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: 9360979
• 负责人: Hideki Ebihara
• 金额: $ 68.43万
• 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/9360979
• 关键词: Aerosols; Africa; Alanine; Amino Acids; Angola; Animal Model; Animals; Appearance; Atlases; Automobile Driving; Basic Science; Binding; Bioinformatics; Case Fatality Rates; Categories; Cavia; Cell Nucleus; Cells; Clinical; Co-Immunoprecipitations; Coagulation Process; Confocal Microscopy; Country; Development; Disease; Disease Outbreaks; Ebola Hemorrhagic Fever; Ebola virus; Event; Exanthema; Filovirus; Frankfurt-Marburg Syndrome Virus; Gene Expression; Genetic Techniques; Genetic Transcription; Goals; Hamsters; Human; Immune response; Immunoprecipitation; Infection; Inflammatory; Inflammatory Response; Interferons; Interleukin-1; Intervention; Knowledge; Length; Licensing; Liver; Medical; Membrane Proteins; Mesocricetus auratus; Modeling; Molecular; Monitor; Mus; Mutate; Mutation; National Institute of Allergy and Infectious Disease; Nucleocapsid; Nucleoproteins; Organ; Organ failure; Outcome; Pathogenesis; Pathology; Play; Process; Proteins; Recombinants; Reporting; Research; Rodent; Rodent Model; Role; Sepsis Syndrome; Series; Signal Transduction; Signaling Molecule; Spleen; System; TLR4 gene; TNF receptor-associated factor 6; TRAF6 gene; Therapeutic Intervention; Translational Research; Tropism; United States; Vaccines; Variant; Viral; Viral Hemorrhagic Fevers; Viral Proteins; Virion; Virulence; Virulent; Virus Replication; Virus-like particle; Work; base; chemokine; cytokine; functional outcomes; genome sequencing; in vivo; interest; mutant; novel; pathogen; protein function; protein protein interaction; receptor; research and development; response; response biomarker; reverse genetics; therapeutic vaccine; transcription factor; transmission process; vaccine efficacy; viral RNA; virus host interaction

(1) 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. 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) and incorporation of viral RNA into viral nucleocapsid suggesting that VP24 plays a critical role in condensing the nucleocapsid, thereby restricting viral replication and transcription and promoting nucleocapsid packaging and egress. Additionally, mutational analyses revealed the 200 amino acids of NP that are critical for its interaction with VP24. Furthermore, in order to examine the significance of the NP-VP24 interaction in the viral replication cycle, recombinant Ebola viruses possessing mutations at the domains that are responsible for NP-VP24 interactions in NP and VP24 will be generated and characterized using our recently developed efficient reverse genetics system. This study, although ongoing, is the first to identify the molecular determinants of the interaction between VP24 and NP, and it suggests that the interaction between these two proteins is critical to the EBOV replication cycle, possibly hinting at new targets for therapeutic intervention. (2) The molecular mechanism for the over-induction of the host pro-inflammatory response by EBOV matrix protein VP40. Several mechanisms have been postulated to contribute to the catastrophic pathology of filoviral hemorrhagic fevers. Induction of an uncontrolled pro-inflammatory response by filoviruses has been proposed as one of the key pathological events that cause coagulation abnormalities, hemorrhagic manifestations, and multi-organ failure in infected humans and animals. For the induction of a pro-inflammatory response by infection, the canonical NF-B signaling cascade is initiated upon stimulation of various receptors (e.g., toll-like receptor 4), which then leads to the activation of a cellular signaling cascade that finally results in the translocation of NF-B to the nucleus where it acts as a transcription factor primarily involved in upregulating the expression of pro-inflammatory cytokines. The filovirus matrix protein, VP40 is a membrane-associated protein and promotes formation, budding, and release of filamentous virus particles from infected cells. In addition, MARV VP40 functions to inhibit host type I IFN signaling, suggesting that VP40 is multifunctional protein, although no IFN antagonist function is assigned to EBOV VP40. We uncovered a putative TNF-receptor associated factor (TRAF) 6 binding motif that overlaps with the late domain in EBOV VP40, and we subsequently demonstrated an interaction between TRAF6 and VP40. Following the identification of a putative TRAF6 binding motif in EBOV VP40, we confirmed the ability of VP40 to interact with TRAF6 by a series of co-immunoprecipitations. We have also demonstrated that expression of VP40 activates NF-B without any stimulation (e.g., LPS, IL-1 etc,), which agrees with our hypothesis that VP40 may, in part, be responsible for driving the uncontrolled expression of pro-inflammatory cytokines during infection. Interestingly, we also have evidence suggesting that TRAF6 expression increases EBOV budding, perhaps implying a novel role for TRAF6 or other downstream signaling molecules in EBOV egress. This finding strongly suggests that VP40 induces the host cellular pro-inflammatory response, and it implies a novel molecular mechanism of Ebola HF pathogenesis. Since the molecular basis of systemic inflammatory response syndrome in filoviral HFs still remains elusive, this finding will facilitate our understanding of pathogenesis and the development of post-exposure interventions targeting the host deleterious response triggered by filovirus infection. (3) 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 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. 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. In addition, by using qRT-PCR for monitoring host cytokine/chemokine gene expression, we revealed that early activation of innate immune responses including pro-inflammatory responses in the target organs (e.g., spleen, liver) in infected animals are host response markers associated with a lethal outcome. 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. Overall, development of novel small animal models that mimic filoviral HFs provides an excellent experimental system that can be used to illuminate the molecular basis of pathogenesis and virus-host interactions in vivo. In addition, these series of studies will certainly facilitate translational research for the development of medical countermeasures.

(1) 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，我们对了解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都是病毒核衣壳的组成部分，是小鼠EBOV毒力的唯一因素，这表明这两种蛋白在诱导疾病中起着关键作用。鉴于它们对EBOV毒力的贡献，我们试图表征NP和VP24之间的物理关系。我们使用共聚焦显微镜和免疫沉淀法来证明野生型NP既与VP24共定位又与VP24相互作用。为了确定VP24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-蛋白质相互作用的区域。基于这一预测，我们产生了一系列VP24突变体，每个突变体中有多达8个连续的氨基酸突变为整个蛋白质的丙氨酸。评估这些突变体与NP相互作用的能力揭示了VP24 c端附近的一个区域，该区域在VP24与NP相互作用的能力中起着关键作用。与野生型VP24不同，不能与NP相互作用的VP24突变体也不能支持转录/复制能力强的病毒样颗粒（trvlp）的形成和病毒RNA进入病毒核衣壳，这表明VP24在压缩核衣壳中起关键作用，从而限制病毒的复制和转录，促进核衣壳的包装和输出。此外，突变分析揭示了NP的200个氨基酸对其与VP24的相互作用至关重要。此外，为了检验NP-VP24相互作用在病毒复制周期中的重要性，我们将利用我们最近开发的高效反向遗传学系统，在NP和VP24中负责NP-VP24相互作用的结构域产生突变的重组埃博拉病毒，并对其进行表征。这项研究虽然仍在进行中，但首次确定了VP24和NP之间相互作用的分子决定因素，并表明这两种蛋白质之间的相互作用对EBOV复制周期至关重要，可能暗示了治疗干预的新靶点。

项目成果

Rodent-Adapted Filoviruses and the Molecular Basis of Pathogenesis.

• DOI: 10.1016/j.jmb.2016.05.008
• 发表时间: 2016-08-28
• 期刊: JOURNAL OF MOLECULAR BIOLOGY
• 影响因子: 5.6
• 作者: Banadyga, Logan;Dolan, Michael A.;Ebihara, Hideki
• 通讯作者: Ebihara, Hideki

Protective efficacy of a bivalent recombinant vesicular stomatitis virus vaccine in the Syrian hamster model of lethal Ebola virus infection.

• DOI: 10.1093/infdis/jir379
• 发表时间: 2011-11
• 期刊: The Journal of infectious diseases
• 作者: Yoshimi Tsuda;D. Safronetz;Kyle Brown;R. Lacasse;A. Marzi;H. Ebihara;H. Feldmann