Developing Transgenic Sindbis Virus as a Tool to Trace Neural Circuitry

描述(由申请人提供)： 通过定义已识别神经元之间的突触连接模式来重建神经系统的解剖结构，是深入理解神经计算的关键一步。基于转基因病毒的工具已经在小鼠身上证明了这一点，但这些病毒跨越突触的机制尚不清楚。更广泛地说，我们对病毒感染如何在大脑内传播导致脑炎的了解有限，目前几乎没有治疗这种感染的治疗选择。这一建议使用了一种新的方法来针对特定神经元产生病毒颗粒，并使用遗传学方法来了解控制病毒传播的调节机制，并合理地设计一种新的电路跟踪工具。狂犬病病毒和伪狂犬病病毒的基因工程衍生品利用了这些病毒通过大脑传播的内在能力，为解剖神经电路提供了宝贵的工具。然而，这些病毒跨越突触连接的机制尚不清楚，这些实验需要物理注射具有感染性的病毒颗粒，从而产生实验变异性并减少吞吐量。此外，这些工具还没有被证明在果蝇中起作用，果蝇是解开微电路功能的重要模型系统。因此，我们提出了一种不同的方法，一种基于另一种病毒Sindbis的方法，在这种方法中，病毒颗粒被遗传编程，专门在神经元的亚群中组装。使用这种方法，我们建议首先使用遗传方法来定义控制病毒感染在大脑中传播的细胞机制。然后，利用病毒表面蛋白的基因工程，我们将专门针对突触释放病毒。最后，通过将宿主防御系统的操作与我们的转基因病毒基因组相结合，我们将进行重建特定神经回路的原则性实验的证明。虫媒病毒是同时感染昆虫和哺乳动物宿主的病毒，是美国病毒性脑炎的一些最常见的原因。目前还不存在用于治疗这些感染的靶向治疗剂或疫苗，临床结果往往很差。其中许多病毒剂与Sindbis密切相关，了解调节其在大脑中传播的细胞防御机制将有助于我们理解这些病原体。通过识别病毒复制和组装所依赖的宿主蛋白，我们的研究将确定新的治疗靶点。此外，定义神经回路接线图的新工具的开发将使对大脑功能和功能障碍的进一步研究成为可能。

DESCRIPTION (provided by applicant): Reconstructing the anatomy of the nervous system by defining the pattern of synaptic connections between identified neurons represents a crucial step toward developing a deep understanding of neural computation. Tools based on genetically modified viruses have demonstrated utility in this regard in the mouse, but the mechanisms by which these viruses cross synapses are unknown. More broadly, our understanding of how viral infections spread within the brain to cause encephalitis is limited, and there are presently few therapeutic options available to treat such infections. This proposal uses a new method to target production of virus particles to specific neurons, and uses genetic approaches to both understand the regulatory mechanisms that control virus spread, and to rationally design a new circuit tracing tool. Genetically engineered derivatives of Rabies virus and Pseudo-rabies virus that exploit the intrinsic ability of these viruses to spread through the brain have provided valuable tools for dissecting neural circuitry. However, the mechanisms by which these viruses cross synaptic connections is unknown, and these experiments require the physical injection of infectious virus particles, creating experimental variability and reducing throughput. Moreover, these tools have not been shown to work in the fruit fly, an important model system for unraveling microcircuit function. We therefore propose a different approach, one based on another virus, Sindbis, in which virus particles are genetically programmed to assemble specifically in subsets of neurons. Using this approach, we propose to use genetic approaches to first define the cellular mechanisms that regulate spread of virus infections in the brain. Then, using genetic engineering of the surface proteins of the virus, we will specifically target viral release to synapses. Finally, by combining manipulations of the host defense repertoire with our genetically modified virus genomes, we will perform proof of principle experiments that reconstruct specific neural circuits. Arboviruses, viruses that infect both insect and mammalian hosts, represent some of the most common causes of viral encephalitis in the United States. No targeted therapeutic agents, or vaccines, presently exist for the treatment of these infections, and poor clinical outcomes are frequent. Many of these viral agents are closely related to Sindbis, and understanding the cellular defense mechanisms that regulate its spread in the brain will inform our understanding of these pathogens. By identifying host proteins that the virus depends on for its replication and assembly, our studies will identify new therapeutic targets. In addition, the development of a new tool for defining the wiring diagram of neural circuits will enable further studies of brain function and dysfunction.