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.
Developing transgenic Sindbis virus as a tool for tracing neural circuitry PI: Thomas R. Clandinin Viral tools have proven invaluable for conducting anatomical reconstruction of neuronal connections in a variety of experimental contexts. However, the existing tools of this type have important technical limitations. At the same time, viral infection of the brain leading to encephalitis can cause severe clinical conditions for which our limited mechanistic understanding precludes the development of effective therapies. This proposal seeks to develop a rationally designed viral circuit-tracing tool, one that exploits genetic approaches to understand the viral and cellular mechanisms that regulate viral spread in the brain.
|Wernet, Mathias F; Klovstad, Martha; Clandinin, Thomas R (2014) Generation of infectious virus particles from inducible transgenic genomes. Curr Biol 24:R107-8|
|Wernet, Mathias F; Klovstad, Martha; Clandinin, Thomas R (2014) A Drosophila toolkit for the visualization and quantification of viral replication launched from transgenic genomes. PLoS One 9:e112092|