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: 8946508
• 负责人: Hideki Ebihara
• 金额: $ 48.51万
• 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/8946508
• 关键词: Affect; Alanine; Amino Acids; Angola; Animal Model; Appearance; Bioinformatics; Biological; Case Fatality Rates; Cavia; Cell Culture Techniques; Cells; Clinical; Clinical Data; Coagulation Process; Communicable Diseases; Confocal Microscopy; Disease; Disease Outbreaks; Ebola Hemorrhagic Fever; Ebola virus; Electron Microscopy; Exanthema; Family; Filoviridae; Filovirus; Foundations; Frankfurt-Marburg Syndrome Virus; Future; Genetic Transcription; Hamsters; Human; Immune; Immunoprecipitation; In Vitro; Infection; Interferons; Length; Licensing; Link; Mesocricetus auratus; Modeling; Molecular; Mus; Mutate; Mutation; Nucleocapsid; Nucleoproteins; Pathogenesis; Pathway interactions; Play; Point Mutation; Process; Proteins; RNA; Research; Rodent; Rodent Model; Role; Serial Passage; Series; Signal Transduction; Syndrome; System; Techniques; Therapeutic; Tropism; Vaccines; Variant; Viral; Viral Hemorrhagic Fevers; Viral Proteins; Virulence; Virulent; Virus; Virus Diseases; Virus-like particle; Work; base; effective therapy; functional outcomes; genome sequencing; in vivo; interest; member; mutant; nonhuman primate; novel; positional cloning; protein function; protein protein interaction; response; structural biology; therapeutic vaccine; vaccine efficacy

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 two ongoing projects: (1) characterization of the pathogenic processes in the Syrian hamster model that recapitulates Marburg hemorrhagic fever (MHF) and (2) characterization of EBOV protein interactions. (1) Characterization of pathogenic processes in the Syrian hamster model, which recapitulates MHF. While the NHP model is used to evaluate the efficacy of vaccines and therapeutics against filoviruses 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 filovirus research. However, rodent models produce only limited and inconsistent coagulation abnormalities, which are a hallmark clinical feature of filoviral HFs. 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. Similarly, we sought to apply our work with EBOV to establishing a rodent model for Marburg virus (MARV) strain Angola, thought to be the most virulent strain of MARV. MARV Angola was lethally adapted to both hamsters and guinea pigs through serial passage. We demonstrated that infection with hamster-adapted MARV produces severe disease in hamsters, including coagulation abnormalities evidenced by the appearance of a petechial rash that mimics that observed on humans and NHPs. Furthermore, to identify the molecular determinants of MARV virulence in rodent models, we determined the full-length genome sequences of several lethal rodent-adapted variants and identified three amino acid mutations in VP35, VP40, and VP24. Although the role that these mutations play in the acquisition of virulence during the course of rodent adaptation is unclear, based on MARV VP40s proven ability to antagonize IFN signaling and the fact that mouse-adaptation of EBOV may be linked to the ability of the virus to subvert the IFN pathway, we speculate that these three point mutations may affect the ability of MARV VP40 to inhibit IFN signaling in a species-specific manner. (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. To determine the region on VP24 responsible for the interaction with NP, we performed bioinformatics analysis to identify the regions 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 plays a critical role in the ability of VP24 to interact with NP. Further mutational analysis allowed us to resolve the interaction domain to the amino acid level. Unlike wild-type VP24, the VP24 mutants that were unable to interact with NP were also unable to support the formation of transcription/replication-competent virus-like particles (trVLPs). Interestingly, however, the ability to interact with NP was not predictive of the ability to support trVLP formation, suggesting the involvement of additional factors. 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 trVLP systems, RNA immunoprecipitations, electron microscopy, and structural biology.

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。为了实现这一点，我们有两个正在进行的项目：（1）在叙利亚仓鼠模型，再现马尔堡出血热（MHF）的致病过程的表征和（2）EBOV蛋白质相互作用的表征。 (1)叙利亚仓鼠模型中致病过程的表征，该模型概括了MHF。虽然NHP模型用于评估疫苗和治疗剂对丝状病毒的效力，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，并已广泛用于丝状病毒研究的许多方面。然而，啮齿动物模型仅产生有限且不一致的凝血异常，这是丝状病毒HF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。同样，我们试图将我们对EBOV的研究应用于建立马尔堡病毒（MARV）安哥拉株的啮齿动物模型，该病毒株被认为是MARV毒力最强的毒株。安哥拉。MARV安哥拉通过连续传代致死性适应仓鼠和豚鼠。我们证明，感染仓鼠适应MARV在仓鼠中产生严重疾病，包括凝血异常，表现为出现类似于在人类和NHP上观察到的瘀点皮疹。此外，为了确定啮齿动物模型中MARV毒力的分子决定因素，我们确定了几种致死啮齿动物适应变体的全长基因组序列，并确定了VP 35、VP 40和VP 24中的三个氨基酸突变。尽管这些突变在啮齿动物适应过程中获得毒力中的作用尚不清楚，但基于MARV VP 40已被证明具有拮抗IFN信号传导的能力以及EBOV的小鼠适应可能与病毒破坏IFN途径的能力有关的事实，我们推测这三个点突变可能影响MARV VP 40以物种特异性方式抑制IFN信号传导的能力。 (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。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，以确定最可能参与蛋白质-蛋白质相互作用的区域。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相互作用的能力揭示了VP 24的C-末端附近的区域，该区域在VP 24与NP相互作用的能力中起关键作用。进一步的突变分析使我们能够将相互作用结构域解析到氨基酸水平。 与野生型VP 24不同，不能与NP相互作用的VP 24突变体也不能支持具有转录/复制能力的病毒样颗粒（trVLPs）的形成。然而，有趣的是，与NP相互作用的能力并不能预测支持trVLP形成的能力，这表明参与了其他因素。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trVLP系统，RNA免疫沉淀，电子显微镜和结构生物学。