ACE inhibitor, captopril, attenuates cytopathic effects of herpes simplex virus 1 in SH-SY5Y cells

Cell culture

The neuroblastoma SH-SY5Y cell line (ATCC, CRL2266, lot # 11C016) was purchased from Sigma-Aldrich (St. Louis, MO). Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Sigma Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (Sigma Aldrich, St. Louis, MO) and 7% Penicillin and Streptomycin (Sigma Aldrich, St. Louis, MO). Cells were seeded into flasks containing supplemented medium and maintained at 37°C in saturated humidity with 5% CO₂. For assays, SY5Y cells were sub-cultured into 96-well plates at a seeding density of 1× 10⁵ cells per well.

Virus

The F strain of HSV-1 was graciously contributed by the Fraser Lab at The University of Pennsylvania. Cells were infected at a multiplicity of infection (MOI) of 0.01. During infection, virus was delivered to cells in serum-free DMEM and cells were administered either captopril (Sigma Aldrich, St. Louis, MO; administered at various concentrations (0, 1pM, 10pM, 1nM, 10nM, 1µM)) or vehicle control (0.1% dimethyl sulfoxide (DMSO)) post-infection. One-hour post-infection, inoculum was aspirated and an equal volume of complete media was added to the cells. Cells were incubated until the cell viability assay or imaging analysis was performed. Andrea Bertke (Virginia Tech) donated the green fluorescent protein-tagged HSV-1 (GFP-HSV-1).

Cell viability

Cell viability was quantified via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay (Sigma-Aldrich, M5655). Cells were cultured with cDMEM in 96-well plates until they were 90% confluent. Next, subsets of wells were infected with HSV-1 (F strain) at a MOI of 0.01 for 24 hours. MTT solution (5mg/ml) was added to each well and incubated for four hours at 37°C. After removing the supernatant from wells, 100 µl DMSO was added to each well. The absorbance was read at 570 nm with a 630nm reference wavelength via a Benchmark Plus (BIO RAD) spectrophotometer. All experiments were repeated in triplicate and processed in parallel.

Time course analysis

Time course analysis was evaluated via the previously described methods used in the cell viability protocol, utilizing the MTT assay at 3, 9, and 24 hpi. All experiments were repeated in triplicate and processed in parallel.

Photomicrographs

Photomicrographs were obtained utilizing an Olympus IX81 microscope equipped with a Hamamatsu camera. MetaMorph software (version 6) was used to image cells, and photomicrographs were taken with the differential interference contrast (DIC) filter at a final magnification of 20x (Micro-Manager is a free alternative if access to MetaMorph is not available). Three distinct fields of view were imaged per well.

GFP-replication assay

Green fluorescent protein-tagged HSV-1 (GFP-HSV-1) was used to infect cells, as previously described. There were three treatment groups: Mock, Infected/Vehicle, and Infected/Captopril. A Mock/Captopril group was not used in this experiment as we wanted to examine the effect of captopril of HSV-1 replication and previous data demonstrated that captopril was non-toxic. Photomicrographs were taken with a DIC filter at 8, 24, and 48 hours post infection (hpi).

Molecular modeling

AutoDock Vina (version 1.1.2), a docking program, was used to investigate the interaction between HSV-1 glycoprotein D (gD) (Protein Data Bank (PDB) ID: 1P7C) and captopril (PDBID: 2X8Z). Captopril was collected from the bound crystal structure of AnCE using PyMOL V1.8.6.2. AutoDock Vina was used to approximate the favorability of HSV-1 gD binding with captopril. The crystal structure of both molecules were obtained from the PDB and used to generate 3D structures. To perform the docking simulations, AutoDock Vina generated a grid box to enclose the active site with dimensions of 126 Å × 126 Å × 126 Å and a grid spacing of 0.375 Å using Autodock Tools. The results are represented by free energy binding.

To render the Ligand Interactions Diagram, Schrodinger Maestro V10.6 was used. Initially, crystal structure of HSV1 gD (receptor) and captopril (ligand) were obtained from the PDB as mentioned above. The imported protein structure obtained from the PDB was not suitable for immediate use in the molecular docking study as it contained excess material (i.e., water molecules, metal ions, cofactors, etc.). To overcome this obstacle, the protein structure was prepared using the protein preparation wizard (preprocessed, optimized, and minimized) in Maestro v10.6^11,12. The ligand structures of the data set were prepared by LigPrep module of Schrodinger v10.6¹².

Quantitative PCR (qPCR)

SH-SY5Y neuroblastoma cells were treated with either vehicle (0.1% DMSO), the F strain of HSV-1 (MOI=0.01), or captopril and HSV-1 (F strain, MOI=0.01) together in media without serum for one hour. After this, an equal volume of complete media was added to the cells. Next, RNA was isolated from treated cells according to the manufacturer’s instructions (Qiagen AllPrep DNA/RNA Mini Kit; Cat. No. 80204, Lot # 154018161). Following RNA isolation, the purity and concentration of resulting RNA were measured using the Thermo Scientific Nano Drop 2000 Spectrophotometer. All samples were stored at -80°C for future use.

cDNA synthesis. The protocol used to synthesize first-strand complementary DNA from purified RNA was followed using the Invitrogen SuperScript III First-Strand Synthesis System for RT-PCR (Thermo Fisher, Cat. No. 18080-051). RNA (100ng) isolated from SH-SY5Y cells was placed in Bio-Rad Low-Profile PCR Tubes containing 10mM dNTP mix and 50ng of random hexamer primers. The final volume in each tube was 10 uL. Positive (HeLa RNA) and negative control (tube containing HeLa RNA but no reverse transcriptase) reactions were also prepared. Samples were placed in a Bio-Rad T100 Thermal Cycler and incubated at 65°C for 5 minutes, cDNA synthesis mix (according to the manufacturer’s instructions) was added to each tube including the positive and negative controls. After following subsequent steps detailed in the SuperScript III manual, the resulting cDNA was stored at -20°C.

qPCR cycle settings. The protocol for qPCR was adapted from Thermo Fisher Scientific PowerUp SYBR Green Master Mix (Catalog No., lot# 1601009). Reactions were run in triplicate in Bio-Rad 96-well PCR plates. Primers for each gene of interest were synthesized by Integrated Data Technologies and diluted to 10uM concentrations with DEPC-treated water. Human GAPDH was used as a reference gene, and ICP0 was utilized to measure HSV-1 viral gene expression. The primer sequences for human GAPDH were: 5’ TGG GCT ACA CTG AGC ACC AG 3’ (forward) and 5’ GGG TGT CGC TGT TGA AGT CA 3’ (reverse). The forward primer sequence for ICP0 was 5’ CCC ACT ATC AGG TAC ACC AGC TT 3’. The sequence for ICP0 reverse primer was 5’ CTG CGC TGC GAC ACC TT 3’. Each analyzed well of the PCR reaction plate contained the following: forward primer (500nM), reverse primer (500nM), PowerUp SYBR Green Master Mix (2X), DEPC-treated water, and cDNA (10ng). The PCR plate was sealed and centrifuged, and inserted into the CFX96 Real-Time System. The qPCR cycling mode was as follows: two minutes at 50°C followed by two minutes at 95°C. Next, a total of 40 denature (15 seconds at 95°C) and anneal (60°C) cycles were utilized. Melt curve analyses were performed with each reaction. ICP0 gene expression levels were normalized to human GAPDH levels.

Data analysis

All analyses for statistically significant differences were performed utilizing a two-way analysis of variance (time x infection status or concentration x infection status). The alpha value was set at 0.05. If warranted, a Tukey’s post-hoc test was performed. For the concentration-response curve, a Dunnett’s multiple comparisons post-hoc test was used, comparing the different concentrations to the vehicle (0) control. Data are expressed as means (± SEM). GraphPad Prism (version 6) statistical analysis software was utilized to perform statistical comparisons and prepare graphical representations of quantitative data.

Concentration response

To determine the concentration dependent response of captopril on SH-SY5Y neuroblastoma cells, we first assessed cell viability and cellular morphology via the MTT assay and DIC microscopy, respectively. SH-SY5Y cells were treated with a range of concentrations (0, 1pM, 10pM, 1nM, 10nM, 1uM) of captopril. To investigate if captopril treatment alone adversely affected cell viability, the experiment was first performed in uninfected cells. The concentration response data in uninfected SH-SY5Y cells demonstrated that captopril (Figure 1) did not impact cell viability as viability remained approximately 100% with no statistically significant differences observed at any concentration.

Figure 1. Captopril confers protective effects on HSV-1 infected SH-SY5Y cells.

The response of uninfected and HSV-1 infected SH-SY5Y neuroblastoma cells to varying concentrations (0, 1pM, 10pM, 1nM, 10nM, 1uM) of captopril was measured via MTT cell viability assay at 24 hours post infection and treatment. Data from uninfected and captopril treated cells showed no significant change in cell viability. Cells infected with the F strain of HSV-1 (MOI=0.01) demonstrated a statistically significant reduction in viability, compared to their uninfected counterparts (F_(1,98)=10.22, p < 0.005). HSV-1 infected, captopril-treated cells had a statistically significant, dose-dependent increase in cell viability from 10pM to a maximum efficacy observed at 10nM, compared to infected, untreated cells (F_(6,98)=23.82, p<0.0001). The asterisk denotes that the HSV-1-infected group was statistically significant from the uninfected group at the 0 concentration. The plus signs represent groups (infected with HSV-1 and treated with captopril) statistically significant from their counterparts that were infected the F strain of HSV-1 but received no captopril treatment. Each bar represents the average of six cell culture replicates.

Alongside the uninfected cells, HSV-1 infected SH-SY5Y cells were treated with a range of concentrations of captopril (0, 1pM, 10pM, 1nM, 10nM, 1uM). Results from the MTT cell viability assay demonstrated captopril treatment caused a significant increase in viable cells at 10pM, 1nM, 10nM, and 1μM (F_(6,98)=23.82, p<0.0001), when compared to the infected, untreated group. In subsequent experiments, we chose to use the 10nM dose of captopril as our results indicated it offered maximum protection, compared to the other doses evaluated, and was less than the 1μM dose.

Time course analysis

Experiments were conducted to measure cell viability at different time points (3, 9, and 24 hpi). Cell viability measurements were equivalent among all the groups at 3 hpi. At 9 hpi (F strain of HSV-1, MOI=0.01), the Virus/Vehicle group had 20% fewer viable cells (F_(3,8) =23.27; p<0.0005) compared to every other treatment group (Figure 2). At 24 hpi there were approximately 25% less viable cells in the Virus/Vehicle group compared to all other groups. At 9 and 24 hpi, the only significant difference observed was between the Virus/Captopril group and the Virus/Vehicle group. Differences due to time alone were not statistically significant.

Figure 2. Time course analysis of HSV-1 infected SH-SY5Y cells treated with captopril (10 nM).

Cell viability was evaluated via MTT assay at 3, 9, and 24 hours post infection (hpi) with the F strain of HSV-1 (MOI=0.01). At 9 and 24 hpi, the Virus/Vehicle group was statistically significantly different compared to the all other groups. Each bar represents the average of six biological replicates. The asterisks indicate groups that were statistically significant from the other groups (not denoted with an asterisk) at their respective time points.

Cellular morphology

Photomicrographs were taken as a qualitative analysis to corroborate the MTT results (Figure 3). The experimental groups (Mock, Mock/Captopril, Virus/Vehicle, and Virus/Captopril) were exposed to the same conditions (24 hpi and 10 nM captopril) prior to obtaining photomicrographs. Cells in Figure 3a (Mock) are representative of healthy SH-SY5Y cells indicated by the confluence and the appearance of intact cellular extensions. Cells in the Mock/Captopril group (Figure 3b) were also very confluent and exhibited similar structural characteristics to the Mock group (Figure 3a). In both of these groups, there were no indicators of substantial cell death such as changes in cellular morphology (i.e., cell rounding) and loss of anchorage dependence (i.e., floating cells). HSV-1 infected cells (Virus/Vehicle) exhibited a reduction in confluence (Figure 3c). Cells in this group also demonstrated multiple instances of cytopathy such as changes in morphology, the loss of anchorage dependence, and a decrease in neurite extensions on cells that remained attached. HSV-1 infected cells treated with captopril (Virus/Captopril, Figure 3d) were more confluent compared to the Virus/Vehicle group and maintained normal characteristic extensions of neuroblastoma cells.

Figure 3. Photomicrographs of SH-SY5Y cells: qualitative comparison of treatment groups.

Photomicrographs show morphological changes 24 hours after captopril (10nM) administration. Uninfected cells administered vehicle (0.1% DMSO) (3a) or captopril alone (3b) were indistinguishable in confluence and structure. In the absence of captopril, HSV-1 infected cells were rounded, an indicator of adverse cell health (3c). Captopril treated, HSV-1 infected cells (3d) maintained their neurite extensions (as seen in 3a and 3b) and were more confluent when compared to HSV-1 infected, untreated cells (3c). (Arrows are intended to illustrate morphological changes.) Additionally, cells treated with captopril preserved the neurite extensions associated with healthy SH-SY5Y neuroblastoma cells. Images were taken at a final magnification of 200x using an IX Olympus 81 inverted microscope and MetaMorph microscopy imaging software.

Replication of GFP-expressing HSV-1

Cells infected with a GFP-expressing HSV-1 strain were analyzed at 1, 3, 8, 24, and 48 hpi. In the Virus/Vehicle group, GFP expression was not observed until 8 hpi. After this time point, the percentage of GFP-positive cells increased in a time-dependent manner. At 8 hpi, an average of 13% of cells were GFP-positive in the Virus/Vehicle group compared to only 1% in the Virus/Captopril group (F _(7,11) =3.58; p<0.05; Figure 4.). At 24 hpi, 30% of cells were GFP-positive in the Virus/Vehicle group compared to 25% of GFP-positive cells in the Virus/Captopril group. This pattern continued at 48 hpi, with 61% of GFP-positive cells observed in the Virus/Vehicle group versus 27% of GFP-positive cells in the Virus/Captopril group (F _(7,11) =2.72; p<0.05).

Figure 4. Viral replication assay based on percentage of GFP-positive cells.

HSV-1 used was constructed to express GFP as a measure of replication. The percentage of GFP positive cells was quantified via manual image analysis at 1, 3, 8, 24, and 48 hpi. At 1 and 3 hpi there were no GFP-positive cells in any of the treatment groups. At later time points (8, 24, and 48 hpi), there was a time-dependent increase in percentage of cells expressing GFP in both the Virus/Vehicle and Virus/Captopril group. Each bar represents the average of six biological replicates. The statistically significant differences in the percentage of GFP positive cells were observed between the Virus/Vehicle and the Virus/Captopril groups at 8 and 48hpi (F_(2,30) =12.82; p<0.0001).

Micrographs of cells infected with the GFP-HSV were imaged at 20X magnification. Cells receiving the Uninfected/Vehicle treatment did not exhibit any GFP fluorescence at any time point (Figure 5a–c). With the administration of the GFP-expressing virus (MOI=0.01), cytopathic effects (e.g., cellular rounding) were noted as well as fluorescent imaging at all three time points (8, 24, and 48 hpi; Figure 5d–f). Co-administration of captopril (10nM) with the GFP-HSV reduced the occurrence of both cytopathy and green fluorescence (Figure 5g–i).

Figure 5. Captopril reduces virus-induced cytopathy and GFP-HSV-1 replication.

Photomicrographs from cells in the Uninfected/Vehicle, Virus/Vehicle, and Virus/Captopril groups were imaged at a final magnification of 200X using an Olympus IX81 microscope. An HSV-1 virus fused with GFP was utilized in this experiment. GFP expression, an indicator of viral replication, was not detected in any cells in the Uninfected/Vehicle group (a–c). GFP expression increased from over time in cells infected with GFP-HSV (MOI=0.01, d–f). With co-administration of captopril (10nM), there was a decrease in green fluorescence and dying cells (indicated by the rounded morphology) in HSV-1 infected cells (g–i).

Molecular modeling

We conducted molecular modeling studies to determine the free binding energy (∆G) between the ACE inhibitor captopril and HSV-1 gD. Results demonstrated that the free binding energy between captopril and HSV-1 gD was favorable and calculated to be -3.9 kCal/mol (Figure 6A). Further evaluation of the interaction between HSV-1 gD and captopril identified the specific amino acid residues responsible for the interaction (Figure 6B).

Figure 6. Molecular modeling: captopril has a favorable interaction with HSV-1 gD.

(A) A ribbon diagram of HSV-1 gD is shown in complex with captopril (small molecule). Molecular modeling via AutoDock Vina determined that captopril has a favorable interaction with HSV-1 gD, indicated by a predicted free binding energy (ΔG) of -3.9 kCal/mol. (B) Captopril-HSV-1 gD predicted interaction with specific amino acid residues of gD color coordinated by charge.

qt-PCR

Transcripts of the viral gene ICP0 were detected at 2 hpi by SH-SY5Y neuroblastoma cells infected with the F strain of HSV-1 (Virus/Vehicle Group). qPCR results demonstrated that captopril co-administration to HSV-1 infected cells decreased ICP0 gene expression by approximately 34%, compared to infected cells not administered captopril (p<0.0001; Figure 7).

Figure 7. Captopril treatment down-regulates ICP0 viral gene expression.

Two hours after being infected with the F strain of HSV-1 (MOI=0.01), cells in the Virus/Vehicle group had transcript levels of the viral gene ICP0 statistically significantly greater than those in the Uninfected/Vehicle condition (F_(2,23)=41.59; p<0.0001). ICP0 was not detected in uninfected SY5Y neuroblastoma cells. Each treatment group consisted of eight biological replicates. qPCR was executed in a parallel fashion and each bar represents the average of three replicates. Transcript levels of the viral gene ICP0 were normalized to levels of human GAPDH.

Underlying data

Figshare: Quantitative and qualitative data assessing the antiviral effects of Captopril, an ACE-1 inhibitor, on HSV-1, https://doi.org/10.6084/m9.figshare.12881441.v1²².

This project contains the underlying data files:

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).