Human fetal glial SVG-A cells were cultured in Minimum Essential Medium (Corning) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals), 1% penicillin/streptomycin (P/S) (Mediatech, Inc.), and 0.2% Plasmocin prophylactic (Invivogen) (cMEM). Cells were maintained in a humidified incubator at 37°C with 5% CO₂. SVG-A cells (Major et al., 1985) were generously provided by the Atwood laboratory (Brown University) and have been authenticated by ATCC through STR profiling. Viral infections were performed with a purified JCPyV strain Mad-1/SVEΔ as previously described (Nelson et al., 2012), or SV40 strain 777. Virus strains were generously provided by the Atwood laboratory.

Commercially available ab34756 (Abcam) and a monoclonal antibody derived from a hybridoma supernatant (PAB597) (generously provided by the Atwood laboratory) (Maginnis et al., 2013) were used to probe for the JCPyV major capsid protein VP1 to quantitate JCPyV infection. PAB597 cross-reacts with the SV40 VP1 protein (Maginnis et al., 2013) and was also used to score SV40 infectivity. Antibodies for ERK1/2 (ERK) (CST #4695), phosphorylated ERK1/2 (pERK) (CST #9101), or GAPDH (Abcam, ab8245) were used for ICW or western blot protein quantifications. For the ICW, LI-COR 800 anti-mouse or anti-rabbit secondary antibodies (LI-COR) were used. For FFU, Alexa Fluor 488 (Thermo Fisher Scientific) anti-mouse or anti-rabbit secondary antibodies were used. CellTag 700 (LI-COR) was used as a cell count normalization stain for ICW assays as indicated. DAPI nuclear counterstain (Thermo Fisher Scientific) was used as a cell count normalization stain for FFU assays.

Chemical inhibitors of MEK, U0126 and PD98059 (Cell Signaling Technology), were used at concentrations of 10 and 50 μM, respectively. Bay43-9006 (Cayman Chemical), a chemical inhibitor of Raf, was used at 15 μM. Retro2 (Sigma–Aldrich), a retrograde trafficking inhibitor, was used at a concentration of 100 μM (Nelson et al., 2013). All chemical inhibitors were reconstituted in DMSO (Cell Signaling Technology), which served as a volume-specific vehicle control. EGFR and ERK1/2 siRNAs (Cell Signaling Technology) were transfected into SVG-A cells with RNAiMax (Thermo Fisher) at 10 pmol per well per manufacturer’s instructions. Successful transfections of the siRNAs were confirmed using BLOCK-iT Red (Thermo Fisher) (DuShane et al., 2018).

SVG-A cells were infected with JCPyV and SV40 at MOIs indicated per figure legend in cMEM at 37°C for 1 h. Cells were then fed with cMEM, and plates were incubated at 37°C for 72 h. At 72 hpi, cells were washed with 1X PBS, fixed in 4% PFA at RT for 10 min, and washed three times in 1X PBS prior to staining for either ICW or FFU.

SVG-A cells were plated to ∼70% confluency in 96-well plates. Cells were pre-treated with cMEM containing DMSO, U0126, PD98059, Bay43-9006, or Retro2 at 37°C for 1 h. Following pre-treatment, cells were infected with JCPyV (MOI = 0.5 FFU/cell) at 37°C for 1 h. Media containing the indicated treatment was then added back to appropriate wells at 37°C for 72 h with the exception of Bay43-9006-treated wells. Bay43-9006-treated wells were incubated for at 37°C for 2 h following infection, aspirated, washed with 1X PBS, and cMEM was added back for the duration of the experiment. At 72 hpi, cells were fixed in 4% PFA, washed with 1X PBS three times and FFU and ICW plates were stained as described.

Prior to siRNA transfection, SVG-A cells were plated to ∼50% confluency in 12-well plates (Greiner Bio-One). Cells were then transfected with an EGFR or ERK1/2 siRNA with RNAiMax. The RNAiMax transfection reagent and siRNAs were diluted in incomplete MEM (lacking FBS and antibiotics), combined, and incubated at RT for 5 min. siRNA complexes were added to SVG-A cells and incubated at 37°C for 72 h. At 72 hpt, cells were infected with JCPyV (MOI = 0.5 FFU/cell) at 37°C for 1 h. Cells were then fed with cMEM and incubated at 37°C for 72 h. At 72 hpi, cells were fixed with 4% PFA, washed with 1X PBS three times, and stained for VP1 for FFU and ICW assays as described.

Following fixation, cells were permeabilized with 1X TBS-1% Triton X-100 at RT for 15 min and were then incubated with TBS Odyssey Blocking Buffer (LI-COR) at RT for 1.5 h. Cells were stained with the primary antibodies PAB597 (JCPyV or SV40 VP1, 1:40) or ab34756 (JCPyV VP1, 1:1000) in TBS Odyssey Blocking Buffer (LI-COR) at 4°C overnight while rocking. After primary incubation, cells were washed with 1X PBS and incubated with either an anti-mouse or anti-rabbit Alexa Fluor polyclonal 488 antibody (Thermo Fisher Scientific) at RT for 1 h while rocking and nuclei were counter stained with DAPI (Thermo Fisher Scientific). Using a Nikon Eclipse Ti epifluorescence microscope (Micro Video Instruments, Inc.), the number of infected cells per 10x visual field was quantitated. Percent infection was determined by dividing the number of infected cells/field by the total number of DAPI-positive nuclei/field as previously described (DuShane et al., 2018), reported as percent infection. As indicated, the average percent infection was normalized to the highest MOI, vehicle control DMSO, or siRNA controls (set at 100%).

To confirm ERK1/2 host-cell protein knockdown by ICW, SVG-A cells transfected with EGFR and ERK1/2 siRNAs (as described) were fixed at 72 hpt with 4% PFA. Cells were washed with 1X PBS three times and stained for total ERK and CellTag for ICW analysis with the LI-COR Odyssey CLx.

Following fixation, cells were incubated with 1X TBS-1% Triton X-100 to permeabilize for 15 min. Cells were then incubated with TBS Odyssey Blocking Buffer (LI-COR) at RT for 1.5 h while rocking. Cells were stained with primary antibodies as indicated: PAB597 (1:40), ab34756 (1:1000), ERK1/2 (1:500), or pERK1/2 (1:500) in TBS Odyssey Blocking Buffer (LI-COR) at 4°C overnight while rocking. After primary incubation, cells were washed with 1X TBS-T and incubated with either an anti-mouse or anti-rabbit LI-COR 800 secondary antibody (1:10,000) and CellTag 700 (1:500) at RT for 1 h while rocking. Secondary-alone wells were treated only with species-appropriate LI-COR 800 secondary antibody (1:10,000). Cells were washed with 1X TBS-T three times and aspirated to remove all liquid prior to scanning. Using a LI-COR Odyssey CLx Infrared Imaging system, plates were immediately scanned to detect 700 and 800 nm channel intensities. Plates were read at a 42 μm resolution, at medium quality, with a 3.0 mm focus offset. After scanning, 700 and 800 nm channels were aligned using the Image Studio software (version 5.2) equipped with the ICW module. After scanning, the ICW analysis grid (Image Studio) was applied to the plate image to outline each well and images were then processed using Image J (NIH).

Western blot analysis of ERK1/2 protein expression was also used to confirm siRNA protein knockdown. SVG-A cells were transfected with either EGFR or ERK1/2 siRNAs as described above. Cells were then washed with 1X PBS and manually scraped from sample wells. Cells were pelleted by centrifugation at 376 ×g 4°C for 5 min. Pellets were resuspended in 50 μl of Tris-HCl lysis buffer containing protease and phosphatase inhibitors and incubated on ice for 15 min. Samples were centrifuged at 18,600 ×g at 4°C for 10 min. Samples were combined with Laemmli sample buffer (Bio-Rad), boiled at 95°C for 5 min, and proteins were then resolved by SDS-PAGE using a 4–15% gel (Bio-Rad). Proteins were transferred onto a nitrocellulose membrane with a Transblot Turbo Transfer System (Bio-Rad). Protein-containing membranes were then blocked with 5% non-fat dry milk/TBS-T (1X TBS/0.1% Tween 20) overnight at 4°C while rocking. Membranes were washed with 1X TBS-T for 15 min, three times. Membranes were then incubated with primary antibodies for total ERK (1:500) and GAPDH (1:2000) in 5% BSA/TBS-T overnight at 4°C while rocking. After primary antibody incubation, membranes were washed in TBS-T at RT for 15 min three times each and then incubated with the secondary anti-rabbit 680 antibody (1:10,000) (LI-COR) and anti-mouse 800 antibody (1:10,000) (LI-COR) at RT for 1 h in 5% milk/TBS-T. Membranes were washed in TBS and then imaged using a LI-COR Odyssey CLx.

SVG-A cells were plated to ∼70% confluency in 96-well plates. Cells were either mock-infected (cMEM only) or infected with JCPyV (MOI = 0.5 FFU/cell) in cMEM at 37°C for 0, 15, 30, or 60 min. At the indicated timepoints, cells were fixed in 4% PFA at RT for 10 min and washed three times in 1X PBS. After fixation, both mock- and JCPyV-infected wells were probed for pERK and CellTag for ICW analysis with the LI-COR Odyssey CLx.

Each plate processed for ICW was scanned using the LI-COR Odyssey CLx imager, and a size-specific plate template was added to the image containing both 700- and 800-channel intensities to define well boundaries. Each image was subsequently loaded into ImageJ for analysis, adapted from previous models (Rueden et al., 2017; Kelley and Paschal, 2018). In brief, RGB ICW image channels were split and a background subtraction was applied to the 8-bit, red and green channels, in accordance to well size, using the rolling ball radius algorithm. To measure signal intensity within each well, a mask of each well was generated from the aforementioned size-specific template as regions of interest (ROIs). The ROIs generated from the template image were then applied to both the 700 (red) and 800 (green) images, and the fluorescence intensities from each well (as characterized per pixel) were generated. To account for background fluorescence intensity from uninfected or untreated wells (mock), the mock sample (as experimentally indicated) intensity values for both the 700 and 800 channels were subtracted from experimental wells to account for non-specific fluorescence. Resultant values were plotted using the ggplot2 R package (version 3.5.1) (Wickham, 2009) and reported as percent response.

Student’s t-test was used to compare means from at least triplicate samples in Microsoft Excel to determine statistical significance. P-values < 0.05 were considered statistically significant. Each experiment was performed in triplicate containing a minimum of triplicate samples. Pearson correlation coefficients, calculated using R, were used to correlate manual microscopy FFU (percent infection) data with corresponding ICW (percent response) data as indicated.

To determine whether the ICW can be used to accurately quantitate JCPyV infection, SVG-A cells were infected with varying JCPyV MOIs and analyzed for infectivity using manual FFU quantitation (percent infection) and ICW (percent response) analyses performed in parallel (Figure 1A,B). The Cell Tag 700 (ICW) and DAPI stain (FFU) were used as total cell number normalization controls to quantify the percentage of infected cells. Infectivity was characterized through measurement of newly synthesized VP1 at 72 hpi, which represents a single replication cycle. Both the percent infection and percent response data demonstrate an increase in VP1 expression with increasing MOIs. Interestingly, the percent infectivity and percent response data were positively correlative across the range of the varying MOIs tested, as demonstrated by a Pearson correlation coefficient of 0.978 (Figure 1C). These data indicate that the ICW is a robust and accurate assay for scoring JCPyV infectivity.

A key protein necessary for productive JCPyV infection is the host-cell protein extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase (MAPK) cascade. It has been previously shown that chemical inhibition of ERK significantly reduces JCPyV infection (DuShane et al., 2018). ERK functions as the terminal kinase of the MAPK cascade and is directly phosphorylated by MEK, which is activated by the kinase Raf (Shaul and Seger, 2007). It has been previously shown that inhibitors of MEK (U0126 and PD98059) can be applied to cultured cells to inhibit the activation of the downstream target ERK (English and Cobb, 2002; Wan et al., 2004), thereby impacting JCPyV infection (Ravichandran et al., 2007; DuShane et al., 2018). Moreover, as Raf is critical for the downstream activation of ERK, chemical inhibition of Raf (Bay43-9006) was also investigated to determine if viral infection was impacted.

To characterize viral infectivity in the presence of the aforementioned MAPK inhibitors, SVG-A cells were pretreated with DMSO (vehicle control), Bay43-9006, PD98059, or U0126 and subsequently infected with JCPyV (Figure 2). A cell viability assay (MTS) was used to confirm that chemical inhibitors were not toxic (data not shown) and total cell numbers are accounted for through CellTag (ICW) and DAPI staining (FFU). Cells were fixed and stained for VP1 using both FFU and ICW assays performed in parallel. SVG-A cells treated with the inhibitor Bay43-9006 demonstrated an ∼60% decrease in infection in the case of both the FFU and ICW analyses, in comparison to controls (Figure 2A). The MEK inhibitor PD98059 decreased JCPyV infectivity by ∼80% as measured by FFU and ICW (Figure 2B). U0126 reduced JCPyV infectivity by ∼60% in both the FFU analysis and the ICW quantitation of JCPyV VP1 expression (Figure 2C). In addition, the retrograde trafficking inhibitor Retro2, which has been previously demonstrated to inhibit JCPyV infection (Nelson et al., 2013), was tested as a JCPyV inhibitor via ICW. Treatment of SVG-A cells with Retro2 significantly reduced JCPyV infection by ∼90% as measured by FFU and ∼80% by ICW (Figure 2D), suggesting that an inhibitor of another component of the viral infectious cycle, viral trafficking, can be assessed by ICW. These data demonstrate the versatility of the ICW analysis in the characterization of viral infection during chemical inhibition.

Treatment of SVG-A cells with an ERK1/2-specific siRNA has been previously shown to significantly decrease JCPyV infection as measured by FFU quantitation (DuShane et al., 2018). To determine whether siRNA-induced protein knockdown can be detected by the ICW, SVG-A cells were transfected with either an irrelevant EGFR- or an ERK1/2-specific siRNA. In parallel, siRNA transfected SVG-A cells under the same conditions were also processed by western blot to confirm that the ICW provides an accurate measure of protein expression. ERK protein expression was reduced in samples transfected with the ERK1/2 siRNA in comparison to the EGFR control for both the ICW and western blot assays (Figure 3A,B), suggesting that the ICW is a viable method for protein expression analysis during siRNA transfection.

To determine if the ICW can be used to quantitate JCPyV infection during siRNA conditions, SVG-A cells were transfected with the aforementioned siRNAs and cells were subsequently infected with JCPyV. Quantification of VP1 by FFU and ICW showed significant decreases of ∼90% for both percent infection and percent response (Figure 3C), suggesting that the ICW can be utilized to quantitate JCPyV infection during siRNA transfection with the same degree of accuracy as traditional FFU assays.

Previous research has demonstrated that early events during JCPyV infection induce alterations to the MAPK pathway resulting in phosphorylation of ERK (Querbes et al., 2004; DuShane et al., 2018). Within 15 min of viral challenge, pERK is highly upregulated in SVG-A cells (Querbes et al., 2004; DuShane et al., 2018). Traditionally, quantitation of protein phosphorylation has been measured through western blotting techniques, which can be laborious and time consuming. However, the ICW has been implemented as an effective tool for the quantitation of both protein expression and signal transduction (Hannoush, 2008; Aguilar et al., 2010; Schnaiter et al., 2014; Cuartas-López et al., 2018), but has had limited use in conjunction with the study of viral infectivity (Cuartas-López et al., 2018). We employed the ICW technique to determine if alterations to pERK levels during JCPyV infection could be quantified using the ICW methodology, SVG-A cells were either mock-infected or infected with JCPyV, and levels of pERK were assessed by ICW over a time course of infection. At 15 min post viral challenge, activation of ERK peaked, followed by a steady decline through 1 hpi (Figure 4). These findings are in line with previously published work demonstrating that levels of pERK are highly upregulated in JCPyV-infected samples in comparison to mock-infected SVG-A cells at early timepoints during infection (Querbes et al., 2004; DuShane et al., 2018). These results demonstrate that the ICW can not only assess viral infectivity, but can also quantify host-cell protein response to viral infection.

SV40 is the most phylogenetically related polyomavirus to JCPyV and has a very similar replication cycle (Perez-Losada et al., 2006). Thus, it was hypothesized that ICW can be utilized to quantify and characterize SV40 infectivity as well. To determine whether the ICW technique could be applied to SV40, SVG-A cells were infected with varying MOIs of SV40. Cells were then incubated for 72 h and stained for SV40 VP1 expression and analyzed by FFU and ICW (Figure 5A,B). Resultant data from both FFU and ICW demonstrated an increase in VP1 expression that correlated with increasing MOI (Figure 5C). Moreover, infectivity quantified by FFU and ICW proved to be positively correlative as per a Pearson correlation coefficient of 0.985, suggesting that the ICW is a viable and accurate measure of SV40 infectivity (Figure 5C).