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. Author manuscript; available in PMC: 2025 Feb 1.
Published in final edited form as: Antiviral Res. 2024 Jan 19;222:105817. doi: 10.1016/j.antiviral.2024.105817

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

Avery CS Bond 1, Mason A Crocker 1, Michael P Wilczek 1,+, Jeanne K DuShane 1, Amanda L Sandberg 1, Lucas J Bennett 1, Nicholas R Leclerc 1, Melissa S Maginnis 1,2,*
PMCID: PMC10922812  NIHMSID: NIHMS1963628  PMID: 38246207

Abstract

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.

Keywords: JC polyomavirus, progressive multifocal leukoencephalopathy (PML), drug screen, NIH Clinical Collection, SV40, BK polyomavirus, SARS-CoV-2, RPTECs, calcium, calmodulin, trifluoperazine, nifedipine, MAPK signaling pathway, ERK

1. Introduction

JC polyomavirus (JCPyV) infects 50–80% of the human population and is the etiological agent of the debilitating neurodegenerative disease progressive multifocal leukoencephalopathy (PML) (Atkinson and Atwood 2020). Infection is thought to occur via fecal-oral route early in life, as JCPyV is shed in urine and can be found in untreated wastewater (Polo, Perez et al. 2004, Vanchiere, Abudayyeh et al. 2009). Immunocompetent individuals harbor an asymptomatic, persistent infection in the kidneys, while severely immunosuppressed individuals are at risk for JCPyV spread to the brain, resulting in PML (Prezioso, Pietropaolo et al. 2023). People with HIV/AIDS or those undergoing immunosuppressive therapies for diseases such as multiple sclerosis are at greatest risk for development of PML (Kaiserman, O’Hara et al. 2023). PML can be fatal within one year if the underlying immunosuppression is left untreated, as there are currently no approved targeted therapies for PML (Atkinson and Atwood 2020). Though patients can live with PML for up to 10 years, symptoms of PML are debilitating and quality of life is poor (Joly, Conte et al. 2023).

Polyomaviridae are non-enveloped, double-stranded DNA viruses, and include JCPyV, simian virus 40 (SV40), BK polyomavirus (BKPyV), and Merkel cell polyomavirus (MCPyV), among others (Assetta and Atwood 2017). Polyomavirus (PyV) capsids consist of three structural proteins, including the major capsid protein viral protein 1 (VP1), which mediates interactions with host cells (Nelson, Stroh et al. 2015). PyV genomes also encode the T-antigen (TAg) protein, which is a major regulatory protein in replication and infection (DeCaprio and Garcea 2013). JCPyV enters cells via clathrin-mediated endocytosis and requires the G-protein coupled receptor (GPCR) 5-hydroxytryptamine 2 subfamily of receptors (5-HT2Rs), while MCPyV, BKPyV, and SV40 enter by caveolin-mediated endocytosis (Anderson, Chen and Norkin 1996, Elphick, Querbes et al. 2004, Becker, Dominguez et al. 2019, Mayberry, Soucy et al. 2019). Once internalized, PyVs transit the endolysosomal route and are deposited into the endoplasmic reticulum (ER) and eventually into the nucleus for transcription and replication (Tsai and Qian 2010). Though it is known that JCPyV uses multiple signaling pathways during infection, including phosphatidylinositol-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK), the intracellular signaling pathways activated during JCPyV infection remain poorly understood (DuShane, Wilczek et al. 2018, Clark, Gee et al. 2020).

Interestingly, SV40 and MCPyV have recently been reported to require calcium channel activation during infection. Verapamil, a transient (T-type) and long lasting (L-type) Ca2+ channel inhibitor, reduces SV40 and MCPyV infection. Additionally, a two-pore Ca2+channel (TPC) inhibitor, tetrandrine, drastically reduced SV40 and MCPyV infections, likely during the endolysosomal fusion step in the viral infectious cycles (Dobson, Mankouri and Whitehouse 2020). Many other viruses also rely on calcium channel activity and calcium signaling at various points during infection, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during entry (Straus, Bidon et al. 2021), human immunodeficiency virus (HIV) during trafficking (Perlman and Resh 2006), and influenza during viral replication (Nugent and Shanley 1984). PyVs require Ca2+ ions for capsid stability, but whether JCPyV relies on calcium pumps and calcium signaling during infection has yet to be studied (Haynes, Chang and Consigli 1993).

This study reports the results of a large-scale drug screen to discover potential antivirals for JCPyV infection and PML that identified calcium-signaling related inhibitors that reduce JCPyV infection. Drug screens represent an effective method for antiviral discovery, and similar screens have been performed for numerous viruses, including Ebola, Zika, and SARS-CoV-2 (Barrows, Campos et al. 2016, Lee, Shum et al. 2018, Garcia, Sharma et al. 2021). The screen was conducted using the NIH Clinical Collection (NCC), and changes in viral infection were assessed using a high-throughput In-Cell Western (ICW) assay for JCPyV infection. Forty-two drugs that reduced JCPyV infection were identified, and 19% of these hits were calcium-related drugs. Hits were further characterized by ICW and validated by fluorescent focus unit (FFU) infectivity assays (DuShane, Wilczek et al. 2019). Calcium- and calmodulin-specific drugs significantly reduced infection, and a selected calmodulin inhibitor, TFP, was investigated for its specific role in the JCPyV infectious cycle. Interestingly, calmodulin inhibitors also significantly impacted BKPyV, SV40, and SARS-CoV-2 infections. Together, results demonstrate that calcium and calmodulin-related pathways are necessary for JCPyV infection and also for BKPyV, SV40, and SARS-CoV-2 infections. TFP is currently an FDA-approved drug, and therefore has potential to be repurposed for treatment of JCPyV infection and PML, and possibly as a broad-spectrum antiviral.

2. Materials and methods

2.1. Cell lines and viruses

SVGA cells (Major, Miller et al. 1985) were maintained in complete minimum essential medium (MEM) (Corning) containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (P/S) (Mediatech, Inc.), and 0.2% Plasmocin (InvivoGen). Renal proximal tubule epithelial cells (RPTEC) were maintained in complete renal epithelial growth medium (REGM) containing a renal epithelial cell growth kit (ATCC) and 1% P/S. Vero E6 and HeLa cells were cultured in complete Dulbecco’s modified Eagle medium (DMEM) (Corning) containing 10% FBS, 1% P/S, and 0.2% Plasmocin. HEK293A cells stably expressing 5-HT2CR (Assetta, Maginnis et al. 2013) in fusion with YFP (HEK-2C) were maintained in DMEM with 10% FBS, 1% P/S, 0.2% Plasmocin, and 1% G418 (MP Biomedicals) to maintain receptor expression. Cell lines were propagated in a humidified incubator at 37°C with 5% CO2 and were passaged 2–3 times weekly. RPTEC and Vero E6 cells were obtained directly from ATCC, while SVGA, HeLa, and HEK-2C cells were generously provided by the Atwood Laboratory (Brown University).

JCPyV strain Mad-1/SVEΔ, SV40 strain 777, and BKPyV Dunlop strain (Atwood Laboratory, Brown University) were generated and propagated as described previously (Vacante, Traub and Major 1989, Nelson, Derdowski et al. 2012). Crude supernatant stock was used in all experiments except when labeled purified virus is indicated. Labeling of JCPyV with Alexa Fluor 647 was described previously (Stroh, Maginnis et al. 2015). JCPyV, SV40, and BKPyV stocks were titered by FFU infectivity assay in SVGA (JCPyV) and Vero E6 cells (SV40 and BKPyV). Reovirus strain T3D was a gift from Pranav Danthi, Indiana University.

SARS-CoV-2 procedures were performed under BSL-3 conditions at the Diagnostic Research Laboratory (University of Maine, Co-Operative Extension). SARS-Related Coronavirus 2, Isolate hCoV-19/USA/OR-OHSU-PHL00037/2021 (Lineage B.1.1.7; Alpha Variant), NR-55461 was obtained through BEI Resources. Propagation of SARS-CoV-2 was performed as described previously (Harcourt, Tamin et al. 2020) with modifications recently reported (Regan, Fong et al. 2022). Titration of SARS-CoV-2 was performed by TCID50 assay as previously described (Stanifer, Kee et al. 2020).

2.2. Antibodies, inhibitors, and plasmids

Antibodies used to detect infectivity by FFU and ICW assays include PAB962, a monoclonal antibody (mAb) derived from a hybridoma supernatant for detection of JCPyV large TAg graciously provided by the Tevethia Laboratory, Penn State University (Maginnis, Haley et al. 2010); PAB597, a mAb obtained from a hybridoma and targeted against JCPyV, SV40, and BKPyV viral protein 1 (VP1), generously provided by Ed Harlow and Walter Atwood; reovirus antisera, generously provided by Pranav Danthi; a primary antibody against SARS-CoV-2 nucleocapsid (NP) (Sino Biological, 40143-MM05); a primary antibody against phosphorylated ERK (pERK) (Cell Signaling Technology, 9101); secondary polyclonal goat anti-mouse and goat anti-rabbit Alexa Fluor 488 or 594 antibodies (Thermo Fisher); and secondary LI-COR 800 anti-mouse or anti-rabbit antibodies (LI-COR). DAPI (Thermo Fisher) was used for FFU assays to stain cell nuclei, while CellTag 700 (LI-COR) was used as a cell count normalization stain for ICW assays.

The NIH Clinical Collection was generously provided by Dr. Bernardo Mainou (Emory University) with inhibitors suspended in DMSO at 10 mM. Chemical inhibitors used in validation experiments possessed a purity of at least 98% and include W-7 (SC-201501) (Santa Cruz Biotechnology), Trifluoperazine HCl (S3201) (Selleckchem), Tetrandrine (SML3048–10MG) (Sigma-Aldrich), Flunarizine (SC-201473) (Santa Cruz Biotechnology), Nifedipine (N7634–1G) (Sigma-Aldrich), Nimodipine (66085-59-4) (Acros Organics), and Nitrendipine (Santa Cruz Biotechnology) (SC-201466). All inhibitors were resuspended in DMSO (Tocris Bioscience), which was used as a volume-specific vehicle control. Concentrations of each inhibitor are listed in figures or figure legends where applicable.

Plasmids used for transfection of the infectious clone, including pUC19 and JC pUC19, were generously provided by the Atwood Laboratory, Brown University. JC pUC19 was created using JCPyV strain JC12 DNA, a subclone of Mad1-SVEΔ, subcloned into pUC19 at a BamHI site (Gee, Tsomaia et al. 2004).

2.3. Drug screen

SVGA cells were plated to 70% confluence in 96-well plates in complete MEM. Inhibitors from the NCC were diluted in MEM containing 10% FBS (10% MEM) to a final concentration of 10 μM. Cells were pre-treated with each inhibitor in respective wells and incubated at 37°C for 1 h. JCPyV (MOI = 0.5 FFU/cell) in 10% MEM was added directly into each well containing inhibitor and incubated for 1 h. Cells were then fed with 10% MEM and incubated for the duration of the 72 h infection. Plates were fixed with 4% paraformaldehyde (PFA), stained for VP1 (1:40), and analyzed via ICW (described below). Each experimental plate also contained the controls: 4 wells of mock-infected cells (no virus), 4 wells of vehicle control DMSO for the drugs in the screen, 4 wells of PD98059 (50 μM) (positive control for viral inhibition), and 4 wells of vehicle control DMSO for the PD98059 control. Three independent replicates were performed. Z-scores were used to identify hits (described below).

2.4. Cell Viability Assays

Cell viability under specified inhibitor concentrations was tested by MTS assay (G3581) (Promega) for each cell type and corresponding infection duration according to manufacturer’s instructions. Cells were plated to 70% confluence in 96-well plates in complete media (MEM or DMEM), and toxicity assays were performed to mimic the experimental design of infectivity assays. Cells were pre-treated with inhibitor or DMSO volume control at 37°C for 1 h, mock-infected with media at 37°C for 1 h, then inhibitor or DMSO control was added back for the duration of the infectious cycle. MTS reagent was added, incubated at 37°C for 1–4 h, then absorbance measurements were taken at 490 nm using an Agilent BioTek Cytation 5 Imaging Reader. Experiments were performed in triplicate. Concentrations that did not induce significant toxicity and maintained >80% cell viability in comparison to the relevant DMSO control were considered usable concentrations.

2.5. JCPyV and SV40 FFU and ICW infections

SVGA, HEK-2C, RPTEC, or Vero E6 cells were seeded in 96-well plates in 10% MEM (SVGA), REGM (RPTEC), or DMEM (HEK-2C and Vero E6) to achieve 70% confluence at time of infection. Inhibitors were diluted in 10% MEM, REGM, or DMEM to concentrations indicated in figures, added to triplicate wells, and incubated at 37°C for 1 h. Cells were infected with JCPyV or SV40 in 10% MEM, REGM, or DMEM (MOIs indicated in figure legends) in absence of inhibitor and incubated at 37°C for 1 h. Infections were fed with 100 μl/well of 10% MEM, REGM, or DMEM containing appropriate concentrations of inhibitors and incubated for 48 h. Cells were fixed in 4% PFA, stained for TAg (JCPyV) (1:5) or VP1 (SV40) (1:40), and analyzed by FFU or ICW assay. All infections were performed in triplicate for a minimum of 3 replicates.

2.6. pERK assays

SVGA or HEK-2C cells were plated to 70% confluence in 96-well plates. Nifedipine or tetrandrine were diluted in 10% MEM or DMEM to concentrations indicated in figures, added to triplicate wells, and incubated at 37°C for 24 h. Cells were fixed in 4% PFA, stained for pERK (1:750) and CellTag (1:500), and analyzed by ICW assay. All experiments were performed in triplicate for a minimum of 3 replicates.

2.7. ICW staining and protein quantification

After fixation, wells were washed 3x with PBS-T for 5 min. Cells were permeabilized with 1% TX-100 at RT for 15 min then incubated in TBS Odyssey Blocking Buffer (LI-COR) at RT for 1 h. PAB962 (1:5), PAB597 (1:40), or pERK (1:750) primary antibody in TBS Odyssey Blocking Buffer were incubated at 4°C overnight while rocking. Cells were washed with PBS-T 3x for 5 min, then LI-COR 800 secondary anti-mouse or anti-rabbit antibody (1:10,000) and CellTag 700 (1:500) were incubated at RT for 1 h. Wells were washed and liquid was removed prior to scanning. Plates were scanned using a LI-COR Odyssey CLx Infrared Imaging system for detection of 700 and 800 nm channel intensities. Settings were as follows; 42 μm resolution, medium quality, and 3.0 mm focus offset (DuShane, Wilczek et al. 2019). Channels were aligned after scanning using Image Studio software with the ICW module. The ICW analysis grid was used to outline each well and intensity values for the 700 and 800 channels within the wells were recorded. Infection was quantified by dividing the 800 channel intensity value by the 700 channel intensity value times 100. Values were then normalized to the relevant volume control.

2.8. BKPyV and reovirus infections

Vero E6 cells or HeLa cells were plated in 96-well plates in 10% DMEM to achieve 70% confluence at time of infection. Inhibitors were diluted in 10% DMEM to concentrations indicated in figures, added to triplicate wells, and incubated at 37°C for 1 h. For BKPyV, Vero E6 cells were infected in 10% DMEM in absence of inhibitor at 37°C for 2 h, then fed with 100 μl/well of DMEM containing appropriate concentrations of inhibitors and incubated for 72 h. For reovirus, HeLa cells were infected in 10% DMEM in absence of inhibitor at RT for 1 h, then fed with 100 μl/well of DMEM containing appropriate concentrations of inhibitors and incubated for 24 h. Cells were fixed in 4% PFA, stained for viral protein, and analyzed by FFU assay.

2.9. SARS-CoV-2 infection

Under BSL-3 conditions, Vero E6 cells were plated in 10% DMEM in 96-well plates to 70% confluence. Inhibitors were diluted in 10% DMEM, added to wells, and incubated for 1 h. Cells were infected with SARS-CoV-2 in serum-free DMEM at 0.025 TCID50/cell in absence of inhibitor and incubated at 37°C for 1 h, then fed with 100 μl/well of 10% DMEM containing inhibitor and incubated at 37°C for 24 h. Cells were fixed with 4% PFA, stained for SARS-CoV-2 NP (1:500), and analyzed by FFU assay. All SARS-CoV-2 infections were performed in triplicate for 3 replicates in a BSL-3 laboratory.

2.10. FFU infectivity assay staining and quantification

JCPyV, SV40, BKPyV, reovirus:

Infection plates were fixed with 4% PFA and washed with 0.1% PBS-Tween (PBS-T) 3x for 5 min each. Cells were permeabilized with 1% Triton X-100 (TX-100) in PBS at RT for 15 min and blocked with 10% goat serum in PBS at RT for 1 h. Primary antibody against JCPyV TAg (PAB962, 1:5), SV40/BKPyV VP1 (PAB597, 1:40), or reovirus (reovirus antisera, 1:500) in PBS were added to wells at RT for 1 h. Wells were washed 3x with PBS-T for 5 min each, then incubated with secondary polyclonal goat anti-mouse (JCPyV, SV40, BKPyV) or goat anti-rabbit (reovirus) Alexa Fluor 594 or 488 antibody (1:1000) in PBS at RT for 1 h. Cells were again washed 3x with PBS-T for 5 min and DAPI (1:1000) in PBS was added at RT for 5 min for visualization of cell nuclei. Plates were washed with PBS-T and PBS was added for storage. Infected cells were visualized by Nikon Eclipse Ti epifluorescence microscope (Micro Video Instruments, Inc.) and percent infection was quantified by dividing the number of TAg- (JCPyV), VP1- (SV40 and BKPyV), or reovirus-positive cells per 10X (reovirus) or 20X (JCPyV, SV40, BKPyV) visual field by the total number of DAPI-positive cells, then multiplying by 100. This was repeated for 5 fields of view (FOV) per well. TAg- or VP1-positive cells were counted manually, while DAPI-positive cells were counted using a binary algorithm in the Nikon NIS-Elements Basic Research software. Cells were separated in the binary algorithm by intensity, diameter, and circularity to achieve an accurate count of the total number of DAPI-positive cells in each FOV.

SARS-CoV-2:

Cells were fixed with 4% PFA, washed with PBS-T, and permeabilized with 1% TX-100. Primary antibody against SARS-CoV-2 NP in PBS (1:500) was added at RT for 1 h. Cells were washed 3x with PBS-T for 5 min, then secondary polyclonal goat anti-mouse Alexa Fluor 488 antibody (1:1000) in PBS was added at RT for 1 h. Wells were washed with PBS-T, and DAPI (1:1000) in PBS was added at RT for 5 min. Plates were washed with PBS-T, PBS was added for storage, and infected cells were visualized using a Nikon Eclipse Ti epifluorescence microscope. Infection was quantified by dividing the number of NP-positive cells per 20X visual field by the total number of DAPI-positive cells for 5 FOV per well and multiplying by 100.

2.11. Inhibitor time of addition assay

HEK-2C cells were plated to 70% confluence in 96-well plates overnight. Cells were either pre-treated (−1 h), treated at the time of infection (0 h), or added back with inhibitor post infection (4, 6, 12, and 24 hpi). For all treatments the inhibitor was diluted in 10% DMEM at 37°C and 100 μl/well was added or 10% DMEM alone was added. Unless indicated, infections were performed in the absence of inhibitor. At 48 hpi cells were fixed with 4% PFA, stained for JCPyV TAg (1:5), and analyzed by ICW assay.

2.12. Flow cytometry

HEK-2C cells were plated to 100% confluence in 12-well plates. Cells were treated with DMSO or TFP at 37°C for 1 h. Plates were washed with 1X PBS and incubated in Cellstripper (Corning) at 37°C for 15 min to remove cells from plate. Cells were pelleted at 2,000 rpm at 4°C for 5 min and washed with 1X PBS. Alexa Fluor 647-labeled JCPyV (JCPyV-647) in DMEM (without phenol red) was added to cells on ice for 1 h with agitation every 15 min (100 μl total volume). Cells were pelleted by centrifugation and fixed in 4% PFA for 10 min on ice, then resuspended in 300 μl of 1X PBS. Analysis was performed by flow cytometry for viral attachment using a LSRII system (BD Biosciences) equipped with a 640 nm AP-C laser line for at least 10,000 events. Data analyses were performed using BD FACSDiva and FlowJo software. Gating was performed to exclude complex and dead cells using FlowJo software.

2.13. Infectious clone

JC pUC19 and pUC19 plasmids were extracted from glycerol stocks using a HiSpeed Plasmid Maxi Kit (Qiagen) and digested with BamHI-HF (New England Biolabs) at 37°C for 2 h. Successful linearization of plasmids was determined by agarose gel electrophoresis.

SVGA cells were plated to 50% confluence in 12-well plates, then pre-treated with TFP or DMSO for 1 h at 37°C. Cells were then transfected with 2 μg/well of DNA containing the linearized plasmids of either JC pUC19 or pUC19 using Fugene 6 at a ratio of 1.5 μl Fugene: 1 μg DNA and incubated at 37°C. After 6 h, media was replaced with 10% MEM and allowed to incubate for 3 or 7 days, when cells were fixed and stained for newly synthesized VP1 (1:40) via FFU infectivity assay.

2.14. Confocal microscopy and image analysis

HEK-2C cells were plated to 70% confluence in number 1.5 96-well glass bottom plates (CellVis). Cells were pre-treated with TFP or DMSO in 10% DMEM at 37°C for 1 h then shifted to 4°C for 45 min to pre-chill. JCPyV-647 (MOI = 4 FFU/cell) was added at 4°C for 1 h to allow for synchronized viral attachment, cells were fed with pre-warmed media containing TFP or DMSO, then plates were shifted to 37°C for 2 h for viral internalization. Cells were fixed with 4% PFA, washed with 1X PBS, and stained with DAPI (1:1000). PBS was added to wells for storage. A Leica SP8 microscope was utilized for sample visualization at 63X magnification (oil immersion) using LAS X software. Images were acquired using diode 405 and white light lasers and cross sections of individual cells were analyzed (at least 30 cells per sample). ImageJ software was used to define regions of interest (ROIs) by using the polygon selection tool and 5-HT2CR channel, excluding the plasma membrane (Mehmood, Wilczek et al. 2022). Relative internalized virus was measured by relative fluorescence units per cell for background-corrected samples. Each experiment was performed 3 times, with graphs representing 3 independent replicates (90 cells per treatment).

2.15. Statistical analyses

Drug screen:

The drug screen was performed with three independent replicates. Statistical significance (z-score) of the effect of each drug on JCPyV infectivity was scored using z = (x − μ) / σ, where x represents the inhibitory effect of a given drug, μ represents the average inhibitory effect of all drugs within a single cell culture plate, and σ represents the standard deviation of the negative control for that plate. Each z-score was compared to a “hit” threshold that required it be lower than the negative value of the number of drugs on the same plate multiplied by the standard deviation of the inhibitory effect of those drugs. Z-scores were normalized to the z-score threshold for the plate containing any particular drug.

Student’s t-test:

Using Microsoft Excel, two-sample Student’s t tests were performed to determine statistical significance, assuming unequal variance, by comparing mean values of triplicate samples.

Standard error of the mean (SEM):

Using Microsoft Excel, SEM was calculated to determine variation in a given population and was done so by calculating standard deviation and dividing by the square root of the sample size.

3. Results

3.1. NCC drug screen

There are currently no approved targeted therapies for JCPyV infection and PML, and the cellular factors required for JCPyV infection remain poorly understood. In order to identify potential therapeutics, a large-scale drug screen was performed using the NCC. The NCC contains over 700 drugs that have been tested in clinical trials, with many of them FDA approved (Mainou, Zamora et al. 2013). To perform the drug screen, SVGA cells were pre-treated with inhibitors for 1 h, infected with JCPyV (MOI = 0.5 FFU/cell), incubated for 72 h, then fixed and stained for VP1 expression and analyzed using a high-throughput ICW assay (Fig. 1A) (DuShane, Wilczek et al. 2019). Results demonstrated that 42 drugs from various drug classes, including receptor agonists and antagonists (36%), calcium signaling-related drugs (19%), enzyme inhibitors (19%), steroids (7%), antifungals (5%), antibiotics (5%), xanthine derivatives (5%), lignans (2%), and flavonoids (2%) were capable of reducing JCPyV infection in SVGA cells (Fig. 1B) (Supplemental Table 1). The largest drug hit category represented was receptor agonists and antagonists, and the majority of the hits target GPCRs, which was of interest given the role of serotonin receptors in JCPyV entry and GPCR-related signaling in JCPyV infection (DuShane, Wilczek et al. 2018, Mayberry, Wilczek et al. 2021). Calcium signaling-related drugs were the second largest category in the drug screen (Fig. 1C), revealing a novel area of inquiry for JCPyV research that also corroborated preliminary data gathered in our lab suggesting that a calmodulin inhibitor reduced JCPyV infection. Furthermore, a recent study had demonstrated that SV40 and MCPyV infections are reliant on calcium channel activity (Dobson, Mankouri and Whitehouse 2020), adding polyomaviruses to a large group of viruses that modulate calcium signaling during infection. Additionally, calcium is known to be required for PyV capsid stability (Haynes, Chang and Consigli 1993), providing further rationale to explore calcium-related hits. Thus, subsequent validation studies focused on calcium-related drugs.

Fig. 1. NIH-CC drug screen reveals several calcium-related drug hits.

Fig. 1.

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(A) SVGA cells were pre-treated with each of >700 inhibitors [10 μM] from the NIH-CC, infected with JCPyV (MOI = 0.5 FFU/cell) at 37°C for 1 h, fed with MEM, then incubated at 37°C for 72 h. Cells were fixed and stained for VP1 and analyzed via ICW. Each drug replicate from the screen is represented by one dot, with calcium-related drugs shown in blue. The statistical significance (z-score) was calculated and normalized to the z-score threshold, represented by the black solid line. Drugs that reduced infection below this threshold were considered hits. (B) Hits from the screen were grouped into 9 categories. (C) Table displaying all calcium-related drug hits from the screen.

3.2. ICW screen of calcium-related drugs hits

To further validate hits from the drug screen, ICW assays were performed in triplicate for three replicates for selected drug hits nimodipine, nifedipine, and TFP and other related calcium and calmodulin inhibitors. Hits chosen represented the major inhibitor classes represented and were further explored in both glial (SVGA) and kidney (HEK-2C) cells, which are targets of JCPyV infection (Fig. 2). Results showed that when SVGA and HEK-2C cells were treated with nimodipine, an L-type Ca2+ channel inhibitor, JCPyV infection was modestly reduced (Fig. 2A). A significant reduction in JCPyV infection was observed in HEK-2C cells when treated with nifedipine, another L-type Ca2+ channel inhibitor, but not in SVGA cells (Fig. 2B). Treatment of SVGA cells with tetrandrine, a NAADP-sensitive TPC inhibitor not included in the screen that blocks MCPyV and SV40 PyV infection (Dobson, Mankouri and Whitehouse 2020), resulted in a slight, but not statistically significant, increase in JCPyV infection (Fig. 2C). However, a significant reduction in infection in HEK-2C cells was observed upon treatment with tetrandrine (Fig. 2C). No significant change in infection was found when SVGA or HEK-2C cells were treated with flunarizine, a T-type Ca2+ channel inhibitor not included in drug screen that blocks infection of MCPyV but not SV40 (Dobson, Mankouri and Whitehouse 2020) (Fig. 2D). A significant reduction in JCPyV infection was observed when SVGA or HEK-2C cells were treated with trifluoperazine (TFP), a calmodulin inhibitor (Fig. 2E). A second calmodulin inhibitor, W-7, that was not represented in the drug screen, was tested for the capacity to reduce JCPyV infection to further support a role for calmodulin during JCPyV infection. Treatment with W-7 resulted in decreased JCPyV infection in both SVGA and HEK-2C cells (Fig. 2F). Other hits from the drug screen, dantrolene sodium and topiramate, did not significantly reduce infection (data not shown). Cytotoxicity profiles for drugs that significantly reduce infection (>50%) and were further characterized are shown in Table 1.

Fig. 2. Calcium channel and signaling inhibitors reduce JCPyV infection in glial and kidney cell lines.

Fig. 2.

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SVGA or HEK-2C cells were pre-treated at 37°C for 1 h with each drug at concentrations listed in figures. Cells were infected with JCPyV (MOI = 1 FFU/cell) at 37°C for 1 h in absence of inhibitor, then 100 μl/well of cell media containing inhibitor was added back and plates were incubated at 37°C. At 48 h, cells were fixed and stained for viral TAg and CellTag, and analyzed by ICW using a LI-COR Odyssey CLx. Determination of % infection was calculated by subtracting background from the 800 nm channel (virus channel), then dividing the 800 nm signal from each well by its respective 700 nm signal (Cell Tag) and normalizing the control values to 100%. Graphs represent 3 replicates performed in triplicate. Error bars represent SEM. Student’s t-test was used to determine statistical significance. *, P < 0.05.

Table 1.

Cell viability at various concentrations and timepoints post-inhibitor treatment. Concentrations that demonstrated < 80% viability were not included in further validation studies.

% Viability
Inhibitor Cell Type & Timepoint ≥ 100% ≥ 95% ≥ 90% ≥ 85% ≥ 80% < 80%
Tetrandrine SVGA 48 h 10 μM - - - - -
HEK-2C 48 h - 1 μM 7 μM - - 10 μM
Nifedipine SVGA 48 h - - - 75 μM 150 μM 200 μM
HEK-2C 48 h 50 μM 100 μM - - - -
Vero 48 h 20 μM 75 μM - 100 μM - -
RPTEC 48 h - - - 50 μM 100 μM -
W-7 SVGA 48 h 10 μM - 15 μM - 30 μM 50 μM
HEK-2C 48 h 30 μM - - 60 μM - 100 μM
Vero 24 h - 30 μM - 60 μM - -
Vero 48 h 45 μM - - - - -
Vero 72 h 45 μM - - 60 μM - -
TFP SVGA 7 days 15 μM - - - - -
SVGA 48 h - - 20 μM - - 30 μM
HEK-2C 48 h 7.5 μM - - 20 μM - 30 μM
Vero 24 h 10 μM - - - - -
Vero 48 h 20 μM - - - - -
Vero 72 h > 15 μM - - - - 30 μM
RPTEC 48 h 4 - 5 μM 7.5 μM - - -
HeLa 24 h 7.5 μM - - - 10 μM 15 μM
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3.3. FFU validation of ICW results

In order to verify hits that significantly reduced infection measured by ICW, focus-forming unit (FFU) assays were conducted. Infections were performed using the same experimental conditions as in ICW, but analyses differed in that cells were stained by indirect immunofluorescence and quantified by epifluorescence microscopy to identify characteristic markers of PyV infection. Results from FFU experiments demonstrated similar results observed in ICW experiments, further validating the ICW assay as an effective tool to quantify JCPyV infection (Fig. 3) (DuShane, Wilczek et al. 2019). Nifedipine did not reduce JCPyV infection in SVGA cells, but significantly reduced JCPyV infection in HEK-2C cells (Fig. 3A). Additionally, nifedipine did not reduce SV40 infection (Fig. 3B). Similar trends were seen upon treatment with tetrandrine, where JCPyV infection was not impacted in SVGA cells, but was significantly impaired in HEK-2C cells (Fig. 3C). Further, tetrandrine potently inhibited SV40 infection, confirming previous findings (Dobson, Mankouri and Whitehouse 2020) (Fig. 3D). Nimodipine and nitrendipine, both inhibitors of L-type Ca2+ channels, did not significantly reduce JCPyV infection in SVGA cells (Fig. 3E and 3F). Interestingly, nifedipine and tetrandrine have been reported to alter MAPK signaling and ERK phosphorylation in a cell type-dependent manner (Jia, Xu et al. 2013, Ma, Zhang et al. 2017, Zhao, Guo et al. 2017). Given the necessary role of MAPK activation and ERK phosphorylation in JCPyV infection (DuShane, Wilczek et al. 2018), pERK levels were measured following cellular treatment with nifedipine and tetrandrine. SVGA and HEK-2C cells treated with nifedipine demonstrated a significant reduction in pERK levels, which correlates with reduced infection (Fig. 3G). Interestingly, while HEK-2C cells treated with tetrandrine also showed a reduction in pERK levels, SVGA cells treated with tetrandrine showed a dramatic increase in pERK (Fig. 3H), and these findings are consistent with outcomes of infection (Fig. 3A–D).

Fig. 3. Tetrandrine and nifedipine exhibit cell type-dependent differences in JCPyV inhibition.

Fig. 3.

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SVGA, HEK-2C, or Vero cells were pre-treated with each inhibitor at 37°C for 1 h. JCPyV (A, C, E, F) (SVGA: MOI = 1 FFU/cell; HEK-2C: MOI = 0.5 FFU/cell) or SV40 (B and D) (MOI = 1 FFU/cell) were then added for a 1 h infection at 37°C in absence of inhibitor. Cells were fed with 100 μl/well of cell media containing inhibitor, then plates were incubated at 37°C for 48 h. PFA was added for fixation, then cells were stained for DAPI and (A, C, E, F) TAg or (B and D) VP1. Infections were quantified by FFU assay with 5 FOV/well counted. Determination of % infection was calculated by dividing the number of infected cells/ the number of DAPI+ cells in each 20X visual field and normalizing to 100%. (G and H) SVGA or HEK-2C cells were treated with (G) nifedipine or (H) tetrandrine at 37°C for 24 h. Cells were fixed with PFA and stained for pERK and CellTag and analyzed by ICW using a LI-COR Odyssey CLx. Determination of % pERK expression was calculated by subtracting background from the 800 nm channel (pERK channel), then dividing the 800 nm signal from each well by its respective 700 nm signal (CellTag) and normalizing the control values to 100%. Graphs represent 3 replicates performed in triplicate. Error bars represent SEM. Student’s t-test was used to determine statistical significance. *, P < 0.05.

3.4. Calmodulin inhibitors reduce JCPyV infectivity

One hit from the drug screen included the calmodulin inhibitor trifluoperazine (TFP). In order to validate this hit from the screen, cells were treated with TFP, infected with JCPyV, and assessed for infection. In addition to TFP, another calmodulin inhibitor, W-7, was evaluated to test the role of calmodulin during JCPyV infection. TFP blocks calmodulin activity by binding directly to calmodulin and inducing a conformational change, while W-7 binds to each of two calmodulin domains to block activity (Vandonselaar, Hickie et al. 1994, Osawa, Swindells et al. 1998). Results showed that treatment of SVGA and HEK-2C cells with calmodulin inhibitors TFP and W-7 resulted in significant reduction of JCPyV infection, suggesting a role for calmodulin during JCPyV infection (Fig. 4A–D).

Fig. 4. Calmodulin inhibitors decrease JCPyV infection.

Fig. 4.

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TFP or W-7 were added to SVGA or HEK-2C cells at 37°C for 1 h for pre-treatment. Cells were then challenged with JCPyV (SVGA: MOI = 1 FFU/cell; HEK-2C: MOI = 0.5 FFU/cell) at 37°C for 1 h in absence of inhibitor. Infections were fed with 100 μl/well of cell media containing inhibitor, and plates were incubated at 37°C for 48 h. Cells were fixed in 4% PFA, then stained for JCPyV TAg and DAPI. Infections were quantified by FFU assay with 5 FOV/well counted. Determination of % infection was calculated by dividing the number of infected cells/ the number of DAPI+ cells in each 20X visual field and normalizing to 100%. Graphs represent 3 replicates performed in triplicate. Error bars represent SEM. Student’s t-test was used to determine statistical significance. *, P < 0.05.

3.5. Calmodulin inhibitors reduce SV40, BKPyV, and SARS-CoV-2 infections

Due to the consistent reduction in JCPyV infection upon treatment with calmodulin inhibitors, it was questioned whether calmodulin may be a key factor during other polyomavirus infections and non-polyomavirus infections. Vero cells were treated with either TFP or W-7, challenged with either SV40 or BKPyV, and assessed for infection. Both SV40 and BKPyV infections were significantly reduced (Fig. 5A–D). These results suggest a potential role for calmodulin in the polyomavirus family of viruses. In addition to polyomaviruses, TFP and W-7 were also tested for their effects on the ssRNA virus SARS-CoV-2, as this is a prominent virus of concern and calmodulin is known to interact with the angiotensin-converting enzyme-2 (ACE2) receptor – the primary receptor involved in SARS-CoV-2 entry (Lambert, Clarke et al. 2008). Since SARS-CoV-2 has been shown to be inhibited by TFP, it was hypothesized that TFP and W-7 would reduce SARS-CoV-2 infection (Yuan, Dong et al. 2022). Treatment with both TFP and W-7 resulted in dramatically impaired SARS-CoV-2 infection, indicating the importance of calmodulin during SARS-CoV-2 infection and suggesting TFP as a potential broad antiviral (Fig. 5E and 5F). Importantly, it was previously reported that reovirus, a dsRNA virus, was not impacted by treatment with TFP (Mainou, Zamora et al. 2013). As a negative control, HeLa cells were treated with TFP and infected with reovirus. Reovirus infection was not impacted by treatment with TFP, indicating that TFP and W7 demonstrate specificity for inhibition of certain viruses (Fig. 5G).

Fig. 5. TFP and W-7 broadly reduce polyomavirus and coronavirus infections.

Fig. 5.

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(A-F) Vero or (G) HeLa cells were pre-treated with (A and B, E and G) TFP or (C and D, F) W-7 at 37°C for 1 h. (A and C) Cells were infected with SV40 (MOI = 1 FFU/cell) at 37°C for 1 h, then fed with 100 μl/well of cell media containing inhibitor and plates were incubated at 37°C for 48 h. (B and D) Cells were infected with BKPyV (MOI = 4 FFU/cell) at 37°C for 2h, then fed with 100 μl/well of cell media containing inhibitor and plates were incubated at 37°C for 72 h. (E and F) Cells were infected with SARS-CoV-2 (TCID50 = 0.025/cell) at 37°C for 1 h, then fed with 100 μl/well of cell media containing inhibitor and plates were incubated at 37°C for 24 h. (G) Cells were infected with reovirus (MOI = 300 FFU/cell) at RT for 1 h, then fed with 100 μl/well of cell media containing inhibitor and plates were incubated at 37°C for 24 h. Cells were fixed with 4% PFA and stained for viral protein, then quantified by FFU assay with 5 FOV/well counted. Determination of % infection was calculated by dividing the number of infected cells/the number of DAPI+ cells per visual field and normalizing to 100%. Samples were analyzed under the following magnifications: 10X (SV40, reovirus, and BKPyV); 20X (SARS-CoV-2). Graphs represent 3 replicates performed in triplicate. Error bars represent SEM. Student’s t-test was used to determine statistical significance. *, P < 0.05.

3.6. JCPyV infection of primary kidney cells

The lack of a tractable animal model for JCPyV infection reduces opportunities to test inhibitors, yet primary cell lines represent an innovative model that more accurately represents the cells in the human host. Renal proximal tubule cells (RPTECs), a primary kidney cell type, were treated with TFP or nifedipine and infected with JCPyV. TFP and nifedipine significantly reduced JCPyV infection in RPTECs (Fig. 6A and 6B). Taken together, these results indicate that the impacts of TFP and nifedipine on JCPyV infection in immortalized vs primary kidney cells are comparable.

Fig. 6. JCPyV infection is significantly impaired in primary RPTECs.

Fig. 6.

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Cells were pre-treated with (A) TFP or (B) nifedipine at 37°C for 1 h. JCPyV (MOI = 2 FFU/cell) was added in absence of inhibitor at 37°C for 1 h, then cells were fed with 100 μl/well of cell media containing inhibitor. Plates were incubated at 37°C for 48 h, fixed with PFA, then stained for JCPyV TAg. Infected cells were quantified by FFU assay with 5 FOV/well counted. Determination of % infection was calculated by dividing the number of infected cells/ the number of DAPI+ cells per 10X visual field and normalizing to 100%. Graphs represent 3 replicates performed in triplicate. Error bars represent SEM. Student’s t-test was used to determine statistical significance. *, P < 0.05.

3.7. Time of addition, attachment, entry, and trafficking of JCPyV during TFP treatment

To understand the step in the JCPyV infectious cycle that is inhibited by TFP, a time of addition assay was performed in HEK-2C cells. Results showed the most significant reduction in JCPyV infection when TFP was added at the time of virus addition, as well as up to 4 hours-post infection (hpi), and no inhibition was observed at times after 6 hpi (Fig. 7A). These time points are consistent with viral attachment, entry, and trafficking, so these steps were further investigated (Nelson, Derdowski et al. 2012, Mayberry, Soucy et al. 2019). Flow cytometry was performed to determine whether TFP was impacting JCPyV attachment. HEK-2C cells were incubated with either DMSO (control) or TFP, then infected with JCPyV-647 in suspension. Results showed no significant difference in JCPyV attachment when cells were treated with TFP, indicating that TFP is not acting upon JCPyV infection during attachment (Fig. 7B). To determine whether TFP was affecting viral internalization or trafficking, confocal microscopy was performed to assess internalization of Alexa 647-labeled JCPyV in HEK-2C cells treated with TFP compared to the DMSO control. Viral internalization was significantly impaired in cells treated with TFP, suggesting that TFP is impacting JCPyV entry (Fig. 7C). To assess viral trafficking, cells were treated with TFP and transfected with the infectious clone of JCPyV, which bypasses viral entry and trafficking. Results showed that there was a modest reduction in JCPyV VP1+ cells, indicating a potential role for TFP in a post-entry step in JCPyV infection (Fig. 7E), yet the greatest reduction in infection was observed during viral entry.

Fig. 7. TFP reduces JCPyV entry in kidney cells.

Fig. 7.

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(A) TFP [7.5 μM] was added to HEK-2C cells 1 h prior to infection with JCPyV (MOI = 1 FFU/cell), simultaneously with JCPyV (t = 0 h), or at times post-infection indicated in figure. At 48 h, cells were fixed and stained for JCPyV TAg and imaged on a LI-COR Odyssey CLx. Determination of % infection was calculated by subtracting background from the 800 nm channel (virus channel), then dividing the 800 nm signal from each well by its respective 700 nm signal (CellTag) and normalizing the control values to 100%. Graphs represent 3 replicates performed in triplicate. Error bars represent SEM. (B) HEK-2C cells were pre-treated with TFP at 37°C for 1 h, infected with JCPyV-647 on ice for 1 h, then fixed with PFA. Samples were analyzed by flow cytometry using a BD LSR II flow cytometer equipped with an APC laser line (640 nm). Data was analyzed using FACSDiva and FlowJo software, with gating performed to exclude complex or dead cells. Data is representative of 3 independent experiments with at least 5,000 events per sample. (C) HEK-2C cells were pre-treated with TFP [7.5 μM] at 37°C for 1 h, pre-chilled to 4°C for 45 mins, infected with JCPyV-647 at 4°C for 1h to allow for synchronized viral attachment, then shifted to 37°C for viral internalization. After 2 h, cells were fixed with PFA and stained with DAPI. Samples were imaged on a Leica SP8 confocal microscope at 63X magnification. ImageJ software was used to draw ROIs around the perimeter of each cell, excluding the plasma membrane, to collect mean intensity values for internalized virus. Graph represents 3 replicates with at least 30 cells analyzed per sample per replicate. (D) Representative images from internalization assay with nuclei (cyan), JCPyV-647 (magenta), and 5HT2CR (yellow). Scale bars = 20 μm. (E) SVGA cells were pre-treated with TFP [15 μM] at 37°C for 1 h, transfected with the JCPyV infectious clone in the presence of TFP, then media was replaced after 6 h. At day 3 or day 7, cells were fixed and stained for JCPyV VP1 and analyzed for % VP1+ nuclei. VP1+ nuclei were quantified by FFU assay with 5 FOV/well counted at 10X magnification. Determination of % VP1+ nuclei was calculated by dividing the number of VP1+ cells/ the number of DAPI+ cells per visual field and normalizing to 100%. Graphs represent duplicate experiments where each duplicate contained three individual plasmid digestions and transfections per treatment condition. Samples were normalized to the average of control-treated samples (100%). Error bars represent SEM. Student’s t-test was used to determine statistical significance. *, P < 0.05.

4. Discussion

In this report, we reveal findings from a large-scale drug screen performed in an attempt to discover potential antivirals for JCPyV infection and describe further characterization of calcium-related drug hits. Results from the drug screen identified 42 hits, with the most substantial drug categories being receptor agonists/antagonists, enzyme inhibitors, and calcium-related drugs. This study focused on the calcium-related drugs and found that TFP, W-7, nifedipine, and tetrandrine are capable of decreasing JCPyV infectivity. Nifedipine and tetrandrine, both calcium channel blockers, exhibited cell type-dependent inhibition of infection, which was partially explained by changes in pERK upon treatment of cells with the inhibitors. Interestingly, TFP, an inhibitor of calmodulin and currently FDA-approved treatment for schizophrenia, may serve as a broad antiviral treatment for polyomavirus infections as well as coronavirus infections. Upon further investigation using time course experiments and targeted analysis of the infectious cycle, it was determined that TFP was impeding viral entry. Taken together, calcium-related drugs inhibited JCPyV infection, with calmodulin inhibitors broadly impacting polyomavirus and coronavirus infections.

Performing a high-throughput NCC drug screen to identify potential antivirals for JCPyV infection led to the discovery that calcium signaling-related inhibitors are capable of reducing JCPyV infectivity. Findings reported by Dobson et al. (Dobson, Mankouri and Whitehouse 2020) that implicated host-cell calcium signaling during MCPyV and SV40 infections further contributed to the rationale to pursue these hits. Dobson et al. demonstrated that tetrandrine, an inhibitor of NAADP-sensitive TPCs, drastically reduced both MCPyV and SV40 infections, and the inhibition specifically impacted endosomal fusion with the endoplasmic reticulum. Although tetrandrine is an inhibitor of TPCs, tetrandrine has also been shown to inhibit calmodulin and act broadly as a calcium channel inhibitor (Dittmar, Lee et al. 2021). Our findings showed that flunarizine, a T-type Ca2+ channel blocker, did not reduce JCPyV infection, yet, interestingly, Dobson et. al found that flunarizine significantly impaired MCPyV, but not SV40 infection. Nifedipine, an inhibitor of L-type Ca2+ channels, reduced JCPyV infection in HEK-2C cells but not SVGA cells. Because nifedipine also reduced pERK, this suggests either an off-target effect or possibly that reducing calcium signaling through L-type Ca2+ channels is impacting the MAPK pathway and therefore reducing JCPyV infection.

Treatment of cells with tetrandrine, the NAADP-sensitive TPC inhibitor, resulted in a cell-type dependent difference in outcomes for JCPyV infection. In SVGA cells, an immortalized glial cell line, tetrandrine slightly increased JCPyV infection. However, in HEK-2C cells, an immortalized kidney cell line, tetrandrine significantly reduced JCPyV infectivity. Interestingly, tetrandrine also reduced SV40 infection in Vero cells, an immortalized African green monkey kidney cell line, indicating a potential tissue-specific mechanism of reduction of polyomavirus infection by tetrandrine in kidney cell types. One possible explanation for this cell-type dependent difference in infection during tetrandrine treatment is a difference in signaling pathway regulation during infection. To delve deeper into this interesting finding, both cell types were treated with tetrandrine in absence of JCPyV and levels of pERK were measured. After 24 hours of treatment with tetrandrine, levels of pERK were significantly increased in SVGA cells, but significantly reduced in HEK-2C cells (Fig. 3H). This difference in relative levels of pERK, a key signaling protein known to be important for JCPyV infection, could help explain the cell-type dependent differences in infection. Of note, HEK-2C cells overexpress the 5-HT2Rs, which contain calmodulin-binding domains (Turner and Raymond 2005, Labasque, Reiter et al. 2008). An additional explanation for these cell-type dependent differences in JCPyV infection may be due to differences in permissivity of SVGAs and HEK-2Cs. SVGAs are transformed with SV40 TAg, rendering them more permissive for JCPyV infection (Major, Miller et al. 1985). HEK-2Cs are poorly permissive for JCPyV infection prior to overexpressing the 5-HT2C receptor (Assetta, Maginnis et al. 2013). Therefore, greater inhibition may be observed in the HEK-2C cell model due to reduced permissivity in this cell type. Although Ca2+ signaling and channel inhibitors seem promising, not all Ca2+ inhibitors proved effective in reducing viral infection. Flunarizine, a T-type Ca2+ channel blocker, did not reduce JCPyV infection, suggesting that JCPyV infection is independent of T-type Ca2+ channel activity. L-type Ca2+ channel inhibitors nimodipine and nitrendipine did not have a major impact on JCPyV infection, but nifedipine significantly reduced JCPyV infection in HEK-2C cells. These results combined with the pERK nifedipine experiments (Fig. 3G) indicate that L-type Ca2+ channels may not be essential for JCPyV infection but rather impact ERK activation. Analysis of other drugs used in this study for pERK activation did not demonstrate a significant difference in pERK levels compared to controls (data not shown).

Inhibitors of calmodulin, TFP and W-7, showed a consistent decrease in JCPyV infection, indicating that calmodulin may play a key role during JCPyV infection. TFP also significantly reduced SV40, BKPyV, and SARS-CoV-2 infections, but not reovirus infection. To define how TFP was impacting the JCPyV infectious cycle, the inhibitor was added over a time course and found to reduce infection the most at 0–4 h, times consistent with viral attachment, entry, and trafficking (Fig. 7A). Further analysis of viral entry by confocal microscopy revealed that TFP significantly reduced viral internalization (Fig. 7C). To confirm our finding that viral entry (and not earlier events) is affected by TFP, viral attachment was assessed by flow cytometry and data showed that attachment was unaffected (Fig. 7B). Points post-entry were also investigated using the viral infectious clone, which bypasses entry and trafficking, and a modest reduction in VP1+ cells was observed. These data suggest that in addition to blocking viral internalization, TFP could also impact post-entry steps such as viral transcription (Fig. 7E). For instance, TFP could impact other components of calcium-signaling pathways such as calcineurin, which is activated by Ca2+ and calmodulin binding and then induces activation of NFAT4, a transcription factor required for JCPyV infection (Manley, O’Hara B et al. 2006). However, the time course experiments demonstrate that while TFP induces a slight decrease at later times in the infectious cycle (6, 12, and 24 hpi), the greatest impact is consistent with the timing of viral entry or trafficking to proper cellular compartments (Fig. 7A). Only the JCPyV infectious cycle was analyzed, and thus TFP could be blocking other steps during BKPyV, SV40, and SARS-CoV-2 infections. Some commonalities between polyomaviruses and coronaviruses during infection include the requirement to enter cells by clathrin-mediated endocytosis and trafficking in the endosomal pathway (Jackson, Farzan et al. 2022), yet the later steps in the viral life cycle differ significantly for the dsDNA polyomaviruses compared to the ssRNA coronaviruses. Thus, the mechanisms of inhibition for other viruses explored in this study require more investigation.

RPTECs, a primary kidney cell line, were used to better recapitulate JCPyV infection in human cells in absence of potentially altered signaling pathways due to immortalization of cells. TFP, an inhibitor of calmodulin, showed great potential to reduce JCPyV, SV40, BKPyV, and SARS-CoV-2 infections in immortalized cell lines. When RPTECs were treated with TFP and challenged with JCPyV, a significant reduction in infection was observed (Fig. 6A). Because nifedipine also showed a significant reduction in JCPyV infection in HEK-2C cells, nifedipine was also tested for the capacity to reduce JCPyV infection in primary RPTECs. A significant decrease in JCPyV infection upon treatment with nifedipine was also noted (Fig. 6B). These findings are important as the kidney is a major target of JCPyV infection. TFP is already an FDA-approved drug and is capable of crossing the blood-brain barrier, so there is a potential for repurposing TFP for treatment of JCPyV infection for PML (Kang, Hong et al. 2017). Although primary cells are more similar to in vivo human infection than immortalized cells, they cannot fully recapitulate the in vivo environment. For this reason, in vivo studies should be performed to test efficacy of TFP on polyomavirus infections in a whole-body system. TFP may serve as one potential treatment for polyomavirus infections, but other possible inhibitors remain to be discovered.

TFP is currently an FDA-approved drug, and thus repurposing TFP as an antiviral would be an efficient means of drug discovery. This method of repurposing FDA-approved drugs is a well-demonstrated tactic and was utilized for the discovery of remdesivir for treatment of SARS-CoV-2 (Lemaitre, Guetard et al. 1990, Murugan, Aruna et al. 2016, Wang, Zhang et al. 2020). Additionally, large-scale screens such as the NCC have led to the identification of other potential anti-coronavirus drugs (Cao, Forrest and Zhang 2015). Given that TFP was demonstrated to inhibit polyomavirus and coronavirus infections and that TFP has previously been shown to inhibit dengue, Zika, hepatitis C virus, influenza, measles, Epstein-Barr, arenaviruses, and coronavirus infections, it may serve as a broad-spectrum inhibitor (Bohn, Rutter et al. 1983, Nemerow and Cooper 1984, Ochiai, Kurokawa and Niwayama 1991, Candurra, Maskin and Damonte 1996, Chockalingam, Simeon et al. 2010, Xiao, Wang et al. 2020, Piccini, Castilla and Damonte 2022). However, infection with reovirus, a dsRNA virus, was not impacted by treatment with TFP, hinting at a possible shared mechanism between multiple virus families, excluding Reoviridae. Many viruses are known to utilize calcium signaling pathways during infection, including HIV, influenza, and SARS-CoV-2, demonstrating a commonality between several virus families. Although polyomaviruses have been shown to rely on host cell calcium signaling for successful infection, the specific roles of calcium signaling during polyomavirus infection are still not well-understood and warrant further exploration.

Conclusions

A screen for inhibitors of JCPyV infection using the NCC led to the identification of 42 hits with antiviral activity against JCPyV. Calcium signaling-related inhibitors comprised a large portion of the hits from the screen. Upon further characterization, various calcium inhibitors reduced JCPyV infection in immortalized brain and immortalized and primary kidney cells. Of these, calmodulin inhibitors showed the greatest capacity to impact infection of JCPyV as well as SV40, BKPyV, and SARS-CoV-2, suggesting that calmodulin inhibitors may serve as broad-spectrum antiviral therapeutics. Further analysis of the JCPyV infectious cycle showed that calmodulin inhibitor TFP disrupted infection at the point of viral internalization. Taken together, this work highlights the utility of repurposing drugs as antivirals and demonstrates that drug screens can effectively identify broad-spectrum antiviral treatments due to conservation in viral interactions with cellular targets.

Supplementary Material

1

Highlights.

  • NCC drug screen reveals several targets for JCPyV infection.

  • JCPyV infection is reduced by calcium signaling-related drugs.

  • Calcium and calmodulin inhibitors have different effects in kidney and brain cells.

  • FDA-approved calmodulin inhibitor trifluoperazine blocks JCPyV entry.

  • Trifluoperazine may serve as a broad spectrum antiviral.

Acknowledgements

We thank members of the Maginnis laboratory for thoughtful discussion and contributions and Sophie Craig for careful review of the manuscript. We also thank Pranav Danthi, Bernardo Mainou, Walter Atwood, Andy Holmes, and Clarissa Henry for providing critical reagents and access to resources for this study. Research reported in this manuscript was supported by the National Institute of Allergy and Infectious Diseases [grant number R15AI144686] (MM) of the National Institutes of Health and by the Maine IDeA Network of Biomedical Research Excellence (INBRE) and the Centers of Biomedical Research Excellence (COBRE) through the National Institute of General Medical Sciences [grant numbers P20GM103423 and P20GM144265] (MM). Critical equipment resources were provided by the UMaine Institute of Medicine and NSF MRI 1726541. This work was also financially supported in part by funding from the University of Maine: Chase Distinguished Research Award (AB), Graduate Student Government Degree-Related Grants (AB), Maine Space Grant Consortium (MC), Institute of Medicine Summer Fellowship (LB), Center for Undergraduate Research Fellowship (AS), and Biomedical Accelerator Summer Research fellowship (AS).

Footnotes

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Data availability

All hits are demonstrated in Supplemental Table 1. The full drug screen data set will be provided upon request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1963628-supplement-1.pdf (122.3KB, pdf)

Data Availability Statement

All hits are demonstrated in Supplemental Table 1. The full drug screen data set will be provided upon request.

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