High-throughput drug screen identifies calcium and calmodulin inhibitors that reduce JCPyV infection.

JC polyomavirus (JCPyV) is a nonenveloped, double-stranded DNA virus that infects the majority of the population. Immunocompetent individuals harbor infection in their kidneys, while severe immunosuppression can result in JCPyV spread to the brain, causing the neurodegenerative disease progressive multifocal leukoencephalopathy (PML). Due to a lack of approved therapies to treat JCPyV and PML, the disease results in rapid deterioration, and is often fatal. In order to identify potential antiviral treatments for JCPyV, a high-throughput, large-scale drug screen was performed using the National Institutes of Health Clinical Collection (NCC). Drugs from the NCC were tested for inhibitory effects on JCPyV infection, and drugs from various classes that reduced JCPyV infection were identified, including receptor agonists and antagonists, calcium signaling modulators, and enzyme inhibitors. Given the role of calcium signaling in viral infection including Merkel cell polyomavirus and simian virus 40 polyomavirus (SV40), calcium signaling inhibitors were further explored for the capacity to impact JCPyV infection. Calcium and calmodulin inhibitors trifluoperazine (TFP), W-7, tetrandrine, and nifedipine reduced JCPyV infection, and TFP specifically reduced viral internalization. Additionally, TFP and W-7 reduced infection by BK polyomavirus, SV40, and SARS-CoV-2. These results highlight specific inhibitors, some FDA-approved, for the possible treatment and prevention of JCPyV and several other viruses, and further illuminate the calcium and calmodulin pathway as a potential target for antiviral drug development.

High-Throughput Characterization of Viral and Cellular Protein Expression Patterns During JC Polyomavirus Infection.

JC polyomavirus (JCPyV) is a ubiquitous human pathogen and the causative agent of a fatal demyelinating disease in severely immunocompromised individuals. Due to the lack of successful pharmacological interventions, the study of JCPyV infection strategies in a rapid and highly sensitive manner is critical for the characterization of potential antiviral therapeutics. Conventional methodologies for studying viral infectivity often utilize the detection of viral proteins through immunofluorescence microscopy-based techniques. While these methodologies are well established in the field, they require significant time investments and lack a high-throughput modality. Scanning imager-based detection methods like the In-cell Western (ICW)TM have been previously utilized to overcome these challenges incurred by traditional microscopy-based infectivity assays. This automated technique provides not only rapid detection of viral infection status, but can also be optimized to detect changes in host-cell protein expression during JCPyV challenge. Compared to traditional manual determinations of infectivity through microscopy-based techniques, the ICW provides an expeditious and robust determination of JCPyV infection. The optimization of the ICW for the detection of viral and cellular proteins during JCPyV infection provides significant time and cost savings by diminishing sample preparation time and increasing resource utilization. While the ICW cannot provide single-cell analysis information and is limited in the detection of quantitation of low-expressing proteins, this assay provides a high-throughput system to study JCPyV, previously unavailable to the field. Thus, the high-throughput nature and dynamic experimental range of the ICW can be applied to the study of JCPyV infection.