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（由申请人提供）：该提案整合了分子病毒学，宿主病原体相互作用和神经元生物学的多个方面，并强调了Matthew P. Taylor博士在科学研究职业中发展的技能和专业知识。研究策略中详细介绍的实验扩展了Taylor博士在神经培养，活细胞成像以及DNA和RNA病毒学方面的研究结果和专业知识。他在学术和工业环境中的17年实验室经验以及分子生物学技术的广泛背景使他能够开发出广泛的实验模型。在泰勒博士的毕业生和博士后研究中，对脊髓灰质炎病毒和疱疹病毒的传播进行了完善。他的20份出版物，包括2项开创性评论和4个病毒 - 细胞相互作用的第一篇作者出版物，证明了对病毒学领域的显着生产力和影响。 研究策略是基于最近出版物“α-疱疹病毒轴突到细胞传播的涉及有限的病毒率传播”，它描述了新颖的观察结果，即疱疹单纯疱疹病毒（HSV）和伪比例病毒（PRV）的方向扩散涉及单个病毒颗粒的传递以启动感染。这种种群瓶颈对病毒感染和人类健康具有重要意义，因为方向扩散是与HSV感染相关的复发性病变的基础。本应用程序中提出的研究基于轴突到细胞扩散定量的方法论，以了解人口瓶颈的性质和机制。 [AIM 1试图确定参与调节感染轴突到细胞传播的重要病毒蛋白。先前表征的病毒突变体和从随机诱变筛选中分离出来的新型突变体将与已发表的测定法相结合，以量化顺行扩散。通过了解哪些蛋白质参与调节病毒粒子传播，我们可以了解种群瓶颈背后的机制。 AIM 2，研究了细胞抗病毒信号传导的作用和干扰反应的激活在限制易感细胞群体中感染的病毒体数量中的作用。无细胞接种后，可以在HSV进入期间激活细胞抗病毒信号，但尚未表征有限数量的病毒体的轴突到细胞的传播。抗病毒信号传导的潜在作用将通过蛋白质定位，荧光报告基因细胞系以及细胞和病毒蛋白的缺失突变体阐明。] AIM 3 WLL确定对感染宿主中疱疹病毒的轴突对细胞种群瓶颈的传播的影响。表达HSV和PRV重组剂的荧光蛋白的混合物将被注入眼睛，以遵循感染的多样性。表达病毒混合物的三色荧光蛋白提供了病毒基因组表达的视觉读出，从而可以在眼睛中原发性感染的离散部位和在大脑中的轴突到细胞散布的良好描述的位点的离散部位可视化和定量共同感染。确定体内扩散瓶颈的存在将允许从先前的目的对感染宿主内病毒感染的扩散进行测试。 该提案中的实验利用了许多泰勒博士的实验强度，并扩展到了实验方法的新领域。为了确保成功的最高水平，他将继续通过他的未来机构的课程和合作来获得神经生物学和成像的经验。他接受了感染，解剖和组织学动物模型的培训，来自Enquist实验室的一位长期合作者J. Patrick Card A博士匹兹堡大学。 Card Dr. Card是PRV和HSV感染动物模型的专家，他执行了描述PRV的高保真神经电路追踪能力的第一项工作 在啮齿动物的眼中。 培训和实验的结合将为泰勒博士成为独立研究人员的目标提供坚实的基础。此处描述的工作对于启动其研究过程至关重要，并提供重要的初步结果，以支持FUR THE FUR THE FUR THE GRAT应用。回答疱疹病毒的轴突到细胞传播基础的基本问题将为有关轴突信号传导，神经免疫调节的病毒相互作用以及对感染宿主中神经性病毒感染的一般调节的新质疑。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。