A principled, novel rabies virus toolbox for tracing synaptic circuits and pinpointing how neuronal innate immunity shapes viral spread in the brain

About This Grant

PROJECT SUMMARY The re-engineering of viral pathogens into vectors for biological discovery and human therapy has driven landmark advancements across biomedicine, including in neuroscience. However, the molecular principles through which viruses interact with diverse types of primary host cells in complex tissue are largely unknown, a knowledge gap that complicates the use and interpretation of virus-based tools. Rabies lyssavirus has played an important role in the defining the synaptic organization of the brain’s neural circuitry: Rabies evolvedto spread from neuronal dendrites to neuronal axons through synapses and has been engineered to restrict spread into just those presynaptic neurons making direct synaptic connections on molecularly-defined postsynaptic neurons, revealing “monosynaptic” networks in the intact brain through expression of fluorescent proteins. Another salient and unique infection property of rabies is the maintenance of “stealth”from innate immunesystem, accomplished in part by re-deployment of rabies proteins to physically sequester and thus antagonize key signaling nodes downstream of viral sensing and anti-viral responses. Virologists and immunologists have detailed the molecular mechanisms of innate immune antagonism by rabies proteins over decades in cell line and have revealed that the potency of antagonism tends to be genetically-eroded as rabies strains are adapted to cell culture. Because the two rabies strains currently used by neuroscientists for monosynaptic tracing are (to different degrees) cell culture adapted, experimentally-identified monosynaptic networks may be influencedby the innate immune state of infected neurons. Here, we propose to use a novel “toolbox” based on the minimally cell culture adapted “Tha” strain to establish 1) a potent novel vector for monosynaptic tracing (Aim 1) and 2) directly test the hypothesis that the innate immune state in postsynaptic neurons controls the degree of rabies presynaptic uptake (Aim 2). To do this, I will leverage a Tha variant carrying two point mutations in the viral phosphoprotein (2P) and four point mutations in the viral matrix protein (4M) that largely abrogate innate immune antagonism while leaving canonical viral functions in fact. By quantitative comparison of monosynaptic networks and single-cell molecular properties across Tha and Tha2P4M infected neurons, I will directly test the hypothesis that upregulated innate immune signaling in postsynaptic neurons actively restricts presynaptic rabies uptake and identify the viral “restriction factors" deployed by neurons to limit rabies uptake. My proposal will not only provide neuroscientists a novel, cutting-edge tool to accurately characterize the synaptic organization of neural circuits in vivo, but will also establish a first-in-kind link between neuronal innate immunity and viral infection properties, with broad implications for how neurotropic infection proceeds across diverse cell types that compromise the central nervous system.