ABSTRACT: JC polyomavirus (JCPyV) infects the majority of the population, establishing a lifelong, asymptomatic infection in the kidney of healthy individuals. People that become severely immunocompromised may experience JCPyV reactivation, which can cause progressive multifocal leukoencephalopathy (PML), a neurodegenerative disease. Due to a lack of therapeutic options, PML results in fatality or significant debilitation among affected individuals. Cellular internalization of JCPyV is mediated by serotonin 5-hydroxytryptamine subfamily 2 receptors (5-HT2Rs) via clathrin-mediated endocytosis. The JCPyV entry process requires the clathrin-scaffolding proteins β-arrestin, adaptor protein 2 (AP2), and dynamin. Further, a β-arrestin interacting domain, the Ala-Ser-Lys (ASK) motif, within the C-terminus of 5-HT2AR is important for JCPyV internalization and infection. Interestingly, 5-HT2R subtypes A, B, and C equally support JCPyV entry and infection, and all subtypes contain an ASK motif, suggesting a conserved mechanism for viral entry. However, the role of the 5-HT2R ASK motifs and the activation of β-arrestin-associated proteins during internalization has not been fully elucidated. Through mutagenesis, the ASK motifs within 5-HT2BR and 5-HT2CR were identified as critical for JCPyV internalization and infectivity. Further, utilizing biochemical pulldown techniques, mutagenesis of the ASK motifs in 5-HT2BR and 5-HT2CR resulted in reduced β-arrestin binding. Utilizing small-molecule chemical inhibitors and RNA interference, G-protein receptor kinase 2 (GRK2) was determined to be required for JCPyV internalization and infection by mediating interactions between β-arrestin and the ASK motif of 5-HT2Rs. These findings demonstrate that GRK2 and β-arrestin interactions with 5-HT2Rs are critical for JCPyV entry by clathrin-mediated endocytosis and resultant infection.IMPORTANCE As intracellular parasites, viruses require a host cell to replicate and cause disease. Therefore, virus-host interactions contribute to viral pathogenesis. JC polyomavirus (JCPyV) infects most of the population, establishing a lifelong asymptomatic infection within the kidney. Under conditions of severe immunosuppression JCPyV may spread to the central nervous system, causing the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). Individuals living with HIV or undergoing immunomodulatory therapies are at risk for developing PML. The mechanisms of how JCPyV uses specific receptors on the surface of host cells to initiate internalization and infection is a poorly understood process. We have further identified cellular proteins involved in JCPyV internalization and infection and elucidated their specific interactions that are responsible for activation of receptors. Collectively, these findings illuminate how viruses usurp cellular receptors during infection, contributing to current development efforts for therapeutic options for the treatment or prevention of PML.
