Progressive Multifocal Leukoencephalopathy (PML) is a life-threatening demyelinating brain disease in immune-compromised individuals caused by the JC polyomavirus (JCV), a ubiquitous human pathogen. No anti-JCV agents are available. PML is a significant complication for patients receiving long-term natalizumab, a humanized monoclonal antibody (mAb) to α4 integrin that blocks binding of the VLA-4 (α4β1) heterodimer expressed by activated T cells to VCAM-1 on CNS endothelial cells, and dramatically reduces relapses in multiple sclerosis (MS) patients. PML is being diagnosed with increasing frequency in patients treated with other immunomodulatory agents (e.g., Rituxamab, Efalixumab, Fingolimod and dimethyl fumarate, and chemotherapy) as well. Drug withdrawal is often complicated by Immune Reconstitution Inflammatory Syndrome (IRIS), a severe inflammatory reaction with paradoxical worsening of demyelination that carries a high mortality rate. Lack of a tractable animal model for PML is a widely recognized hurdle to defining pathogenesis of demyelination in PML and PML-IRIS. Using mouse polyomavirus (MuPyV), my laboratory developed a robust model of polyomavirus-associated demyelinating leukoencephalitis, with viral infection and T cell infiltration. This proposal addresses the lack of direct experimental evidence to mechanistically understand how immunosuppression predisposes patients to CNS infection by JCV and whether JCV has a host-range for CNS-resident glial cells beyond oligodendrocytes that can contribute to PML. In Specific Aim 1, I hypothesize that VLA-4 blockade during persistent MuPyV infection causes dysfunction and/or loss of brain CD8+ (tissue-resident memory)TRM cells, which results in loss of virus control and brain demyelination. To test this hypothesis, I will administer a highly specific small molecule inhibitor of α4β1 during persistent MuPyV infection to determine whether systemic blockade of VLA-4 impairs the maintenance and/or function of MuPyV- specific CD8+ T cells in the brain. Histologic and immunohistologic evaluation of MuPyV-infected brains will be complemented by ImageJ analysis to detect and quantify multifocal lesions in brain sections. For Specific Aim 2, I hypothesize that MuPyV infection of oligodendrocytes and/or astrocytes disrupts cellular metabolism leading to glial cell dysfunction. To test this hypothesis, we will use cutting-edge approaches to evaluate viral- RNA expression in infected oligodendrocytes and/or astrocytes and determine the consequences of MuPyV infection on mitochondrial respiration and glycolysis. Success of the proposed studies will advance our understanding of the mechanisms of immune control of the gliatropic JC polyomavirus and the impact of polyomavirus infection on glial cell biology.