征NP和VP 24之间的物理关系。我们使用共聚焦显微镜和免疫沉淀来证明野生型NP与VP 24共定位并相互作用。然后构建了一系列NP缺失突变体来完善这种相互作用的决定因素。最终，NP的N-末端被鉴定为与VP 24相互作用所必需且足够的区域。为了确定VP 24上负责与NP相互作用的区域，我们进行了生物信息学分析以确定最有可能参与蛋白质-蛋白质相互作用的氨基酸。基于这一预测，我们产生了一系列VP 24突变体，每个突变体在整个蛋白质中具有多达8个连续的氨基酸突变为丙氨酸。评估这些突变体与NP相互作用的能力发现，VP 24 C末端附近的区域可能在VP 24与NP相互作用的能力中发挥关键作用。NP和VP 24之间的物理关系的阐明工作为理解这两种蛋白质之间的功能关系奠定了基础。事实上，基于这项和其他工作，我们假设VP 24在浓缩核衣壳中起关键作用，从而限制病毒复制和转录，并促进核衣壳包装和排出。未来的工作将集中在验证这一假设，使用各种技术，包括电子显微镜，反向遗传学，和微型基因组或转录/复制能力的病毒样颗粒系统。