Coronaviruses (CoVs), single-stranded RNA viruses with a crown-shaped morphology, cause diseases in humans and animals. Human coronaviruses (HCoVs) include endemic alpha-coronaviruses (HCoV-229E and HCoV-NL63) and beta-coronaviruses (HCoV-OC43 and HCoV-HKU1) that infect the upper respiratory system and cause the common cold (Fehr and Perlman, 2015). From 2002-December 2003, Severe Acute Respiratory Syndrome CoV (SARS-CoV) emerged as a fatal zoonotic pathogen that caused 8,096 infections and 774 deaths globally, according to a report by the World Health Organization (WHO) (WHO, 2015). SARS-CoV infects the lower respiratory tract in humans and causes bronchitis and pneumonia symptoms (Holmes, 2003; Peiris
COVID-19 patients have common symptoms, such as fever, cough, and fatigue, along with sputum production and lymphopenia. Severe pneumonia has been diagnosed in COVID-19 patients based on clinical features of chest CT scans. Additionally, abnormal features, such as RNAemia, acute respiratory distress syndrome (ARDS), acute cardiac injury, incidence of grand-glass opacities, and significantly high levels of serum cytokines and chemokines, have also been observed in COVID-19 patients (Rothan and Byrareddy, 2020). Investigation of immune responses in COVID-19 patients will guide proper COVID-19 treatment (McKechnie and Blish, 2020). Due to high expression of interferons and interferon-stimulated genes (ISGs) in patients with severe cases of COVID-19, finely tuned interferon therapy should be considered (Park and Iwasaki, 2020). SARS-CoV, MERS-CoV, and SARS-CoV-2 infections have led to severe health hazards involving high global spread and high mortality. However, effective therapeutics and vaccines against these viral infections are currently unavailable.
SARS-CoV-2 was isolated from human airway epithelial cells and amplified in Vero E6 and Huh7 cell lines (Datta
STAT3 is a two-faced antiviral or proviral regulator, depending on the virus type and the host cell type (Kuchipudi, 2015; Chang
STAT1 and STAT2 are involved in interferon (IFN) signaling, which is stimulated by viral infections (Nan
In this study, we investigated the cellular responses and virus production in Vero cells and Calu-3 cells after infection with SARS-CoV-2. We found different patterns of STAT1 and STAT3 phosphorylation in the two cell lines after virus infection. We also investigated the effects of inhibitors that target STAT3 phosphorylation (JAK inhibitor I) and dimerization (STA-21, S3I-201) on SARS-CoV-2 production in Calu-3 cells.
African green monkey kidney Vero and Vero E6 cells and human airway epithelial Calu-3 cells were obtained from the Korean Cell Line Bank (Seoul, Korea). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 25 mM HEPES, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were incubated in 95% air and 5% CO2 at 37°C. SARS-CoV-2 (NCCP No. 43326) was obtained from the National Culture Collection for Pathogens (Osong, Korea).
Vero cells (2×105 cells/well in 6-well plates) were cultured in DMEM containing 10% FBS at 37°C in a CO2 incubator overnight (Park
Vero E6 cells (7×105 cells/well) were cultured in 6-well plates (Corning, NY, USA) for 12 h. The cells were washed with PBS and infected with 10-fold serial dilutions of SARS-CoV-2. After 1 h of incubation, supernatants were removed, and the wells were replenished with 3 mL DMEM/F12 medium (Thermo Fisher Scientific) containing 2% Oxoid agar and N-
Antibodies to STAT1 (Catalog No. 14994S), phospho-STAT1 (Tyr-701, Catalog No. 9167S), STAT3 (Catalog No. 12640S), phospho-STAT3 (Tyr-705, Catalog No. 9145S), poly-ADP ribose polymerase (PARP, Catalog No. 9542S), cleaved Caspase-3 (Catalog No. 9661S), c-Myc (Catalog No. 5605S), and cyclin D1 (Catalog No. 2978S) were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-β-actin monoclonal antibody was obtained from Sigma-Aldrich (Saint Louis, MO, USA).
MAPK/ERK kinase (MEK) inhibitor PD 98059 (Catalog No. P215), p38 inhibitor PD 169316 (Catalog No. P9248), stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) inhibitor SP 600125 (Catalog No. S5567), JAK2 kinase inhibitor AG490 (Catalog No. T3434), STAT3 dimerization inhibitor S3I-201 (Catalog No. SML0330), and STAT3 dimerization inhibitor STA-21 (Catalog No. SML2161) were purchased from Sigma-Aldrich. JAK inhibitor I (Catalog No. 420099), a potent ATP-competitive inhibitor of JAK1, JAK2, and JAK3, was purchased from Calbiochem (San Diego, CA, USA). All the inhibitors were dissolved in dimethyl sulfoxide (DMSO, Catalog No. 10378-73, Kanto Chemical, Tokyo, Japan). In the inhibitor assays, cells were preincubated with each inhibitor for 30 min before infection with SARS-CoV-2 at 0.1 or 0.5 MOI for 1 h and then the cells were cultured in DMEM containing 2% FBS for 48 h or 72 h.
Cell lysates from mock-infected Vero and Calu-3 cells and SARS-CoV-2-infected cells were prepared with cell lysis buffer (20 mM Tris-HCl pH 8.0, 5 mM EDTA, 150 mM NaCl, 100 mM NaF, 2 mM Na3VO4, 1% NP-40) and centrifuged at 14,000 rpm at 4°C for 20 min. Equal amounts of protein were separated on 4-12% Bis-Tris gradient gels (Thermo Fisher Scientific) and transferred onto nitrocellulose membranes. The membranes were blocked and incubated with antibody overnight at 4°C. Then, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody and immunoreactive bands were detected using an enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific).
Induction of STAT3 phosphorylation and phospho-STAT3 nuclear localization by SARS-CoV-2 were observed by indirect immunofluorescence and confocal microscopy. Calu-3 cells were cultured on glass coverslips in 12-well plates overnight. The cells were pretreated with DMSO, 25 μM AG490, 1 μM JAK inhibitor I, 20 μM S3I-201, or 10 μM STA-21 for 30 min and then infected with SARS-CoV-2 in PBS (0.5 MOI) for 1 h at 37°C. The cells were cultured in DMEM containing 2% FBS for 48 h and then treated with Leptomycin B (LMB, Cell Signaling Technology, 20 nM), an inhibitor of the nuclear export receptor CRM1, for 3 h. The cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100, and then blocked with 3% BSA. To detect STAT3 phosphorylation and phospho-STAT3 nuclear localization, cells were stained with phospho-STAT3 antibody (Cell Signaling Technology) for 2 h at room temperature. The cells were washed with PBST (0.1% Triton X-100 in PBS) containing 1% BSA and stained with Alexa Flour 488-conjugated secondary antibody (Thermo Fisher Scientific) for 1 h. Nuclei were stained with Hoechst 33258 (Thermo Fisher Scientific). The samples were observed by confocal laser scanning microscopy (CLSM, LSM 710, Carl Zeiss, Jena, Germany).
Vero cells (5×104 cells/well in 12-well plates) and Calu-3 cells (5×104 cells/well in 12-well plates) were cultured overnight. To investigate the effect of pathway-specific kinase inhibitors and STAT3 dimerization inhibitors (S3I-201 and STA-21) on SARS-CoV-2 production, cells were pretreated with DMSO, 1 μM JAK inhibitor I, 20 μM S3I-201, or 10 μM STA-21 for 30 min prior to virus infection. After cells were infected with SARS-CoV-2 in PBS (0.1 MOI) for 1 h at 37°C, 2 mL of DMEM containing 2% FBS was added to each well and the plates were incubated for an additional 72 h. Supernatants of virus-infected cells were collected and virus replication was quantified using real-time RT-PCR and the plaque formation assay.
Viral particles were collected from virus-infected cell culture supernatants (100 µL) and viral RNAs were isolated from the supernatants using the QIAamp Viral RNA Mini Kit (Catalog No. 52904, Qiagen, Hilden, Germany) according to the manufacturer’s instructions. cDNA (50 µL) was synthesized using the Reverse Transcription System Kit (Catalog No. A3500, Promega, Madison, WI, USA). To quantify expression of the RNA-dependent RNA polymerase (
Results are shown as the mean ± standard deviation. Differences between the samples were analyzed using an unpaired, 2-tailed nonparametric
The cellular response to virus infection can differ depending on the infected cell type, therefore we used two different host cell lines expressing SARS-CoV-2 receptor ACE2, Vero and Calu-3 (Shang
Higher production of virus can be associated with higher apoptosis of host cells. Therefore, we investigated expression of proliferation and apoptosis markers. We infected Vero cells and Calu-3 cells with SARS-CoV-2 and measured the levels of cleaved PARP and cleaved Caspase-3 proteins up to 72 h post infection. In Vero cells infected with SARS-CoV-2, the levels of cleaved PARP and cleaved Caspase-3 proteins increased significantly at 72 h (Fig. 2A). Compared with the mock-infected control, expression of c-Myc and cyclin D1, which are required for cell proliferation, decreased in Vero cells upon virus infection (Fig. 2A). The Vero E6 cell line is a derivative of the Vero cell line. Like Vero cells, the levels of cleaved PARP and cleaved Caspase-3 proteins increased and c-Myc and cyclinD1 proteins were decreased in SARS-CoV-2-infected Vero E6 cells during the culture period (Supplementary Fig. 1). In Calu-3 cells infected with SARS-CoV-2, the level of cleaved PARP protein increased only slightly and no cleaved Caspase-3 protein was present (Fig. 2B). Furthermore, c-Myc and cyclin D1 expression was maintained in SARS-CoV-2-infected Calu-3 cells and mock-infected Calu-3 cells (Fig. 2B). Therefore, apoptosis occurred in Vero cells, but not in Calu-3 cells, upon SARS-CoV-2 infection.
Because STAT1 and STAT3 are associated with cellular apoptosis and also known to contribute to virus production and pathogenesis, we determined phosphorylation levels of STAT1 (Tyr-701) and STAT3 (Tyr-705) in Vero and Calu-3 cells in response to SARS-CoV-2 infection. During the culture period, phosphorylation of STAT1 and STAT3 increased in mock-infected Vero cells. When we infect the cells with SARS-COV-2 or perform mock infection, we wash the cells with PBS, mock-infect or infect the viruses in PBS for 1 h. Then, the cells were cultured in a fresh DMEM medium containing 2% FBS. Therefore, this procedure can be a kind of factor-starvation for cells. We suppose that some unidentified changes resulting in STAT phosphorylation autonomously occurs in the mock-infected Vero cells. However, phosphorylation of STAT1 and STAT3 decreased drastically at 48 h in Vero cells infected with SARS-CoV-2 (Fig. 3A). These results are similar with the previous data published by other investigators regarding SARS-CoV infection in Vero E6 cells (Mizutani
Because STAT family members are phosphorylated by several kinases, including Janus kinases (JAKs) and mitogen-activated protein kinases (MAPKs) (Rawlings
Previously, small-molecule inhibitors that target STAT3 dimerization (e.g., STA-21, S3I-201) were evaluated as antivirals
To evaluate the effects of inhibitors that target STAT3 activation and function on SARS-CoV-2 production, we pretreated Vero and Calu-3 cells with DMSO, JAK inhibitor I, S3I-201, or STA-21 and then measured SARS-CoV-2 production using a plaque assay and qRT-PCR. In Vero cells, none of the inhibitors affected SARS-CoV-2 production. However, treatment with JAK inhibitor I, S3I-201, or STA-21 reduced SARS-CoV-2 production 30-100-fold in Calu-3 cells (Fig. 5). Therefore, the inhibition of STAT3 phosphorylation by JAK inhibitor I and the inhibition of pSTAT3 dimerization by S3I-201 or STA-21 hampered SARS-CoV-2 production suggesting that STAT3 phosphorylation and nuclear translocation is required. Cytotoxicity of the compounds at the concentrations used was minimal in Calu-3 cells (Supplementary Fig. 3).
STAT3 is known to regulate virus infection positively or negatively. The biology of STAT3 activation and regulation in response to SARS-CoV-2 infection has not yet been investigated in detail. By understanding the role of STAT3 in CoV pathophysiology, targeted therapeutics against STAT3 may be effective against CoVs, including the current pandemic SARS-CoV-2. In this study, we found that STAT3 is activated in Calu-3 cells upon SARS-CoV-2 infection and that STAT3-targeted inhibitors suppressed SARS-CoV-2 production.
SARS-CoV and SARS-CoV-2 enter host cells via ACE2 (Datta
Eventually, it is necessary to study cellular responses to virus infection using primary cells
Because the p38 MAPK inhibitor, PD169316, and JAK inhibitor I decreased phosphorylation of STAT1 and/or STAT3 at tyrosine residues after SARS-CoV-2 infection in Calu-3 cells, p38 MAPK, JAK1, and JAK3 are likely involved in STAT activation. Therefore, unidentified SARS-CoV-2 viral proteins are likely involved in activation of p38 MAPK, JAK1, and JAK3 leading to activation of STAT1 and STAT3 in Calu-3 cells. Because MAPK phosphorylates serine or threonine residues (Biondi and Nebreda, 2003; Matsuyama
The proviral functions of STAT3 have been reported for many viruses (Waris
In summary, we investigated the regulation of STAT1 and STAT3 upon SARS-CoV-2 infection in two different cell lines and found that cellular responses to SARS-CoV-2 differ depending on the host cell type (Fig. 6). SARS-CoV-2 enters host cells, such as Vero cells and Calu-3 cells, by binding to the cell surface receptor ACE2. SARS-CoV-2 proteins may be involved in the regulation of STAT signaling pathways. Upon SARS-CoV-2 infection of Vero cells, STAT1 and STAT3 are dephosphorylated and apoptosis occurs with high virus production. In contrast, upon SARS-CoV-2 infection of Calu-3 cells, phosphorylation of STAT1 and STAT3 persists for days after infection and proliferation-associated proteins, such as Cyclin D1 and c-Myc, are expressed with no apparent apoptosis and much lower virus production. In addition, phosphorylation and dimerization of STAT3 are required for virus production in Calu-3 cells. However, the inhibitors that target STAT3 had no effect on Vero cells. Although Vero cells and the derivative Vero E6 cells are widely used for SARS-CoV-2 amplification and drug screening studies, we propose a necessity to reevaluate the utility of the cells comparing with other cell lines because most appropriate cells must be used
This research was supported by grants from the National Research Foundation (NRF-2016M3A9B6916708, NRF-2020R1A2B5B02001806, NRF-2020M3A9I2107294) funded by the Ministry of Science and ICT in the Republic of Korea.