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: 8556041
• 负责人: Hideki Ebihara
• 金额: $ 49.11万
• 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/8556041
• 关键词: Address; Adult; Amino Acid Substitution; Animal Model; Animals; Antibodies; Biological; Biological Assay; Case Fatality Rates; Cavia; Cell Culture Techniques; Cell Line; Cells; Cessation of life; Characteristics; Clinical; Clinical Data; Co-Immunoprecipitations; Coagulation Process; Communicable Diseases; Complementary DNA; Computer Simulation; Depressed mood; Development; Disease; Disease Outbreaks; Ebola Hemorrhagic Fever; Ebola Vaccines; Ebola virus; Epitopes; Event; Family; Filoviridae; Filovirus; Future; Genes; Genome; Growth; Hamsters; Human; Immune; Immune response; Immunoblotting; Immunocompetent; Immunofluorescence Immunologic; Immunoprecipitation; In Vitro; Infection; Inflammatory Response; Interferon Type I; Length; Licensing; Maps; Mesocricetus auratus; Minor; Modeling; Molecular; Mouse Cell Line; Mus; Mutation; Nucleocapsid; Nucleoproteins; Nucleotides; Oryctolagus cuniculus; Pathogenesis; Pathogenicity; Peptides; Phase; Physiological; Point Mutation; Process; Production; Proteins; Publishing; Recombinants; Research; Reston; Rodent; Rodent Model; Role; Series; Structure; Sudan; Syndrome; System; Testing; Therapeutic; Tropism; Vaccines; Vero Cells; Viral; Viral Hemorrhagic Fevers; Viral Proteins; Virulence; Virus; Virus Diseases; Wild Type Mouse; Work; base; effective therapy; functional outcomes; hammerhead ribozyme; improved; in vivo; interest; member; mouse model; mutant; nonhuman primate; novel; polyclonal antibody; positional cloning; protein function; recombinant virus; research study; response; three-dimensional modeling; virus pathogenesis

Viral hemorrhagic fevers caused by viruses belonging to the genus Ebolavirus and Marburgvirus, both members of the Filoviridae family, are known to be 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 severe hemorrhagic syndrome. However, 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. Therefore, our research is focused on elucidating the roles of viral proteins in the viral replication cycle and pathogenesis. To accomplish this, we have four ongoing projects: (1) the characterization of pathogenic processes in the Syrian hamster model that recapitulates EHF, (2) the development of efficient reverse genetics systems for generating recombinant EBOV from cDNA, (3) the elucidation of the molecular mechanisms of EBOV adaptation in mice, and (4) the characterization of EBOV viral protein interactions. (1) The 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. Therefore, we have developed and characterized a novel lethal Syrian hamster model of EHF based on infection with mouse-adapted (MA)-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 characterized and compared host responses induced as a result of lethal (MA-EBOV) versus non-lethal infection (WT-EBOV). Our comprehensive virological, pathological, physiological, and immunological analyses revealed that suppression of type I interferon responses and induction of coagulation abnormalities by MA-EBOV are, along with the uncontrolled pro-inflammatory response, key events contributing to the ultimate death of the animals. Our novel hamster model will facilitate research on pathogenesis and countermeasure development. (2) The development of efficient reverse genetics systems for generating recombinant EBOV from cDNA. We have been developing a more efficient rescue system to generate recombinant EBOV. Full-length wild-type and mouse-adapted EBOV genome clones have been constructed with the hammerhead ribozyme sequence inserted at the 5 terminus, resulting in the production of an authentic 5 terminus in the subsequent viral cRNA. Using this system, we have significantly improved the rescue efficiency of recombinant EBOV over the previous system lacking the hammerhead ribozyme. Additionally, by comparing the rescue efficiencies of this system in a variety of different cell lines, we have determined that Huh7 cells produce significantly higher titers of virus than any other cell line tested, including Vero cells, which have been traditionally used in EBOV rescue systems. In fact, we found that the genomes of EBOV rescued in Vero cells were unstable, accruing significantly more nucleotide substitutions and insertions than the genomes of viruses rescued in Huh7 cells. Overall, we have succeeded in developing an EBOV reverse genetics system that is significantly improvedin both efficiency and fidelityover previous systems. Not only has this system improved our ability to rescue recombinant wild-type and mouse-adapted EBOV, but we anticipate that it will also improve our ability to rescue and characterize mutant viruses that may have depressed growth characteristics. (3) The elucidation of the molecular mechanisms of EBOV adaptation in mice. While adult immunocompetent mice resist EBOV infection, MA-EBOV causes lethal infection in mice. Previously, a study using EBOV reverse genetics identified that amino acid substitutions in VP24 and nucleoprotein (NP) were primarily responsible for the acquisition of virulence in mice. In order to uncover the molecular mechanisms underlying EBOV pathogenesis in the mouse model, we have generated a series of recombinant viruses possessing various combinations of wild-type and mouse-adapted genes, with particular focus on mutations in the NP and VP24 genes. Growth comparison of these mutants in mouse cell lines suggested that mutations in NP and VP24 enhanced the growth ability of EBOV in target cells. We will examine the ability of the mutations in NP and VP24 to enhance viral replication and modify the host response in target cells at the early phase of infection in mice. (4) The characterization of EBOV viral 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. For example, it is thought that NP, the EBOV nucleoprotein, relies in part on an interaction with VP24, the so-called minor matrix protein, for the efficient production of nucleocapsids. Moreover, we have previously demonstrated that two point mutations, one each in NP and VP24, are necessary and sufficient for conferring pathogenicity to EBOV in mice, perhaps suggesting a further functional relationship between these two proteins. Nevertheless, the molecular basis for this interaction is not known. To address this, we have raised a series of rabbit and guinea-pig polyclonal antibodies against several VP24 peptides and demonstrated that these antibodies recognize VP24 by immunoblot, immunoprecipitation, and immunofluorescence. Considering that no commercial antibody exists for detecting VP24 and that epitope-tagging this protein is difficult, possessing an antibody that robustly recognizes VP24 is key to all further downstream experiments. Accordingly, we have used this antibody in co-immunoprecipitation assays to repeat and confirm the interaction between NP and VP24, and we are currently in the process of mapping the regions of NP and VP24 that are critical for this interaction. To this end, we have produced a 3D model of EBOV VP24 based on the recently published structures of Sudan and Reston VP24. This model, and future in silico work with NP, will aid in elucidating the molecular determinants of the NP-VP24 interaction.

病毒性出血热由属于丝状病毒科成员的埃博拉病毒属和马尔堡病毒属的病毒引起，是人类和非人类灵长类动物 (NHP) 中最严重的传染病之一，目前尚无获得许可的疫苗或有效的治疗方法。尤其是埃博拉病毒 (EBOV)，导致多起埃博拉出血热 (EHF) 疫情，病死率高达 65% 至 90%。对动物模型的研究和 EHF 爆发的有限临床数据表明，由 EBOV 感染引起的相互依赖的致病过程，包括宿主免疫和病理生理反应，会引发严重的出血综合征。然而，为了开发有效的 EHF 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作用。为了实现这一目标，我们有四个正在进行的项目：(1) 表征 EHF 的叙利亚仓鼠模型中的致病过程；(2) 开发有效的反向遗传学系统，用于从 cDNA 生成重组 EBOV；(3) 阐明小鼠中 EBOV 适应的分子机制；(4) 表征 EBOV 病毒蛋白相互作用。 (1) 叙利亚仓鼠模型中致病过程的表征，概括了 EHF。 NHP 模型由于可以准确地概括疾病而被用来评估 EBOV 疫苗和治疗的功效，而啮齿动物模型（小鼠和豚鼠）则方便且适合阐明特定病毒蛋白在致病过程中的作用，并已广泛应用于 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。因此，我们开发并表征了一种基于小鼠适应性（MA）-EBOV 感染的新型致死叙利亚仓鼠 EHF 模型，该模型表现出在 EBOV 感染的 NHP 和人类中观察到的许多临床和病理结果，包括凝血异常。为了确定该模型的发病机制，我们对致死（MA-EBOV）与非致死感染（WT-EBOV）引起的宿主反应进行了表征和比较。我们全面的病毒学、病理学、生理学和免疫学分析表明，MA-EBOV 对 I 型干扰素反应的抑制和凝血异常的诱导，以及不受控制的促炎症反应，是导致动物最终死亡的关键事件。我们的新型仓鼠模型将促进发病机制的研究和对策的开发。 (2) 开发高效的反向遗传学系统，用于从 cDNA 产生重组 EBOV。 我们一直在开发更有效的救援系统来产生重组埃博拉病毒。全长野生型和小鼠适应的 EBOV 基因组克隆已构建，并在 5 末端插入锤头核酶序列，从而在后续病毒 cRNA 中产生真正的 5 末端。使用该系统，与之前缺乏锤头核酶的系统相​​比，我们显着提高了重组埃博拉病毒的救援效率。此外，通过比较该系统在各种不同细胞系中的救援效率，我们确定 Huh7 细胞产生的病毒滴度明显高于任何其他测试的细胞系，包括传统上用于 EBOV 救援系统的 Vero 细胞。事实上，我们发现在 Vero 细胞中拯救的 EBOV 基因组不稳定，比在 Huh7 细胞中拯救的病毒基因组产​​生明显更多的核苷酸取代和插入。总体而言，我们成功开发了 EBOV 反向遗传学系统，该系统在效率和保真度方面比以前的系统都有显着提高。该系统不仅提高了我们拯救重组野生型和小鼠适应型埃博拉病毒的能力，而且我们预计它还将提高我们拯救和表征​​可能具有抑制生长特性的突变病毒的能力。 (3)阐明小鼠埃博拉病毒适应的分子机制。 虽然成年免疫功能正常的小鼠可以抵抗 EBOV 感染，但 MA-EBOV 会导致小鼠致命的感染。此前，一项使用 EBOV 反向遗传学的研究发现，VP24 和核蛋白 (NP) 中的氨基酸取代是小鼠获得毒力的主要原因。为了揭示小鼠模型中埃博拉病毒发病机制的分子机制，我们生成了一系列具有野生型和小鼠适应基因的各种组合的重组病毒，特别关注 NP 和 VP24 基因的突变。这些突变体在小鼠细胞系中的生长比较表明，NP 和 VP24 的突变增强了 EBOV 在靶细胞中的生长能力。我们将研究 NP 和 VP24 突变在小鼠感染早期增强病毒复制并改变靶细胞宿主反应的能力。 (4) EBOV病毒蛋白相互作用的表征。 关于控制埃博拉病毒蛋白之间相互作用的分子细节的信息相对较少。因此，我们对了解埃博拉病毒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。例如，人们认为 NP（EBOV 核蛋白）部分依赖于与 VP24（所谓的次要基质蛋白）的相互作用来有效生产核衣壳。此外，我们之前已经证明，两个点突变（NP 和 VP24 各一个）对于赋予小鼠 EBOV 致病性是必要且充分的，这可能表明这两种蛋白质之间存在进一步的功能关系。 然而，这种相互作用的分子基础尚不清楚。为了解决这个问题，我们制备了一系列针对几种 VP24 肽的兔和豚鼠多克隆抗体，并通过免疫印迹、免疫沉淀和免疫荧光证明这些抗体可识别 VP24。考虑到目前尚无用于检测 VP24 的商业抗体，并且对该蛋白进行表位标记很困难，因此拥有能够可靠识别 VP24 的抗体是所有进一步下游实验的关键。因此，我们在免疫共沉淀测定中使用了这种抗体来重复和确认 NP 和 VP24 之间的相互作用，并且我们目前正在绘制对于这种相互作用至关重要的 NP 和 VP24 区域的图谱。为此，我们根据最近发布的苏丹和雷斯顿 VP24 的结构制作了 EBOV VP24 的 3D 模型。该模型以及未来与 NP 的计算机工作将有助于阐明 NP-VP24 相互作用的分子决定因素。