Viral encephalitis is an important cause of morbidity and mortality in children and adults. Key knowledge gaps about the pathogenesis of viral encephalitis include how neurotropic viruses target the central nervous system (CNS), internalize into neurons, and disseminate in the brain. The proposed research uses reovirus, a genetically tractable double-stranded RNA virus that causes encephalitis and shows promise as an oncolytic agent, to dissect mechanisms of viral tropism, cell entry, and spread in the CNS. Reovirus strains display serotype-specific patterns of systemic dissemination and neurologic disease. Serotype 3 (T3) reoviruses are exquisitely neurotropic, internalize into neurons using macropinocytosis, and spread intracellularly by fast axonal transport. Reovirus uses β1 integrin to internalize into some types of cells, but its function in neural cell entry is unknown. Other previously identified reovirus receptors are dispensable for reovirus infection in the murine CNS. We recently identified a new reovirus receptor, paired immunoglobulin-like receptor B (PirB), that allows serotype-independent reovirus infection of cultured cells but is required for efficient T3 infection of neurons and full neurovirulence. Three integrated specific aims are proposed to enhance knowledge of reovirus neuropathogenesis that may have broader applications to other neurotropic viruses. In Specific Aim 1, functions of host receptors in reovirus neuropathogenesis will be determined. Biophysical interactions between PirB and reovirus will be assessed using cryo-electron microscopy, atomic-force microscopy, and structure- guided mutagenesis. The function of PirB in reovirus disease will be elucidated by comparing infection and virulence in wild-type (WT) and PirB-null mice. T3 reovirus-specific receptors that couple with PirB for neurotropism will be identified using CRISPR activation screening. In Specific Aim 2, mechanisms of reovirus entry into CNS neurons will be defined. The functions of PirB and β1 integrin in reovirus neural entry will be determined using mutant receptors and receptor-blind viruses. Receptor-dependent control of macropinocytosis will be elucidated using super-resolution, live-cell, and electron microscopy. Mechanisms of dynein motor recruitment for reovirus transport within axons will be dissected by investigating interactions between WT and mutant reovirus receptors and dynein subunits. In Specific Aim 3, the basis of reovirus dissemination in the CNS will be elucidated. The role of host receptors in reovirus neural transit will be defined using whole-brain imaging and three-dimensional reconstruction to trace reovirus infection and neural spread in WT and receptor- null mice. Mechanisms of reovirus transsynaptic transmission will be elucidated using in vitro and in vivo neural circuits. The effect of altering synaptic activity on reovirus neural transit will be tested using pharmacologic intervention and chemogenetically altered mice. ...