ROCK1 inhibitor-Thiazovivin and Y27632 (Supplementary Fig. 7), MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] and dimethyl sulfoxide (DMSO) were procured from Sigma (Steinheim, Germany).
African green monkey kidney (Vero) and HeLa cells were received from National Center for Cell Science (NCCS), Pune, India. Cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 5–10% fetal calf serum (FBS) (Sigma, St. Louis, USA) and antibiotics.
Vero cell adapted BPXV (Accession Number, VTCC-AVA90) was available at NCVTC Hisar. Vaccinia virus (VACV) was procured from American Type Culture Collection (ATCC). Both viruses were amplified and quantitated by plaque assay in Vero cells, as described previously55. The viral titers were determined as plaque forming units/ml (pfu/ml).
ROCK1 Rabbit monoclonal antibody, p-MLC2 Mouse monoclonal antibody, CNOT7 Rabbit Monoclonal antibody, β-actin Mouse monoclonal antibody, PABP1 Rabbit Monoclonal antibody and p44/42 MAPK (ERK1) Rabbit Monoclonal antibody USA were procured from Cell Signallingusetts Technology (Mass) . Hyperimmune serum that reacts with 23 kDa and 37 kDa BPXV proteins were raised in rabbits and has been previously described by our group55.
Cytotoxicity and virucidal activity
The cytotoxic55 and virucidal11 effects of Thiazovivin (Supplementary Fig. 1a) and Y27632 (Supplementary Fig. 1b) were determined as previously described.
In vitro antiviral efficacy of Thiazovivin (EC50)
Vero cells, in triplicates were infected with BPXV at an MOI of 0.1 for 1 h followed by washing with PBS, and in addition of fresh DMEM containing either DMSO or three-fold serial dilutions of Thiazovivin (50 µg/ml to 0.002 µg/ml). Viral titers in the infected cell culture supernatants at 48 hpi were determined by plaque assay. Effective concentration 50 (EC50), concentration of the inhibitor required to reduce 50% virus yield) was determined by the Reed-Muench method.
Confluent monolayers of Vero cells, in triplicates were infected with BPXV at MOI of 5, followed by addition of Thiazovivin (1 µg/ml) or vehicle-control at 1 hpi, 6 hpi, 12 hpi, 18 hpi, 24 hpi, 30 hpi and 36 hpi. Supernatant from the infected cells was collected at 48 hpi and quantified by plaque assay29,32.
Confluent monolayers of Vero cells, in triplicates, were pre-incubated with 1 µg/ml Thiazovivin or 0.05% DMSO for 30 min followed by BPXV infection at MOI of 5 for 2 h at 4 °C. The cells were then washed 5 times with PBS and the cell lysates were prepared by rapid freeze–thaw method. The viral titers in cell lysates were quantified by plaque assay29,32.
Confluent monolayers of Vero cells, in triplicates, were pre-chilled at 4 °C and infected with BPXV at MOI of 5 in Thiazovivin-free medium for 1.5 h at 4 °C, which allowed virus attachment to the host cells but restricted viral entry . Thereafter, the cells were washed with PBS and incubated with fresh DMEM containing 1 µg/ml Thiazovivin or 0.05% DMSO. To permit viral entry, cells were incubated at 37 °C for 1 h. Thereafter, cells were washed with PBS and grown in fresh DMEM without any inhibitor. Virus yield in the infected cell culture supernatants at 48 hpi was determined by plaque assay29,32.
Virus release assay
Confluent monolayers of Vero cells, in triplicates, were infected with BPXV at MOI of 5 for 1 h. Thereafter, cells were washed with PBS and fresh DMEM was added. At 36 hpi, cells were washed 5 times with chilled PBS followed by addition of fresh DMEM containing 1.0 µg/ml Thiazovivin or equivalent volume of DMSO. Virus yield in the infected cell culture supernatant was quantified by plaque assay at 30 min and 2 h post-drug treatment29,32.
The amount of viral DNA/mRNA(cDNA) in infected cells was measured by quantitative real-time PCR (qRT-PCR). Confluent monolayers of Vero cells, in triplicates, were infected with BXV (MOI of 5) for 1 h followed by washing with PBS and addition of fresh DMEM. Thiazovivin (1 µg/ml) or DMSO (0.05%) were added at 6 hpi. Cells were scraped at 36 hpi to quantify the viral (M gene forward primer: 5′-AACACACATTATTCAGATACGTC-3′ and reverse primer: 5′-TTGTACGTCGCTCTTTGTTAG-3′) and house-keeping control gene (β-actin) by qRT-PCR as previously described29. The levels of viral DNA, expressed as a threshold cycle (Ct) values, were normalized with β-actin housekeeping control gene. Relative fold-change in viral DNA copy number was determined by ΔΔ Ct method56.
Effect of Thiazovivin on synthesis of viral proteins
Confluent monolayers of Vero cells were grown in 30 cm2 tissue culture dishes and infected with BXV at MOI of 5. Inhibitors or vehicle-control were added at 6 hpi. Cells were scraped at 36 hpi to analyze the levels of viral-and house-keeping control proteins (β-actin) in Western blot analysis. Anti-BPXV serum was available at NCVTC Hisar and has been described elsewhere29.
Selection of potential Thiazovivin-resistant virus variants
Vero cells were infected with BXV at MOI of 0.1 in medium containing 0.05% DMSO or 0.2 µg/ml Thiazovivin. At 48–72 hpi, supernatant was collected from the virus infected cells [named passage 1 (P1)] and quantified by plaque assay. Fifty such sequential passages were carried out. The original virus stock (P0), P50-Thiazovivin and P50-Control viruses were used to infect Vero cells at an MOI of 0.1 with either 1.0 µg/ml Thiazovivin or 0.05% DMSO. At 48 hpi, viral titers in the infected cell culture supernatant were quantified by plaque assay29,32.
Ligation-mediated Poly(A) tail length (LM-PAT)
LM-PAT assay was employed to measure the changes in poly(A) tail length by specifically targeting the oligo(dT) anchor at the end of the poly(A) tail as per the previously described method57. Briefly, at 9 h following BPXV infection, the cells were treated with Thiazovivin/Y27632- or DMSO in the presence of Actinomycin D. The RNA was isolated at 2 h and 4 h post-drug treatment. The poly(A) tail of mRNA was allowed to saturate with phosphorylated ATPs and then ligated with adaptor oligo(dT) primers (5′-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTT-3′) at 42 °C in the presence of T4 DNA ligase. The ligated mRNA was subjected to cDNA synthesis by using anchor primer 2 [specifically binds with ligated adaptor oligo(dT) primer; 5′-GGCCACGCGTCGACTAGTAC-3′]. Finally, the length of poly(A) tail was determined by PCR by using an anchor (primer 3; 5′-CUACUACUACUAGGCCACGCGTCGACTAGTAC-3′) and M gene-specific primer (5′-AGTATCAAAGATATAGATCACC-3′). Likewise, the length of poly(A) tail of endogenous/cellular mRNA was determined by PCR by using an anchor (primer 3; 5′-CUACUACUACUAGGCCACGCGTCGACTAGTAC-3′) and β-actin-specific primer (5′-CTTACCTGTACACTGACTTGA-3′).
Measurement of mRNA stability
The stability of the mRNA was estimated as per the previously described method58. Briefly, Vero cells were infected with BPXV at MOI of 5. At 9 hpi, the cells were treated with Thiazovivin (1 µg/ml), Y27632 (1.5 µg/ml) or equivalent volume of DMSO in the presence of Actinomycin D (5 µg/ml). RNA was isolated at 0 h, 1 h, 2 h and 4 h following addition of the inhibitors and examined for the levels of BPXV M gene by qRT–PCR. The half‐lives of the targeted mRNA was calculated as described previously58.
Chromatin immunoprecipitation (CHIP) assay
CHIP assay was carried out to evaluate the interaction of viral mRNA with a cellular poly(A) tail binding protein (PABP) by employing a previously described method59 along with some modifications. Briefly, Vero cells, in triplicates, were infected with BPXV at MOI of 5. At 9 hpi, when the RNA levels were expected to be at its peak, the cells were treated with ROCK1 inhibitors (Thiazovivin and Y27632) or DMSO. At 2 h and 4 h-post drug treatment, the cells were treated with 1% formaldehyde for 10 min to covalently cross-link interacting proteins and nucleic acid. Thereafter, the cross-linking reaction was stopped by addition of 125 mM glycine (final concentration) by washing the cells with ice-cold PBS. The cell lysates were prepared in immunoprecipitation (IP) buffer [150 mMNaCl, 50 mMTris-HCl (pH 7.5), 5 mM EDTA, 0.5% NP-40, 1% Triton X-100 plus protease and phosphatase inhibitor cocktail] and sonicated in a QsonicaSonicator Q500 (Qsonica, Newtown, CT, USA) (6 pulse of 15 s at amplitude of 40%). The cell lysates were then centrifuged for 10 min at 12,000 g. The clarified cell lysates were mixed with 10 units of RiboLock RNase Inhibitor (Thermo Scientific, USA) and then incubated with the PABP antibody (reactive antibody), phospho ERK antibody (non-reactive antibody) or equivalent volume of IP buffer (beads control) for 45 min at room temperature. Thereafter, 40 μl (5 ng/μl) of Protein A Sepharose® slurry, prepared as per the instruction of the manufacturer (Abcam, USA) was added into each reaction and incubated overnight at 4 °C on a rotary platform. The beads were then washed 5 times in the IP buffer (without protease inhibitors). To reverse the cross-linking, the complexes were then incubated with Proteinase K (20 mg/ml final concentration) at 56 °C for 40 min. Finally, the reaction mixtures were centrifuged at 12,000 g for 1 min and the supernatant was subjected to RNA isolation. The RNA was converted into cDNA and then the levels of BPXV M gene was quantified by qRT-PCR.
Vero cells were grown in chamber slides at ~20% confluency and infection with BPXV at MOI of 10 for 2 h, followed by washing with PBS and replacement with fresh medium. Thiazovivin was applied at 5 hpi. The intracellular localization of viral and/or cellular (p-MLC2) proteins in the virus-infected cells was detected by immunofluorescence assay at 15 hpi as described previously51.
siRNA knockdown of ROCK1
In order to further confirm the role of ROCK1 in BPXV replication, as well as to avoid the possibilities that ROCK1 inhibitor may have some off-target effects in the host cells, ROCK1 was knockdown from the cells in a sequence dependent manner by using small interfering RNA (siRNA). Briefly, Vero cells were grown in 96 well plates. When the cells were at ∼75% confluency, 50 nmol and 100 nmol of ROCK1 (FlexiTube siRNA, Qiagen, Germany]or control siRNA (AllStars Neg Control siRNA, Qiagen, Germany]were transfected using Lipofectamine 3000 as per the instruction of manufacturer (Invitrogen, Carlsbad, USA) At 48 h post-transfection, cells were infected with BPXV at MOI of 1 and virus released in the infected cell culture supernatant at 48 hpi was quantified by plaque assay.
In ovo antiviral efficacy of Thiazovivin against BPXV
Egg lethal dose 50 (LD50)
Specific pathogen free (SPF) embryonated chicken eggs were procured from Indovax Pvt Ltd, Hisar, India. LD50 of CGP57380 was determined by inoculating fivefold serial dilutions of Thiazovivin (concentration ranging from 50 to 0.0.02 µg/egg) or DMSO (vehicle-control), in 10 day old SPF eggs, in a total of 100 µl volumes via chorioallantoic membrane (CAM) route. Eggs were examined for the viability of the embryos up to five days post-inoculation to determine the LD50 by the Reed-Muench method29,32.
In ovo antiviral efficacy (EC50)
SPF embryonated chicken eggs, in triplicates, were inoculated with fivefold serial dilutions (25 to 0.1 µg/egg) of Thiazovivin or equal volume of vehicle control via CAM route, followed by infection with BPXV at 100 EID50. On 5–7 dpi, eggs were examined for pock lesions and/or death of the embryos. EC50 was determined by the Reed-Muench method29,32.
Statistical analysis was performed using the Graph Pad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). The group difference was evaluated by student’s t test, error bars represent mean ± SD and the difference was statistically significant when *p < 0.05; **p < 0.01; or ***p < 0.001.
This study does not involve any experiment on humans or animals.
Consent to participate
All authors agreed to participate.