Understanding the bottleneck on axon-to-cell spread of alphaherpesviruses

了解α疱疹病毒轴突到细胞传播的瓶颈

• 批准号: 8700008
• 负责人: Matthew P. Taylor
• 金额: $ 16万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-15 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8700008
• 关键词: Affect; Animal Model; Antiviral Agents; Antiviral Response; Applications Grants; Area; Axon; Biological Assay; Biology; Brain; Cell Line; Cells; Chickenpox; Chimeric Proteins; Collaborations; Color; DNA; Defect; Dissection; Experimental Models; Eye; Eye Infections; Fluorescence; Foundations; Future; Goals; Health; Herpes Labialis; Herpes zoster disease; Herpesviridae; Herpesvirus 1; Herpesvirus Type 3; Histology; Human; Human poliovirus; Image; Imagery; Immune; Immune response; In Vitro; Infection; Institution; Interferons; Investigation; Knock-out; Laboratories; Lead; Lesion; Life; Location; Methodology; Modeling; Molecular Biology Techniques; Molecular Virology; Monitor; Mus; Nature; Nervous system structure; Neurobiology; Neurons; Pathogenesis; Pathway interactions; Polioviruses; Population; Postdoctoral Fellow; Productivity; Proteins; Publications; Publishing; RNA; Reading; Recombinants; Recurrence; Regulation; Reporter; Research; Research Personnel; Retinal Ganglion Cells; Rodent; Role; Scientist; Seminal; Signal Pathway; Signal Transduction; Simplexvirus; Site; Skin; Solid; Suid Herpesvirus 1; System; Test Result; Testing; Time; Training; Transgenic Mice; Universities; Viral; Viral Genome; Viral Proteins; Virion; Virus; Virus Diseases; Virus-Cell Membrane Interaction; Visual; Work; career; cellular imaging; combat; detector; experience; in vivo; insight; mutant; neural circuit; neurotropic; neurotropic virus; next generation; novel; particle; pathogen; public health relevance; research study; response; skills; success; transmission process; virology

DESCRIPTION (provided by applicant): This proposal integrates multiple aspects of molecular virology, host-pathogen interactions, and neuronal biology and highlights the skills and expertise that Dr. Matthew P. Taylor has developed during a career of scientific investigation. The experiments detailed in the research strategy extend upon Dr. Taylor's research findings and expertise in neuronal culture, live cell imaging, and DNA and RNA virology. His 17 years of laboratory experience in both academic and industrial settings and a broad background in molecular biology techniques allow him to develop a wide range of experimental models. This experience was refined during Dr. Taylor's graduate and post-doctoral research into the viral replication and spread of both poliovirus and herpesvirus. His 20 publications, including 2 seminal reviews and 4 first author publications in virus-cell interaction, are demonstrative of significant productivity and impact in the field of virology. The research strategy builds off of the recent publication, "Alphaherpesvirus axon-to-cell spread involves limited virion transmission" in PNAS, which describes the novel observation that directional spread of Herpes Simplex Virus (HSV) and Pseudorabies virus (PRV) involves the transmission of a single viral particle to initiate infection. This population bottleneck has important implications on viral infection and human health, as directional spread is fundamental to the formation of recurrent lesions associated with HSV infection. The research proposed in this application builds off the methodologies of axon-to-cell spread quantification to understand the nature and mechanism of the population bottleneck. [Aim 1 seeks to identify important viral proteins involved in regulating axon-to-cell spread of infection. Previously characterized viral mutants and novel mutants isolated from a random mutagenic screen will be combined with published assays to quantify anterograde spread. By understanding which proteins are involved in regulating virion transmission, we may understand the mechanism behind the population bottleneck. Aim 2, investigates the role of cellular antiviral signaling and activation of interfern responses in restricting the number of virions that initiate infection in susceptible cell populations. Cellular antiviral signaling can be activated during HSV entry upon cell-free inoculation, but has not been characterized for axon-to-cell transmission of limited numbers of virions. A potential role of antiviral signaling will be elucidated through protein localization, fluorescent reporter cell lines, and deletion mutants of both cellular and viral proteins.] Aim 3 wll determine the impact on spread of the axon-to-cell population bottleneck of herpesviruses in an infected host. A mixture of fluorescent protein expressing HSV and PRV recombinants will be injected into the eye to following the diversity of infection. The three-color fluorescent protein expressing mixture of viruses provides a visual read-out of viral genome expression, allowing the visualization and quantitation of co-infection at the discrete sites of primary infection in th eye and at well-described sites of axon-to-cell spread in the brain. Determining the presence of a spread bottleneck in vivo will allow the testing of results from the prior aims on the spread of viral infection within an infected host. The experiments in this proposal utilize a number of Dr. Taylor's experimental strengths and expand into new areas of experimental methodology. To assure the highest level of success, he will continue to gain experience in neurobiology and imaging through coursework and collaboration at his future institution. He has received training in animal models of infection, dissection and histology is from a long-time collaborator of the Enquist lab, Dr. J. Patrick Card a the University of Pittsburgh. Dr. Card is an expert in animal models of PRV and HSV infection, having performed the first work describing the high fidelity neural circuit tracing capacity of PRV in the rodent eye. The combination of training and experimentation will provide a solid foundation in support of Dr. Taylor's goal to be an independent researcher. The work described here will be vital to the initiation of his research pro- gram and provide important preliminary results in support of furthe grant applications. Answering the fundamental questions that underlie axon-to-cell transmission of herpesviruses will open up new lines of questioning regarding the viral interactions with the axonal signaling, neuro-immune regulation, and the general regulation of neurotropic viral infection in an infected host. Dr. Taylor is excited to share his training and expertise with the next generation of scientists while answering fundamental questions of viral infection.

描述(申请人提供)：这项建议综合了分子病毒学、宿主-病原体相互作用和神经生物学的多个方面，并突出了马修·P·泰勒博士在科学研究生涯中发展起来的技能和专业知识。该研究战略中详细介绍的实验基于泰勒博士在神经元培养、活细胞成像以及DNA和RNA病毒学方面的研究成果和专业知识。他在学术和工业环境中拥有17年的实验室经验，在分子生物学技术方面具有广泛的背景，这使他能够开发各种实验模型。在泰勒博士对脊髓灰质炎病毒和疱疹病毒的病毒复制和传播的研究生和博士后研究期间，这一经验得到了提炼。他发表的20篇论文，包括2篇关于病毒-细胞相互作用的开创性评论和4篇第一作者论文，证明了他在病毒学领域的显著生产力和影响力。 这一研究策略建立在最近发表在PNAS上的《甲型疱疹病毒轴突到细胞的传播涉及有限的病毒粒子传播》的基础上，该文章描述了新的观察结果，即单纯疱疹病毒(HSV)和伪狂犬病病毒(PRV)的定向传播涉及单个病毒颗粒的传播以启动感染。这一人口瓶颈对病毒感染和人类健康具有重要影响，因为定向传播是形成与HSV感染相关的复发病变的基础。本申请中提出的研究建立在轴突到细胞扩散量化的方法学基础上，以了解种群瓶颈的性质和机制。[目标1]寻求确定参与调节感染轴突到细胞传播的重要病毒蛋白。从随机诱变筛选中分离出的先前表征的病毒突变体和新突变体将与已发表的分析相结合，以量化顺势传播。通过了解哪些蛋白质参与调节病毒粒子的传播，我们可能了解种群瓶颈背后的机制。目的2，研究细胞抗病毒信号和干扰素反应的激活在限制易感细胞群中引起感染的病毒粒子数量中的作用。当无细胞接种时，细胞抗病毒信号可以在HSV进入时被激活，但尚未被表征为有限数量的病毒粒子的轴突到细胞的传播。抗病毒信号的潜在作用将通过蛋白质定位、荧光报告细胞系以及细胞和病毒蛋白的缺失突变来阐明。]目的3确定疱疹病毒在感染宿主中对轴突到细胞种群瓶颈扩散的影响。表达HSV和PRV重组体的荧光蛋白混合物将被注射到眼睛内，以跟踪感染的多样性。表达混合病毒的三种颜色的荧光蛋白提供了病毒基因组表达的可视读出，使得能够可视化和定量地显示在眼睛中的初始感染的离散位置和在大脑中描述的轴突到细胞扩散的位置的联合感染。确定体内存在传播瓶颈将允许对先前关于病毒感染在受感染宿主内传播的结果进行测试。 这项提议中的实验利用了泰勒博士的许多实验优势，并扩展到实验方法论的新领域。为了确保最高水平的成功，他将继续通过课程学习和未来学院的合作获得神经生物学和成像方面的经验。他从恩奎斯特实验室的长期合作者匹兹堡大学的J·帕特里克·卡德博士那里接受了感染、解剖和组织学的动物模型培训。卡德博士是PRV和HSV感染动物模型方面的专家，他完成了描述PRV高保真神经回路追踪能力的第一项工作 在啮齿动物的眼睛里。 培训和实验的结合将为支持泰勒博士成为一名独立研究员的目标提供坚实的基础。这里描述的工作将对他的研究计划的启动至关重要，并为支持FURTH赠款申请提供重要的初步结果。回答疱疹病毒轴突到细胞传播的基本问题将为病毒与轴突信号的相互作用、神经免疫调节以及感染宿主中嗜神经性病毒感染的一般调节开辟新的问题路线。泰勒博士很高兴能与下一代科学家分享他的培训和专业知识，同时回答病毒感染的基本问题。