p38-MAPK is prerequisite for the synthesis of SARS-CoV-2 protein

Abstract

The inhibition of p38 mitogen-activated protein kinase (p38-MAPK) by small molecule chemical inhibitors was previously shown to impair severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, however, mechanisms underlying antiviral activity remains unexplored. In this study, reduced growth of SARS-CoV-2 in p38-α knockout Vero cells, together with enhanced viral yield in cells transfected with construct expressing p38α, suggested that p38-MAPK is essential for the propagation of SARS-CoV-2. The SARS-CoV-2 was also shown to induce phosphorylation (activation) of p38, at time when transcription/translational activities are considered to be at the peak levels. Further, we demonstrated that p38 supports viral RNA/protein synthesis without affecting viral attachment, entry, and budding in the target cells. In conclusion, we provide mechanistic insights on the regulation of SARS-CoV-2 replication by p38 MAPK.

Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which emerged in 2019, has resulted in more than 767.5 million infections and 6,947,192 fatalities as of September 2023. (WHO 2023). Vaccine is available but emergence of the antigenic variants has raised concerns about the immunity induced due to vaccination or prior infection [62].

Remdesivir, Favipiravir, Itolizumab, Tocilizumab, Hydroxychloroquine, and Ivermectin are among antiviral medications used to treat COVID-19. These medications decrease the number of patients hospitalized and the disease severity [8, 15, 57]; [53]. However specific and effective antiviral medication against COVID-19 are still lacking.

Till date, more than 184 antiviral drugs have been approved by the Food and Drug Administration (FDA) to combat with viral infections. The mechanism of action of most of these approved drugs is based on directly targeting viral protein expression [7]. However, even when successful, the drugs can eventually fail because of the emergence of drug-resistant mutants [21, 23, 39, 40, 47].

To facilitate replication, viruses interact with numerous factors by RNA–protein, protein–protein and protein-lipid interactions [42]. Upon infection, viruses exploit the host cells by subverting the host factors, remodeling subcellular membranes, modulation of cellular proteins and ribonucleoprotein complexes and usurping cellular metabolic pathway [58]. Recent progress through transcriptomics studies, genome-wide knockdown/knockout studies have enabled identification of numerous cellular factors which are essential for virus replication [55]. The host cell factors which are critically required for virus replication but are dispensable for the host may be targeted for antiviral drug development [5, 35, 36]. Since the genetic variability of the host is quite low as compared to the viral genome, host-directed therapies should have fewer tendencies in inducing antiviral drug resistance.

Recently we demonstrated that p38 MAPK inhibitor SB203580 block SARS-CoV-2 yield in Vero cells (In-press, paper entitled p38 MAPK Inhibitor SB203580 suppresses SARS-CoV-2 Replication in Annals of biology, Ref. No. AOB/2023/14). However, the precise mechanism of the antiviral action remains elusive. In this study we provided mechanistic insights on the regulation of SARS-CoV-2 replication by p38-MAPK.

Materials and methods

Cells and viruses

Vero (African green monkey kidney cells), T-293 (human embryonic kidney) and p38-α MAPK knockout Vero cells, available at National Centres for Veterinary Type Culture (NCVTC), Hisar were grown in Dulbecco’s Modified Eagle’s Medium high glucose (Lonza, cat number-12-604F BE12-604F, USA) supplemented with antibiotics and 10% heat-inactivated fetal bovine serum (FBS) (D6429, Sigma, St. Louis, USA) and antibiotic (Penicillin–Streptomycin-Amphotericin B Suspension 100X) (A5955, Sigma, USA). SARS-CoV-2 (NCVTC Accession Number, VTCCAVA 294; SARS-CoV-2/India/2020/tc/Hisar/4907) was available at NCVTC, Hisar. Virus was propagated in Vero cells in the Biosafety level 3 (BSL-3) laboratory of ICAR-National Research Centre on Equines (NRCE), Hisar, India [24]. The virus was quantified by plaque assay and viral titres were measured as plaque forming unit per millilitre (PFU/ml) [33].

Inhibitor

Adezmapimod (SB203580), is a pyridinyl imidazole inhibitor was procured from Biogems (1,524,762, Ariano Irpino, Italy). These inhibitors were dissolved in Dimethyl sulfoxide (DMSO), thereby DMSO was used as a vehicle control in the experiments [18]

Antibodies

SARS-CoV-2 Nucleocapsid antibody (produced in mouse) as a primary antibody was procured from (MA5-35,943, Invitrogen, 1:1000–1:10,000), Rabbit phospho-p38 MAPK and total p38 MAPK Antibody was procured from (9211S and 9212S, Cell Signalling technology, Massachusetts, USA. Mouse anti-β actin primary antibody, Goat Anti-mouse IgG HRP conjugate (665,739 /Merck/Massachusetts, USA/1: 10,000), Goat Anti-Rabbit IgG HRP conjugate (632,131/Merck/Massachusetts, USA, 1:10,000 were procured from Merck (USA). Mouse ß-Actin (3700S/ 1:1000/Cell Signalling technology/Massachusetts, USA) used as primary antibody.

Cell viability assay

Cell viability of SB203580 inhibitor was assessed using MTT dye based on previously described protocol [56]. Briefly, confluent monolayers of Vero cells were grown in 96 well plates and incubated with SB203580 at indicated concentrations for 72 h in triplicates, followed by treatment with 20 mg/ml MTT dye for 1 h at 37°C. After incubation, 100 µl DMSO per well was added to solubilize the formazan crystals and cell viability was assessed by calorimetric detection at 570 nm in a microplate reader.

Overexpression of p38-α to rescue the inhibitory effect of p38-α depletion SARS-CoV-2

Plasmid constructs expressing p38 MAPK (pDEST40-p38-α) was available at NCVTC, Hisar. To further confirm the SARS-CoV-2 supportive role of p38-α, 293T cells were either transfected with pDEST40-p38-α (5 µg) or with the empty vector (control) using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA). At 24 h post-transfection, the cells were infected with SARS-CoV-2 at MOI of 10 and the virus released in the infected cell culture supernatant at 24 hpi was quantified by Quantitative Real time PCR (qRT-PCR).

Attachment assay

Confluent monolayer of Vero cells, in triplicates, were pre-incubated with SB203580 (2.5 µg/ml) or vehicle control for 30 min followed by SARS-CoV-2 infection (MOI of 5) at 4 °C for 1.5 h. This will allow the virus to attach to the cells but restrict virus entry since virus entry is an enzymatic process and require physiological temperature. Thereafter, the cells were extensively washed with cold ice PBS to clear unattached virus particles, and cell lysates were prepared by freeze–thaw method to recover attached viruses in the suspension. After removal of cellular debris by a brief centrifugation step, the virus attached to cell in presence and absence of inhibitor was quantified by plaque assay [34].

Entry assay

The confluent monolayer of Vero cells, in triplicates were prechilled at 4 °C and infected with SARS-CoV-2 (MOI-5) at 4 °C for 1 h which allowed the virus attachment to the cells and restricted the viral entry. Thereafter, the cells were washed with ice-cold PBS and add inhibitor (SB203580, 2.5 µg/ml) or vehicle controls were added, and cells were incubated at 37^OC for 1 h to permit the viral entry. Cells were again washed with PBS, followed by addition of DMEM without any inhibitor. The cells were further incubated at 37°C. Supernatant was collected at 16 hpi and the infectious virus particles were quantified by plaque assay [27].

Virus release assay

Confluent monolayer of Vero cells, in triplicates, were infected with MOI of 5 for 1 h at 37°C followed washing with PBS and addition of fresh DMEM. At 8 hpi, when virus presumably starts budding, cells were again washed with PBS followed by addition of fresh medium having inhibitor (2.5 µg/ml) or vehicle control. Supernatant was collected at 0.5 h and 1 h post-drug treatment and the virus released was quantified by plaque assay.

qRT-PCR

Confluent monolayer of Vero cells was infected with SARS-CoV-2 (MOI = 5) for 1 h at 37 °C, followed by washing with cold ice PBS and addition of fresh DMEM. At 3 hpi, when early stages of life cycles like attachment, entry and uncoating had occurred and RNA synthes is likely to start, 2.5 µg/ml of SB203580 or 0.05% DMSO was added. Cells were scraped at 18 hpi to quantify the viral gene and house-keeping control gene (GAPDH) by qRT-PCR. All the values were normalized with GAPDH housekeeping control gene. Relative fold-change in viral RNA copy number were determined by ΔΔ Ct method [26].

Effect on the synthesis of viral proteins

Confluent monolayers of Vero cells in 30 cm tissue culture dishes were infected with SARS-CoV-2 at MOI of 5 followed by addition of SB203580 (2.5 µg/ml) (at 3 hpi). The cells were scrapped at 24 hpi and cell lysate were prepared in RIPA buffer. The levels of viral and house-keeping control proteins were analyzed in Western blot. SARS-CoV-2 Nucleocapsid antibody (produced in mouse) was procured from (MA5-35,943/Invitrogen/Massachusetts, USA/1:1000–1:10,000) used as primary antibody. Goat Anti-mouse IgG HRP conjugate (665,739 /Merck/Massachusetts, USA/1: 10,000) and its substrate (DAB) were used as per the manufacturer’s protocol.

Activation of p38 MAPK p38 phosphorylation

Confluent monolayers of Vero cells, in 100 cm tissue culture dishes were infected with SARS-CoV-2 at MOI of 10 for 1 h, followed by washing with ice cold PBS and addition of fresh DMEM. Cells were scrapped at 1hpi, 4hpi, 8hpi and 12hpi, and cell lysates were prepared. The levels of phosphorylated p38 MAPK, total p38 MAPK and house-keeping (β-actin) control proteins were analyzed in Western blot.

Results

p38 MAPK is essential for SARS-CoV-2 replication

To evaluate the role of p38 MAPK on SARS-CoV-2 replication, we measured yield of SARS-CoV-2 in WT- and p38-knockout (KO) Vero cells. As shown in Fig. 1A, p38-knockout cells had significantly lower titer as compared to the Wt Vero cells, suggesting p38 MAPK supports SARS-CoV-2 replication.

Fig. 1.

p38 is prerequisite for the propagation of SARS-CoV-2. A Growth of SARS-CoV-2 in p38-α KO cells. The confluent monolayers of WT Vero cells and p38 knockout Vero cells, in triplicates, were infected with SARS-CoV-2 at MOI 1. At 24 hpi, supernatant was collected and yields of infectious progeny virus particles were determined by plaque assay. Values are means ± SD and representative of the result of at least 3 independent experiments. Pair-wise statistical comparisons were performed using Student’s t test (*** =  P < 0.001). B Overexpression of p38. 293 T cells were transfected with plasmid construct that express p38 MAPK or with the empty vector. At 24 h post-transfection, the cells were infected with SARS-CoV-2 at MOI of 10 and the virus released in the infected cell culture supernatant at 24 hpi was quantified by qRT-PCR. Threshold cycle (Ct) values were analysed to determine relative fold-change in copy numbers of total RNA and mRNA. Values are means ± SD and representative of the result of at least 3 independent experiments. Pair-wise statistical comparisons were performed using Student’s t test (*** =  P  < 0.001)

To further confirm, we measured the yield of SARS-CoV-2 in 293 T cells transfected with the construct that express p38 (pDEST40-p38-α). As shown in Fig. 1B, the cells transfected with p38 expressing protein had significantly higher titre as compared to the cells that received empty vector, which further confirmed that p38 is essential for the propagation of SARS-CoV-2.

p38 MAPK inhibition impairs SARS-CoV-2 RNA and protein synthesis

We performed virus step-specific assays to evaluate the role of p38 MAPK in SARS-CoV-2 life cycle. The p38 inhibitor did not affect SARS-CoV-2 attachment (Fig. 2a), entry (Fig. 2b) and budding (Fig. 2c) in the target cells.

Fig. 2.

p38 inhibitor does not inhibit virus attachment, entry and budding. A Cell viability assay. Vero cells in triplicates were incubated with SB203580 at indicated concentrations for 72 h, followed by treatment with 20 mg/ml MTT dye for 1 h at 37 °C. After incubation, DMSO was added to solubilize the formazan crystals and cell viability was assessed by calorimetric detection at 570 nm in a microplate reader. B Attachment assay. Vero cells were pre-incubated with SB2203580 or vehicle control for 30 min followed by SARS-CoV-2 infection (MOI 5) at 4 °C for 1.5 h. The cells were then washed 5 times with PBS and the cell lysates were prepared by rapid freeze–thaw method. Virus attached to the cells in the presence of inhibitor or vehicle control was quantified by Plaque assay (ns = non- significant). C Entry assay. Confluent monolayer of Vero cells, in triplicates were infected with SARS-CoV-2 infection (MOI 5) at 4 °C for 1 h, followed by washing with ice-cold PBS. The attached virus was allowed to enter at 37 °C in the presence of inhibitor or vehicle control. The virus released in the supernatant was quantified at 16 hpi by plaque assay. D Budding assay. Confluent monolayer of Vero cells was infected with SARS-CoV-2 at MOI 5 for 1 h followed by washing with PBS. At 8 hpi, cells were again washed with PBS and fresh medium with inhibitor was added. Supernatant was quantified by plaque assay

To determine the effect of p38 inhibition on viral RNA/protein synthesis, SB203580 was applied at 3 hpi, a time when early steps of SARS-CoV-2 life cycle (attachment, entry) are expected to occur. As shown in Fig. 3A, as compared to the Control, there was significantly less RNA in cells treated with SB203580, which suggested that p38 may be required for efficient synthesis of SARS-CoV-2 RNA. Like RNA, we also observed significantly less SARS-CoV-2 proteins in cells treated with SB203580, as compared to the control-treated cells (Fig. 3B, C).

Fig. 3.

Effect of SB203580 on levels of viral RNA and protein. A RNA level. Confluent monolayers of Vero cells, in triplicates, were infected with SARSCoV-2 for 1 h at MOI 5. SB203580 was added at 3 hpi and cells were harvested at 12 hpi to determine the levels of SARS-CoV-2 RNA by qRT-PCR. Threshold cycle (Ct) values were analysed to determine relative fold-change in copy numbers of total RNA and mRNA. Values are means ± SD and representative of the result of at least 3 independent experiments. Pair-wise statistical comparisons were performed using Student’s t test (*** =  P < 0.001). B Protein levels. Confluent monolayers of Vero cells were infected with SARS-CoV-2 at an MOI of 5. The inhibitor or DMSO was applied at 3 hpi and the cells were scrapped at 24 hpi to examine the levels of viral proteins by immunoblotting. C Quantitation of protein levels. The blots were quantified by densitometry (ImageJ) and the data are presented as mean with SD. The levels of viral proteins (upper panel), along with housekeeping GAPDH protein (lower panels) is shown. Pair-wise statistical comparisons were performed using Student’s t-test. *** = P < 0.001. Values are means ± SD and representative of the result of at least tree-independent experiments

SARS-CoV-2 induces activation of p38 MAPK

To further examine whether the SARS-CoV-2 induces p38 activation (phosphorylation), the cell lysate was collected at different time points and subjected to western blotting analysis. There were minimal or no detectable level of p-p38 in mock-infected cells as well as in cell lysates collected at 1 hpi, 4 hpi, and 12 hpi. The highest level of p-p38 was observed at ~ 8 hpi, while the total level of p38 as well as level of ß-actin (housekeeping gene) were at comparable level in all the samples (Fig. 4A, B), indicating elevated levels of protein synthesis at this time-point. This also corroborate with our previous findings of one-step growth curve [24], suggesting SARS-CoV-2 induces phosphorylation of p38 at later stage of its life-cycle.

Fig. 4.

SARS-CoV-2 activates p38 MAPK. Confluent monolayers of Vero cells, were infected with SARS-CoV-2 at MOI of 10 for 1 h, followed by washing with ice cold PBS and addition of fresh DMEM. Cells were scrapped at 1 hpi, 4 hpi, 8 hpi and 12 hpi to prepare the cell lysates. The levels of p38 phosphorylation at different time points and house-keeping control proteins were analysed in Western blot (immunoblotting) (A). The levels of phosphorylation-p38 (upper panel), along with total p38 MAPK (middle panels) and housekeeping β-actin gene (lower panels) are shown. The blots were quantified by densitometry (ImageJ) and the data are presented as mean with SD (B)

Discussion

Drug repositioning or repurposing is an effective strategy to immediately respond to the emerging diseases [2]. Based on virtual screen and in vitro effects, several therapeutics drugs were repurposed to treat COVID19. These includes, Remdesivir, Favipiravir, Itolizumab, Tocilizumab, Hydroxychloroquinine and Ivermectin [48, 54]. These medications decrease the number of patients hospitalized and the disease severity. [8, 15, 57], [53] However specific and effective antiviral medication against COVID-19 is lacking.

An alternative approach to developing antiviral strategies which has been minimally explored is to design drugs that target host cell proteins needed for virus replication. The family members of mitogen-activated protein kinase (MAPK) are the key kinases involved in most signal transduction pathways [4, 11, 27]. p38 is usually activated in response to stress therefore, it is also considered as a stress-activated MAPK [25]. p38 has four splice variants (isoforms) which include p38α (MAPK14), p38β (MAPK11), p38γ (MAPK12) and p38δ (MAPK13) [22]. A wide variety of viruses are known to directly interact with p38 or its substrates [41]. While some of the interactions are proviral others are inhibitory to virus replication. The previous studies have demonstrated that inhibitor targeting p38 MAPK suppresses SARS-CoV-2 replication [19]. However, the mechanisms underlying regulation of SARS-CoV-2 replication remains elusive. In this study, we provided the mechanistic insights on regulation of SARS-CoV-2 replication by p38 MAPK.

In order to examine the role of p38 signaling in SARS-CoV-2 infections, initially we propagated SARS-CoV-2 in p38 MAPK knockout cells. The reduction in the virus yield in p38-depleted (CRISPR/Cas9 knockout) cells and enhanced growth in cells transfected with the plasmid construct that expresses p38-ἀ MAPK, suggested that p38 MAPK signaling is a prerequisite for SARS-CoV-2 replication. Further, it was also demonstrated that SB203580 inhibitor-mediated suppression of SARS-CoV-2 replication is primarily due to the reduced levels of viral proteins and partly due to reduced levels of viral RNA but without any significant effect on viral attachment, entry, and budding. Our finding are in agreement with other studies on wherein p38 MAPK was shown to promote synthesis of viral proteins [1, 12, 19, 44, 51, 52, 61]. However p38 may also supports RNA synthesis [respiratory syncytial virus (RSV) and influenza A virus mRNA synthesis] [10] and viral assembly of HCV [9], which seems to be due to the involvement of different downstream effector molecules [5, 13].

A major risk factor in death of COVID-19 patients is hyper-induction of cytokine secretion. Since the blockade of p38 also dampens virus induced cytokine storm [3, 5, 14, 16, 17, 38, 43, 46, 49], the drugs targeting p38 may have dual effects, first in limiting virus production in the target cells and secondly, in dampening the cytokine storm.

Due to mutations at the drug-binding sites, viruses rapidly acquire drug-resistant variants, therefore, developing antiviral therapeutics is a major challenge [6, 29, 30, 45]. Whereas directly acting antiviral agents rapidly induce generation of drug-resistant viral mutants [30], host-directed therapies are less prone to generate drug-resistant mutants [6, 27, 28, 31, 32, 35, 36, 59] because viruses cannot easily regain the missing cellular functions by mutation [31, 36]. However, recent evidence suggests that resistance to host-directed antiviral agents can occur at a relatively low magnitude upon long-term limiting availability of the targeted cellular factor [20]. Based on these evidences, it seems unlikely that the host-directed agent (SB203580) used in the present study would generate potential drug-resistant SARS-CoV-2 mutants, however, further investigations are warranted.

Since host-directed agents interfere with the host cell metabolism, their use may be associated with side effects [37]. However, the large number of the host-directed agents licensed to treat cardiovascular and inflammatory diseases or cancers have minimal or no adverse side effects [50, 60]. However, further validation and in vivo efficacy of SB203580 in COVID-19 patients is essential before actually introducing it from the research into the clinical settings.

In conclusion, p38 MAPK serves as an essential cellular factor for the synthesis of SARS-CoV-2 proteins, and may serve as a novel target for antiviral drug development against COVID-19.

Funding

This work was supported by Indian Council of Agricultural Research, New Delhi (grant number IXX14586 to N-Ku and NASF/ABA-8027/2020–21 to N-Ku and B.R.G.)

Declarations

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Data availability

The original contributions presented in the study are included in the article further inquiries can be directed to the corresponding authors.